Hexatoxin variant peptides

Abstract

New insecticidal peptides, insecticidal proteins and polynucleotides—and their expression in culture and plants—are disclosed. In addition, the present disclosure provides methods of producing the peptides, proteins and polynucleotides; new processes; new production techniques; new formulations; and new organisms. The present disclosure is also related to and describes novel peptides called hexatoxin variant peptides (HVPs), we describe: genes encoding HVPs; combinations, mixtures, and compositions comprising HVP peptides and / or insecticidal proteins; and methods using the same that are useful for the control of pests.

Description

Attorney Docket No. 277702-582798HEXATOXIN VARIANT PEPTIDESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of and priority to, United States Provisional Application No. 63 / 775,466 filed on March 21, 2025, the contents of the aforementioned application are incorporated herein by reference in their entireties.SEQUENCE LISTING

[0002] This application incorporates by reference in its entirety the Sequence Listing XML entitled “277702-582798.xmT’ it was created on March 9, 2026, and is 257,824 bytes, and filed electronically herewith.TECHNICAL FIELD

[0003] New and useful peptide intermediates and insecticidal proteins, nucleotides, peptides, their expression in plants, methods of producing the peptides, new processes, production techniques, new formulations, and new combinations of insecticidal agents are provided for the control of insects are described and claimed.BACKGROUND

[0004] Deleterious insects represent a worldwide threat to human health and food security. Insects pose a threat to human health because they are a vector for disease. One of the most notorious insect-vectors of disease is the mosquito. Mosquitoes in the genus Anopheles are the principal vectors of Zika virus, Chikungunya virus, and malaria — a disease caused by protozoa in the genus Trypanosoma. Another mosquito, Aedes aegypti, is the main vector of the viruses that cause Yellow fever and Dengue. And, Aedes spp. mosquitos are also the vectors for the viruses responsible for various types of encephalitis. Wuchereria bancrofti and Brugia malayi, parasitic roundworms that cause filariasis, are usually spread by mosquitoes in the genera Culex, Mansonia, and Anopheles.

[0005] Similar to the mosquito, other members of the Diptera order have likewise plagued humankind since time immemorial. In addition to producing painful bites, Horseflies and deerflies transmit the bacterial pathogens of tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as a parasitic roundworm (Loa loa) that causes loiasis in tropical Africa.1641601282Attorney Docket No. 277702-582798

[0006] Blowflies (Chrysomya megacephala) and houseflies (Musca domes tica) will in one moment take off from carrion and dung, and in the next moment alight in our homes and on our food — spreading dysentery, typhoid fever, cholera, poliomyelitis, yaws, leprosy, and tuberculosis in their w ake.

[0007] Eye gnats in the genus Hippelates can carry7the spirochaete pathogen that causes yaws (Treponema pertenue), and may also spread conjunctivitis (pinkeye). Tsetse flies in the genus Giossina transmit the protozoan pathogens that cause African sleeping sickness (Trypanosoma gambiense and T. rhodesiense). Sand flies in the genus Phlebotomus are vectors of a bacterium (Bartonella bacilliformis) that causes Carrion's disease (Oroyo fever) in South America. In parts of Asia and North Africa, they spread a viral agent that causes sand fly fever (Pappataci fever) as well as protozoan pathogens (Lelshmania spp.) that cause Leishmaniasis.

[0008] Human food security is also threatened by insects. Insect pests indiscriminately target food crops earmarked for commercial purposes and personal use alike; indeed, the damage caused by insect pests can run the gamut from mere inconvenience to financial ruin in the former, to extremes such as malnutrition or starvation in the latter. Insect pests also cause stress and disease in domesticated animals. And, insect pests once limited by geographical and climate boundaries have expanded their range due to global travel and climate change.SUMMARY

[0009] The present disclosure describes novel, recombinant hexatoxin variant peptides (HVPs) having insecticidal activity against one or more insect species, and agriculturally acceptable salts thereof.

[0010] In addition, the present disclosure describes a hexatoxin variant peptide (HYP) having insecticidal activity7against one or more insect species, said HYP comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HYP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HYP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L. Y or S;? is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; X8is D,2641601282Attorney Docket No. 277702-582798G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K. L, N. P, Q. R, S, T, V or D; X15 is G, N. P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or an agriculturally acceptable salt thereof (SEQ ID NO: 131).

[0011] In addition, the present disclosure describes a combination comprising, consisting essentially of, or consisting of, two or more HVPs, each HVP independently having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (I): Xi-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that each of the HVPs in the combination does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3 is Y, A. or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7is Q or R,; X8is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or an agriculturally acceptable salt thereof.

[0012] In another aspect, the present disclosure describes a hexatoxin variant peptide (HVP) and a composition, comprising, consisting essentially of, or consisting of, one or more HVPs, and at least one agriculturally acceptable excipient, wherein the one or more HVPs each independently comprises or consists of an amino acid sequence that is at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an ammo acid sequence according to Formula (II): X1-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-X8-R-N-X9-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 132); wherein Xi is G, L, N, A, or E; X2 is S, L. or A; X3 is N or D; X4 is D, S, K, or E; X5 is D or P; Xe is A or N; X7 is T, V, or F; X8is E, A, or K; and X9 is E, A, or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.3641601282Attorney Docket No. 277702-582798

[0013] In another aspect, the present disclosure describes a hexatoxin variant peptide (HVP) and a composition, comprising, consisting essentially of, or consisting of. one or more HVPs, and at least one agriculturally acceptable excipient, wherein the one or more HVPs each independently comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (III): G-S-Q-Y-C-V-P-V-Xi-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-X2-R-N-X3-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 133); wherein Xi is N or D; X2 is E, A, or K; and X3 is E, A, or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3. 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0014] In another aspect, the present disclosure describes a hexatoxin variant peptide (HVP) and a composition, comprising, consisting essentially of, or consisting of, one or more HVPs, and at least one agriculturally acceptable excipient, wherein the one or more HVPs each independently comprises or consists of an ammo acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (IV): X1-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-E-R-N-E-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 134); wherein Xi is G, L, N, A. or E; X2 is S, L, or A; X3 is N or D; X4 is S. K, or E; X5 is D or P; Xe is A or N; X7 is T, V, or F; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1. 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0015] In another aspect, the present disclosure describes a hexatoxin variant peptide (HVP) and a composition, comprising, consisting essentially of, or consisting of, one or more HVPs, and at least one agriculturally acceptable excipient, wherein the one or more HVPs each independently comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (V): G-S-Q-Y-C-V-P-V-D-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-E-R-N-Xi-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 135); wherein Xi is A or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise 4641601282Attorney Docket No. 277702-582798the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.

[0016] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D. E, L, N, Q. S, T or G; X2is L, Y or S; X3 is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I. Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or a complementary polynucleotide sequence thereof.

[0017] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%. 91%. 92%. 93%. 94%. 95%. 96%.97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (II): X1-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-X8-R-N-X9-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 132); wherein Xi is G, L, N, A, or E; X2 is S, L, or A; X3 is N or D; X4 is D, S, K, or E; Xs is D or P; Xe is A or N; X7 is T, V, or F; Xs is E, A, or K; and X9 is E, A, or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0018] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 97%. 98%. 99% or 100% identical to an amino acid sequence according to Formula (III): G-S-Q-Y-C-V-P-V-X1-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-X2-R-N-X3-N-G-H- 5641601282Attorney Docket No. 277702-582798T-V-Y-Y-C-R-A (SEQ ID NO: 133); wherein Xi is N or D; X2is E, A, or K; and X3is E, A, or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0019] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (IV): X1-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-E-R-N-E-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 134); wherein Xi is G, L, N, A, or E; X2 is S, L, or A; X3 is N or D; X4 is S, K, or E; X5 is D or P; Xe is A or N; X7 is T, V, or F; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0020] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%. 91%. 92%. 93%. 94%. 95%. 96%.97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (V): G-S-Q-Y-C-V-P-V-D-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-E-R-N-Xi-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 135); wherein Xi is A or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0021] In addition, the present disclosure describes a vector comprising a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid 6641601282Attorney Docket No. 277702-582798sequence set forth in SEQ ID NO: 1. with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1: and wherein Xi is D. E, L. N, Q. S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4is I, T or V X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; Xi4is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D. K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E. or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or a complementary polynucleotide sequence thereof

[0022] In addition, the present disclosure describes a method of producing an HVP, the method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of. a polynucleotide operable to encode an HVP, or a complementary nucleotide sequence thereof, said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L. Y or S; X3 is Y, A. or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R.; X8is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; Xi4is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N. or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; (b) introducing the vector into a host cell; and (c) growing the host cell in a growth medium under conditions operable to enable expression of the HVP and secretion into the growth medium.

[0023] In addition, the present disclosure describes a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an HVP, said HVP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1,7641601282Attorney Docket No. 277702-582798with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q. S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4 is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N. or L; X25 is T, V, P, L. W. or G; X26 is Y. or K; or a complementary nucleotide sequence thereof.

[0024] In addition, the present disclosure describes a method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the hexatoxin variant peptide (HVP) as described herein, a combination comprising two or more HVPs as described herein, or a composition comprising at least one HVP and at least one excipient as described herein, to: the pest, a locus of the pest, a food supply of the pest, a habitat of the pest, or a breeding ground of the pest; a plant, a seed, a plant part, a locus of a plant, or an environment of a plant that is susceptible to an attack by the pest; an animal, a locus of an animal, or an environment of an animal susceptible to an attack by the pest; or a combination thereof.

[0025] In addition, the present disclosure describes a polynucleotide operable to hybridize under stringent hybridization conditions with a polynucleotide segment encoding an HVP, said HVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4 is I, T or V; X5 is M or V; X6is G, H, N, Q or D; X7 is Q or R,; X8is D, G, N or S; X9 is P, W. Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I. K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W. or G: X26 is Y, or K; or a complementary polynucleotide sequence thereof.8641601282Attorney Docket No. 277702-582798

[0026] In addition, the present disclosure describes a method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the hexatoxin variant peptide (HVP) as described herein, a combination comprising two or more HVPs as described herein, or a composition comprising at least one HVP and at least one excipient as described herein, to: the pest, a locus of the pest, a food supply of the pest, a habitat of the pest, or a breeding ground of the pest; a plant, a seed, a plant part, a locus of a plant, or an environment of a plant that is susceptible to an attack by the pest; an animal, a locus of an animal, or an environment of an animal susceptible to an attack by the pest; or a combination thereof.

[0027] In addition, the present disclosure describes a polynucleotide operable to hybridize under stringent hybridization conditions with a polynucleotide segment encoding an HVP, said HVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4 is I, T or V; X5 is M or V; X6is G. H, N. Q or D; X7 is Q or R,; X8is D, G. N or S; X9is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W. or G; X26 is Y. or K; or a complementary polynucleotide sequence thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 depicts a point-mutation heat map illustrating how a given mutation affects expression. The original sequence is show n on the x-axis on the bottom of the graph. The mutation and characteristics of said mutation are shown on the y-axis on the left of the graph. The effect of expression is color coded, with the key shown on the upper right of the graph.

[0029] FIG. 2 depicts a multiple-mutations heat map illustrating how a given mutation affects expression. The original sequence is shown on the x-axis on the bottom of9641601282Attorney Docket No. 277702-582798the graph. The mutation and characteristics of said mutation are shown on the y-axis on the left of the graph. The effect of expression is color coded, with the key shown on the upper right of the graph.

[0030] FIG. 3 depicts the activity of Hybrid+2 determined by fly injection. The controls from 20 different injection experiments were graphed as replicated using GraphPad. The data was analyzed using anon-linear regression and the LDso was determined to be 27.62 ng / ul with a R2 of 0.7732. The 90% confidence interval for the data is shown with the dotted lines.

[0031] FIG. 4 depicts LD50 insecticidal activity graphs for the fly injection dose responses for HVPs. Each variant with two or more doses has an LD50 calculated using a non-linear regression with the top constrained to 100, bottom constrained to 0 and hill slope constrained to 2.151. The LD50 values generated are then used to compare to the LD50 90% CI values to determine the impact of the mutation on the HVP’s activity vs Hybrid+2.

[0032] FIG. 5 depicts LD50 insecticidal activity graphs for the fly injection dose responses for HVPs with two or more changes from Hybrid+2. Each variant with two or more doses has an LD50 calculated using a non-linear regression with the top constrained to 100, bottom constrained to 0 and hill slope constrained to 2.151. The LD50 values generated are then used to compare to the LD5090% CI values to determine the impact of the mutation on the HVP’s activity vs Hybnd+2 (SEQ ID NO: 1).

[0033] FIG. 6 depicts expression curves for select HVPs. the number of gene copies inserted were compared to the overall expression observed for each strain. This is done for at least 6 up to 12 independent for each of the HVPs. A line of best fit with a slope forced through zero is used to determine the improvement of the HVPs expressibility per gene copy.

[0034] FIG. 7 shows the results for the injections of Hvla (“Omega,” SEQ ID NO: 247), Hvla-R38Y (SEQ ID NO: 256) and Hvlh (“Hybrid,” SEQ ID NO: 2). M. domestica (houseflies) were injected with hexatoxins to calculate a dose response curve. Mortality was observed approximately 24 hours after injection. The dose response for Hvla are the solid circles with the non-linear regression plotted as a solid black line, giving an LD50 of 1392 pmol / g. The dose response for Hvla-R38Y are the solid squares with the non-hnear regression plotted as a dashed black line, giving an LD50 of 1035 pmol / g. The dose response for Hvlh are the open circles with the non-hnear regression plotted as a solid grey line, giving an LD50 of 841 pmol / g.

[0035] FIG. 8 shows the average dose responses and fitted curves for variants of Hvla that were injected into houseflies. Here, AT. domestica (houseflies) were injected with 10641601282Attorney Docket No. 277702-582798Hv 1 a variants to determine a dose response. Mortality was observed approximately 24 hours after injection. The data points were fitted with a non-linear regression to calculate LD50.

[0036] FIG. 9 shows the resistance ratios obtained from the KO lines. Here, Drosophila melanogaster nAChR knockout (KO) lines were tested with hexatoxins using a topical assay. Resistance ratios were calculated by dividing the LD50 for the KO line by the LD50 for the control line. The a4 homozygous KO line was lethal therefore a point mutation that conveys neonic resistance, T227M, was tested with the hexatoxins except Hvlc. The 01 KO flies resulted in incalculable resistance ratios for Spear and Hvla because an LD50 was not reached with the highest peptide dose against the KO lines. Although some of the lines have a resistance ratio greater than 1, they are lower than what would be considered significant in commercial application as many chemical resistant ratios are greater than 25x.

[0037] FIG. 10 shows resistance ratios for spinosyn and the hexatoxin variant peptides (HVPs) in Drosophila melanogaster (Dm). Here, the graph shows the effect of Spinosyn and the hexatoxins against the a6 KO Drosophila line. Spinosyn has a resistance ratio of 71 where as the hexatoxins are no more than 2.

[0038] FIG. 11 shows the results from the injection assay of hexatoxins in Drosophila melanogaster nAChR 01 homozygous knockout (KO) lines. Resistance ratios were calculated by dividing the LD50 for the KO line by the LD50 for the control line. A) shows the resulting curves with standard error for three reps of flies injected with Spear. The 01 KO line is completely resistant Spear at doses greater than 106pmol / g. B) shows the resulting curves with standard error for three reps of flies injected with Hvl a. The 01 KO line is completely resistant Hvla at doses greater than 106pmol / g. C) shows the resulting curves with standard error for three reps of flies injected with Hvlc. The 01 KO line has an LD50 of 37,640 pmol / g whereas the control had an LD50 of 26 pmol / g, resulting in a resistance ratio of 1,448.

[0039] FIG. 12 shows a 3D model representation of hexatoxin Hvla and nAChR-01. The light grey ribbon structure represents the predicted structure for D. melanogaster nAChR-01 with the hexatoxin binding pocket represented as a surface. The dark grey ribbon structure represents the hexatoxin Hvla bound with nAChR-01. The toxin in the binding pocket is called out with the solid arrow. The open arrow points to the loop C of the nAChR-01.

[0040] FIG. 13 shows an interaction diagram for the amino acids in hexatoxin Hvla and nAChR-01 based on the predicted binding model. As shown here, the hexatoxin (Hxtx) amino acids and positions are listed on the left and the nAChR- 01 amino acids with position 11641601282Attorney Docket No. 277702-582798are listed on the right. Lines connect the amino acids between the two proteins represent predicted interactions.

[0041] FIG. 14 shows how in vivo data was used to validate computation AAG predictions. Here, using the AAG values calculated using the bound model of Hvla and Dm nAChR- / ? 1 and LD50S from houseflies injected with Hvla variants, a correlation can be calculated. This data has an R2of 0.3742.

[0042] FIG. 15 shows two-electrode voltage clamp (TEVC) results in a single cell after the addition of 1 pM of peptide. Here, recombinant nAChR a4 / pi was expressed in Xenopus oocytes, and the channel's function was probed by two-electrode voltage clamp (TEVC). Response signal was normalized to the maximum Acetylcholine signal.Acetylcholine (ACh) was pulsed resulting in inward current (dark box indicates when it was added, light grey line is normalized response). Hvla was added prior to ACh followed by the addition of Ach (grey). The application of Hvla resulted in an inward current without the presence of ACh (dark filled arrow ), followed by a substantial inward current enhancement in the presence of Ach (light filled arrow). The same was conducted for Spear and a similar inward current w as observed (black line). For Hvlc (medium grey line), the direct activation of nAChR a4 / pi was barely observed, however, an enhancement of inward current in the presence of Ach was overserved as the other two toxins.

[0043] FIG. 16, shows the results of select mutations to residues in pi receptor within the binding interface. Mutations were made at sites E206 and 1231. i.e.. E206R, 1231R, and E206N. The samples were then tested with 100 nM of peptide as described herein, and the ACh induced current w as measured and compared to the no peptide controls for three replicates. The average Ach induced current enhancement was plotted (student's t test) and analyzed. For both Hvla and Spear, E206R significantly reduced the ACh induced enhancement. For the mutation 1231R, the ACh enhancement w as significantly altered for both peptides; Hvla showed a significant reduction whereas Spear showed an increase in signal. For the mutation E206N, only Hvla showed a significant decrease in ACh induced enhancement. *** = 0.001, **= 0.01.

[0044] FIG. 17 shows the results of an injection assay inzl / jzs mellifera. Here, Apis mellifera were injected with hexatoxins and the LD50 was calculated based on three replicates. Hvla had an LD50 of 3232 pmol / g whereas Hvlh and Hvla-R38Y had LD50S of 735 and 762 pmol / g, respectively. The mutation R38Y on Hvla makes position 38 more like Hvlh and results in Hvlh-like activity.12641601282Attorney Docket No. 277702-582798

[0045] FIG. 18 shows a phylogenetic tree of the identified nAChR subunits analyzed.(A) Protein sequences were collected for genes annotated as acetylcholine receptors from 22 species of insects of scientific and / or commercial interest distributed throughout 8 different taxonomic orders using the Uniprot database. The collected sequences were aligned with the MAFFT algorithm version 7.511 and manually examined to remove muscarinic receptors and other non-nicotinic receptor sequences, sequence duplications, and truncated sequence isoforms, leaving 204 non-duplicated nicotinic receptor subunit sequences. Additionally, 42 genes annotated as nAChR beta 1 or beta-1 like subunits were collected from the NCBI protein database from a partially overlapping set of species. Both sets of sequences were combined and realigned with MAFFT algorithm version 7.511, method FFT-NS-i and used to generate a phylogenetic tree of all identified nAChR subunits found in these species using a neighbor joining method. The tree was rooted between the Drosophila melanogaster nAChR beta 1 subunit and outgroup sequences, leading to a well-resolved cluster of nAChR beta 1 subunits that were all within 0.2 substitutions / position of each other and more than 0.25 substitutions / position away from other nAChR subunits. (B) Phylogenic tree detail focus on nAChR beta 1, alpha 1, and alpha 8 / beta 2 clusters. Horizontal branch length is given in substitutions / position. Sequences for the nAChR beta 1 are partially duplicated, as some were obtained as annotated single sequences found in the NCBI databases and others as part of a full collection of all nAChR genes from more extensively sequenced insects available in the Uniprot database. The nAChR beta 1 sequence members are all within 0.2 substitutions / position of each other and are well-annotated as either a “beta 1” or “beta 1-like” nAChR. The nAChR alpha 1 sequences are a mostly well annotated and similar in sequence, but the nAChR alpha 8 I beta 2 cluster is more divergent in general and has a greater number of annotation mistakes. These clusters differ from one another by approximately 0.10 substitutions / position, but all are 0.3 to 0.4 substitutions / position different from the nAChR beta 1 cluster. (C) Phylogenic tree detail focus on nAChR alpha 4, alpha 3, and alpha 2 clusters. Horizontal branch length is given in substitutions / position. All three clusters are reasonably well annotated with moderate sequence conservation between species. The clusters differ from one another by approximately 0.15 substitutions / position, but all are 0.3 to 0.4 substitutions / position different from the nAChR beta 1 cluster. (D) Phylogenic tree detail focus on nAChR alpha 7, alpha 6, and alpha 5 clusters. Horizontal branch length is given in substitutions / position. All three clusters less well annotated with a lower sequence conservation between species than for alpha subunits 1-4. The alpha 6 and alpha 7 clusters differ from one another by approximately 0.2 substitutions / position, and the 13641601282Attorney Docket No. 277702-582798alpha 5 is quite different with about 0.5 substitutions / position different from alpha 6 and 7. All three clusters are > 0.5 substitutions / position different from the nAChR beta 1 cluster. (E) Phylogenic tree detail focus on divergent nAChR subunits. These are often annotated as alpha 9, alpha 10, or beta 3 subunits, but are also frequently mis-annotated as well. Horizontal branch length is given in substitutions / position. As their name suggests, these subunits have not only a quite a different sequence from both the other nAChR sequences, but also from each other. These sequences vary by 1-2 substitutions / position with nAChR beta 1 subunit, often sharing only the core residues required to form a functional receptor.

[0046] FIG. 19 shows the alignment of nAChR pi subunits from 42 insect species within 13 different taxonomic orders. Residue numbers correspond to the Drosophila melanogaster sequence. Residues indicated with an arrow correspond to the binding site for the insecticidal peptide of the present disclosure. The residues on the nAChR pi subunit are T153, 1154, D155, V156, T157, Y158, Q163, T165, 1167, KI 69, 1205, E206, V207, P208, Y210, N212, Y214, E223, Y229, 1231, T292, L294, V295, P503, H504, E507, Y508, and V509.DETAILED DESCRIPTION

[0047] DEFINITIONS

[0048] The term “5’-end” and “3’-end” refers to the directionality, i.e., the end-to-end orientation of a nucleotide polymer (e.g., DNA). The 5 ’-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.

[0049] “5’- and 3 ’-homology' arms” or “5’ and 3’ arms” or “left and right arms” refers to the polynucleotide sequences in a vector and / or targeting vector that homologously recombine with the target genome sequence and / or endogenous gene of interest in the host organism in order to achieve successful generic modification of the host organism’s chromosomal locus.

[0050] “ACTX” or “ACTX peptide” or “atracotoxin” or “hexatoxin” or “HXTX” or “HXTX peptide” (all used interchangeably) refer to venom toxins isolated from spiders belonging to the Hexathelidae, Atracidae. Macrothelidae, and Porrhothelidae family of spiders, or variants or mutants thereof. The Hexathelidae family of spiders formerly contained the Atracidae, Macrothelidae, and Porrhothelidae families of spiders; however, molecular phylogenetics revealed that Hexathelidae was not monophyletic, thus the genera Atracidae, Macrothelidae and Porrhothelidae were split off into new families. See Hedin et al., Phylogenomic reclassification of the world’s most venomous spiders (Mygalomorphae,14641601282Attorney Docket No. 277702-582798Atracidae), with implications for venom evolution. Sci Rep. 2018; 8: 1636. One such spider that produces these venom toxins is known by its common name as the Australian Blue Mountains Funnel-web Spider, which includes three genera: Atrax, Hadronyche, and Illciwarra, comprising 35 species, e.g., the species Hadronyche versuta and Atrax robustus. For the sake of brevity, as used herein, venom toxins isolated from the Hexathelidae, Atracidae, Macrothelidae, and Porrhothelidae families of spiders are all referred to herein as “ACTX” or “ACTX peptide7’ or "atracotoxin” or "hexatoxin" or " HXTX or " HXTX peptide.” Examples of ACTX peptides isolated from Atracidae family species are the Omega-ACTX, Kappa-ACTX, and U-ACTX peptides. See “HXTX” below.

[0051] ACTX peptides, such as Hvla (“Omega,” SEQ ID NO: 247), Hvlh (“Hybrid.” SEQ ID NO: 2), Spear (SEQ ID NO: 1). and Hvlc (“Kappa,” SEQ ID NO: 248). are Cysteine Rich Insecticidal Peptides (CRIPs). CRIPs are peptides rich in cysteine residues that, in some embodiments, are operable to form disulfide bonds between such cysteine residues. A “cysteine residue” refers to a cysteine amino acid, and a “disulfide bond” (or “disulfide bridge”) refers to a covalent bond between two cysteine residues derived by the coupling of two thiol groups on their side chains. As used herein, the term “cystine” refers to an oxidized cysteine-dimer. Cystines are sulfur-containing amino acids obtained via the oxidation of two cysteine molecules, and are linked with a disulfide bond. In some embodiments, a disulfide bond occurs via the oxidative folding of two different thiol groups (-SH) present in a polypeptide. In some embodiments, a polypeptide can comprise four, six, or eight different thiol groups (i.e., four, six, or eight cysteine residues each containing a thiol group); thus, in some embodiments, a polypeptide can form two, three, or more intramolecular disulfide bonds. The disulfide bonds found within a CRIP contribute to the folding, three-dimensional structure, and activity of the hexatoxins. The cysteine-cysteine disulfide bonds, and the three-dimensional structure they form, play a significant role in the insecticidal nature of these insecticidal peptides.

[0052] In some embodiments, a CRIP may comprise an inhibitor cystine knot (ICK) motif. As used herein, an “ICK motif’ refers to a disulfide bond structural motif comprising three disulfide bonds. As used herein, a “disulfide bond structural motif’ refers to the three-dimensional spatial arrangement of disulfide bonds that is shared between two or more proteins (e.g., an ICK motif). Typically, the ICK motif occurs when two disulfide bonds and their connecting subunits form an internal ring structure, and that structure is then threaded by the third disulfide bond to form an interlocking and cross braced structure; i.e., an ICK comprises an embedded ring formed by two disulfide bonds and their connecting subunits,15641601282Attorney Docket No. 277702-582798which is threaded by a third disulfide bond. In some embodiments, a protein having an ICK motif has at least 6 motif-forming cysteine residues (i.e., 3 pairs of motif-forming cysteine residues), wherein the 3 pairs of motif-forming cysteine residues are operable to form the three disulfide bonds. Note: there may be other cysteine residues in a protein having an ICK motif, but the motif-forming cysteine residues are those residues that contribute to the disulfide bond structural motif (i.e., the ICK motif).

[0053] Non-limiting examples of hexatoxins comprising a disulfide bond structural motif that is an inhibitor cystine knot (ICK) motif include Hvla (" Omega." SEQ ID NO: 10), Hvlh (“Hybrid,” SEQ ID NO: 8), Spear (SEQ ID NO: 9), and Hvlc (“Kappa,” SEQ ID NO: 11), wherein the ICK motif comprises at least 6 half-cy stine core amino acids having at least three disulfide bridges, wherein the 3 disulfide bridges are covalent bonds, and of the six half-cystine residues the covalent disulfide bonds are between the first (C1) and fourth (C4), the second (C2) and fifth (C5), and the third (C3) and sixth (C6), half-cystines, of the six core half-cystine amino acids starting from the N-terminus amino acid. In general, this type of peptide comprises a beta-hairpin secondary structure, normally composed of residues situated between the fourth and sixth core half-cystines of the motif, the hairpin being stabilized by the structural crosslinking provided by the motifs three disulfide bonds.

[0054] “Administering” or “administration” or “administer” means to dispense and / or otherwise provide, and refers to any method of application or route of administration. For example, administering can refer to. e.g., application of a peptide of the present disclosure, or a composition comprising the peptide of the present disclosure and one or more excipients, e.g., a sprayable composition, a foam; a burning formulation; a fabric treatment; a surfacetreatment; a dispersant; a microencapsulation, and the like. By “co-application” or “coadminister” it is meant that two or more components are applied or administered at the same time; or a one or more components are applied or administered just prior to, or just after the application the other one or more components. For example, in some embodiments, a first peptide of the present disclosure and a peptide of the present disclosure, wherein the first and second peptide of the present disclosure are the same or different, can be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

[0055] “ADN1 promoter” refers to the DNA segment comprised of the promoter sequence derived from the Schizosaccharomyces pombe adhesion defective protein 1 gene.

[0056] “Affect” refers to how a something influences another thing, e.g., how a peptide, polypeptide, protein, drug, or chemical influences an insect, e.g., a pest.16641601282Attorney Docket No. 277702-582798

[0057] “Agent"’ refers to one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryote organisms, or eukaryote organisms, and agents produced therefrom.

[0058] “Agonize” or “agonism” or “agonistic"’ generally refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor. In some embodiments, an agonist may trigger (e.g., initiate or promote), partially or fully enhance, stimulate, increase, or activate one or more biological activities. An agonist may mimic the action of a naturally occurring substance (e g., a naturally occurring ligand). For example, in some embodiments, an agonist refers to a binding molecule that binds to a receptor and is capable of initiating, mimicking, or stimulating a reaction or activity that is less than, similar to or the same as, or greater than, that initiated, mimicked, or stimulated by the receptor’s natural ligand. In some embodiments, an agonist can act in a similar manner to how a ligand engages and activates its cognate receptor. In other embodiments, an agonist is capable of inducing, augmenting, enhancing, and / or stimulating the activation of a receptor or a signal transduction pathway associated with the receptor.

[0059] “Agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation.

[0060] “Agriculturally acceptable salt” is synonymous with pharmaceutically acceptable salt, and as used herein refers to a compound that is modified by making acid or base salts thereof. In some embodiments, agriculturally acceptable salts, hydrates, solvates, crystal forms and individual isomers, enantiomers, tautomers, diastereomers and prodrugs of the WT Omega peptide described herein can be utilized. Non-limiting examples of agriculturally acceptable salts include: acid addition salts, formed with inorganic acids; acid addition salts formed with organic acids; or salts formed when an acidic proton present in the parent compound is replaced by a metal ion. e.g., an alkali metal ion, aluminum ion; or coordinates with an organic base such as ethanolamine, and the like. Other non-limiting examples of agriculturally acceptable salts include: hydrochloride; sodium; sulfate; acetate; phosphate or diphosphate; chloride; potassium; maleate; calcium; citrate; mesylate; nitrate; tartrate; aluminum; gluconate; any organic or inorganic addition salt; alkali metal salts (sodium salts, potassium salts, etc.); alkaline earth metal salts (calcium salts, magnesium salts, etc.); salts derived from inorganic or organic bases (e.g., sodium, potassium, lithium,17641601282Attorney Docket No. 277702-582798ammonium, calcium, magnesium, ferrous, zinc, copper, manganous, aluminum, ferric, manganic salts, isopropylamine, diethylamine, ethanolamine, piperidine, tromethamine, choline, and the like).

[0061] '‘Agroinfection” means a plant transformation method where DNA is introduced into a plant cell by using Agrobacteria A. tumefaciens or A. rhizogenes.

[0062] “Alignment” refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide or protein sequences) for the purpose of determining their relationship to each other. Alignments are typically performed by computer programs that apply various algorithms, however it is also possible to perform an alignment by hand. Alignment programs typically iterate through potential alignments of sequences and score the alignments using substitution tables, employing a variety of strategies to reach a potential optimal alignment score. Commonly-used alignment algorithms include, but are not limited to, CLUSTALW, (see, Thompson J. D., Higgins D. G., Gibson T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research 22: 4673-4680. 1994); CLUSTALV. (see, Larkin M. A., et al., CLUSTAL W2, ClustalW and ClustalX version 2, Bioinformatics 23(21): 2947-2948, 2007); Jotun-Hein, Muscle et al., MUSCLE: a multiple sequence alignment method with reduced time and space complexity, BMC Bioinformatics 5: 113, 2004); Mafft, Kalign, ProbCons, and T-Coffee (see Notredame et al., T-Coffee: A novel method for multiple sequence alignments, Journal of Molecular Biology 302: 205-217, 2000). Exemplary programs that implement one or more of the above algorithms include, but are not limited to MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis. 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio from Accel rys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif. 92121). In some embodiments, an alignment will introduce “phase shifts” and / or “gaps” into one or both of the sequences being compared in order to maximize the similarity7betw een the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.

[0063] “Allosteric site” as used herein refers to a binding site that is topographically distinct from the orthosteric binding site. The term “allosteric modulator” refers to a molecule, moiety or substance that binds predominantly to a site on a molecule other than the active site or orthosteric binding site and causes conformational change in the molecule. “Allosteric modulators” as used herein, refer to “positive allosteric modulators” and “negative allosteric modulators.” “Positive allosteric modulators” refer to modulating18641601282Attorney Docket No. 277702-582798molecules that increase, induce, stimulate, open, activate, facilitate, enhance activation, sensitize or up regulate a receptor or pathway of interest caused by the binding of a different compound to the receptor. '‘Negative allosteric modulators" refer to modulating molecules that reduce, decrease, block, prevent, delay activation, inactivate, desensitize or dow n regulate the biological activity and / or expression of a receptor or pathway of interest caused by the binding of a different compound to the receptor. In some embodiments, a “positive allosteric modulator’7can refer to an agent that binds a receptor (e.g., the nAChR), at a site other than that bound by the natural ligand (e.g., ACh) and enhances the activity of the receptor in response to the natural ligand.

[0064] “Alpha-MF signal’’ or “aMF secretion signal” refers to a protein that directs nascent recombinant polypeptides to the secretory pathway.

[0065] '‘Applying” or “application” or “apply” or '‘administering” or '‘administration” or “administer” means to dispense and / or otherwise provide, and refers to any method of application or route of administration. For example, apply ing can refer to, e.g., application of an HVP or an agriculturally acceptable salt thereof; or application of an HVP-insecticidal protein or agriculturally acceptable salt thereof, as a combination, a mixture, or a composition further comprising one or more excipients, e g., a sprayable composition, a foam; a burning formulation; a fabric treatment; a surface-treatment; a dispersant; a microencapsulation, and the like. By “co-application” or “co-administer” it is meant that two or more components are applied or administered at the same time; or a one or more components are applied or administered just prior to, or just after the application the other one or more components. For example, in some embodiments, a first HVP and a second HVP, wherein the first and second HVP can be the same or different, can be applied or administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

[0066] “BAAS” means barley alpha-amylase signal peptide, and is an example of an ERSP. One example of a BAAS is a BAAS having the amino acid sequence of SEQ ID NO: 101 (NCBI Accession No. AAA32925.1).

[0067] “Binary vector” or “binary expression vector” means an expression vector which can replicate itself in both E. coli strains and Agrobacterium strains. Also, the vector contains a region of DNA (often referred to as t-DNA) bracketed by left and right border sequences that is recognized by virulence genes to be copied and delivered into a plant cell by Agrobacterium.

[0068] “Binding” typically refers to an association (e.g., non-covalent or covalent) between or among two or more entities (e.g., a first molecule and a second molecule or target 19641601282Attorney Docket No. 277702-582798molecule). “Direct binding” involves a direct association between two molecules, due to, e.g., covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water badges; “indirect binding” involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can ty pically be assessed in any of a variety of contexts — including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). With regard to the binding of a first molecule to a second molecule or a target molecule, the term “specific binding” or “specifically binds to” or “binds specifically to” means binding that is measurably different from a non-specific interaction. Specific binding can be measured, e.g., by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. In some embodiments, the ability of a first molecule to specifically bind to a second molecule or a target molecule can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance (SPR) technique, and traditional binding assays known by those having ordinary skill in the art. The degree of specific binding or “affinity” between a first molecule and second molecule or target molecule can be measured by common methods known in the art, including those described herein. The affinity' of a first molecule to a second molecule (e.g., a target molecule) can generally be represented by the dissociation constant (“KD”). The term “KD,” as used herein, is intended to refer to the dissociation equilibrium constant of a particular compoundprotein or protein-protein interaction (e.g., a first molecule and a second molecule or target molecule). In some embodiments, the affinity of a first molecule is described in terms of the KD for an interaction between such first molecule and its target, e.g., a second molecule. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction. Thus, the terms “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target or “binds specifically to” the same, as used herein, can be exhibited, for example, by a molecule having a KD for the target of 104M or lower, alternatively 105M or lower, alternatively 106M or lower, alternatively 107M or lower, alternatively 10sM or lower, alternatively 109M or lower, alternatively 10 M or lower.20641601282Attorney Docket No. 277702-582798alternatively 1011M or lower, or alternatively 1012M or even lower, as determined by, e.g., an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance (SPR) technique, and traditional binding assays known by those having ordinary skill in the art.

[0069] “Bioavailability” refers to refers to the concentration of a molecule (e.g., enzyme, peptide, polypeptide, or protein) available for delivery to, and uptake by, a cell, tissue, and / or biological compartment. In some embodiments, increased and / or prolonged bioavailability refers to the enhanced ability of a peptide, polypeptide, protein, or composition containing the same, to be delivered to and / or or taken up by a cell, tissue, or biological compartment (e.g., enhanced and / or increased absorption into the blood or hemolymph; or enhanced and / or increased delivery to the brain). Thus, in some embodiments, bioavailability refers to the rate and extent to which the active ingredient or active moiety' is absorbed from a drug product, and becomes available at the site of action. In some embodiments, the methods and / or peptides, polypeptides, proteins and / or HVPs of the present disclosure provide increased bioavailability of an HVP. In some embodiments, bioavailability is affected by the extent and rate at which the active moiety (drug or metabolite) enters systemic circulation (e.g., in a pest, such as a deleterious insect that consumes commercial and / or valuable crops, and / or affects human or commercially relevant animals), thereby accessing the site of action. In some embodiments, bioavailability for a given formulation provides an estimate of the relative fraction of the orally administered dose that is absorbed into the systemic circulation. For example, in some embodiments, low bioavailability is most common with oral dosage forms of poorly water-soluble, slowly absorbed drugs. Insufficient time for absorption in the gastrointestinal tract is a common cause of low bioavailability. If the drug does not dissolve readily or cannot penetrate the epithelial membrane (e.g., if it is highly ionized and polar), time at the absorption site may be insufficient. In some embodiments, orally administered drugs must pass through the intestinal w all, which is a common site of first-pass metabolism (metabolism that occurs before a drug reaches systemic circulation). Thus, many drugs may be metabolized before adequate plasma concentrations are reached.

[0070] “Biomass” refers to any measured plant product.

[0071] “bp” or “base pair” refers to a molecule comprising tw o chemical bases bonded to one another forming a. For example, a DNA molecule consists of two winding strands, wherein each strand has a backbone made of an alternating deoxyribose and phosphate groups. Attached to each deoxyribose is one of four bases, i.e., adenine (A),21641601282Attorney Docket No. 277702-582798cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.

[0072] “Bt -resistant” or “Bt-resi stance” or “Bt-resistant insect” or “Bacillus thuringiensis-toxin-resis ant insects” refers to a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product (e.g., Bt) to achieve the expected level of control when used against that pest species.

[0073] "cDN A” or “copy DNA” or “complementary DNA” refers to a molecule that is complementary to a molecule of RNA. In some embodiments, cDNA may be either singlestranded or double-stranded. In some embodiments, cDNA can be a double-stranded DNA synthesized from a single stranded RNA template in a reaction catalyzed by a reverse transcriptase. In yet other embodiments. “cDNA” refers to all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3’ and 5’ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the protein. In some embodiments, “cDNA” refers to a DNA that is complementary to and derived from an mRNA template.

[0074] “CEW” refers to Com earworm.

[0075] ‘Cleavable Linker” see Linker.

[0076] “Cloning” refers to the process and / or methods concerning the insertion of a DNA segment (e.g., usually a gene of interest) from one source and recombining it with a DNA segment from another source (e.g., usually a vector, for example, a plasmid) and directing the recombined DNA, or “recombinant DNA” to replicate, usually by transforming the recombined DNA into a bacteria or yeast host.

[0077] “Coding sequence” or “CDS” refers to a polynucleotide or nucleic acid sequence that can be transcribed (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) into a peptide, polypeptide, or protein, when placed under the control of appropriate regulatory sequences and in the presence of the necessary transcriptional and / or translational molecular factors. The boundaries of the coding sequence are determined by a translation start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A transcription termination sequence will usually be located 3’ to the coding sequence. In some embodiments, a coding sequence may be flanked on the 5’ and / or 3’ ends by untranslated regions. Generally, those having ordinary skill in the art distinguish the terms “coding sequence from the terms “open reading frame” and “ORF,” based upon the fact that the broadest definition of “open reading frame” simply contemplates a series of codons that 22641601282Attorney Docket No. 277702-582798does not contain a stop codon. Accordingly, while an ORF may contain introns, the coding sequence is distinguished by referring to those nucleotides (e.g., concatenated exons) that can be divided into codons that are actually translated into amino acids by the ribosomal translation machinery (i.e., a coding sequence does not contain introns); however, as used herein, the terms “coding sequence”; “CDS”; “open reading frame”; and “ORF,’ are used interchangeably, and all refer to a polynucleotide or nucleic acid sequence that can be transcribed (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) into a peptide, polypeptide, or protein, when placed under the control of appropriate regulatory sequences and in the presence of the necessary transcriptional and / or translational molecular factors.

[0078] “Codon optimization” refers to the production of a gene in which one or more endogenous, native, and / or wild-type codons are replaced with codons that ultimately still code for the same amino acid, but that are of preference in the corresponding host.

[0079] “Combination” refers to the result of combining two or more separate components (e.g., a first component and one or more additional components). Thus, as used herein, a “combination” refers to an association of two or more separate components, e.g., the association of a first HVP, and one or more additional HVPs; wherein the first HVP and one or more additional HVPs are the same or different.

[0080] In some embodiments, a combination can be a “mixture.” For example, in some embodiments, a mixture refers to a combination of a first component, and one or more additional components, wherein the first component and the one or more additional components are present together in a single entity (e.g., a single unit). Thus, in some embodiments, a mixture can comprise a first component, and one or more additional components, wherein the first component and the one or more additional components are present in admixture for simultaneous administration. Accordingly, in some embodiments, a combination can refer to the association of a first HVP, and one or more additional HVPs; wherein the first HVP and one or more additional HVPs are the same or different; and wherein the first HVP and one or more additional HVPs are present in a single entity (e.g., an admixture for simultaneous administration).

[0081] In some embodiments, a combination can comprise a first component, and one or more additional components, wherein the first component and the one or more additional components, are present separately (e.g., more than one unit). For example, in some embodiments, a combination can comprise a first component, and one or more additional components, wherein the first component and the one or more additional components may be23641601282Attorney Docket No. 277702-582798administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

[0082] Accordingly, in some embodiments, a combination can refer to the association of a first HVP, and one or more additional HVPs; wherein the first HVP and one or more additional HVPs are the same or different; and wherein the first HVP and one or more additional HVPs are present separately (e.g., different units for separate, sequential, simultaneous, concurrent or chronologically-staggered administration).

[0083] In some embodiments, a combination can refer to the separate, sequential, simultaneous, concurrent or chronologically-staggered application of two or more separate components (e.g., a first HVP, and one or more additional HVPs; wherein the first HVP and one or more additional HVPs are the same or different).

[0084] For example, in some embodiments, a '‘combination” refers to the result of a simultaneous application of both a first HVP, and one or more additional HVPs; wherein the first HVP and one or more additional HVPs are the same or different.

[0085] In another embodiment, a ‘'combination” refers to the result of a separate application of a first HVP, and one or more additional HVPs; wherein the first HVP and one or more additional HVPs are the same or different.

[0086] In a further embodiments, a “combination” refers to the result of a sequential application of two or more separate components, e.g., a first application of a first HVP, followed by a second application of one or more additional HVPs (wherein the first HVP and one or more additional HVPs are the same or different), or vice versa. Where the application is sequential or separate, the delay in applying the second component should not be such as to lose the beneficial effect of the combination.

[0087] “Complementary” refers to the topological compatibility’ or matching together of interacting surfaces of two polynucleotides as understood by those of skill in the art. Thus, two sequences are '‘complementary” to one another if they are capable of hybridizing to one another to form a stable anti-parallel, double-stranded nucleic acid structure. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions. Thus, the polynucleotide whose sequence 5'-TATAC-3' is complementary to a polynucleotide whose sequence is 5’-GTATA-3’.24641601282Attorney Docket No. 277702-582798

[0088] “Conditioned medium'’ means the cell culture medium which has been used by cells and is enriched with cell derived materials but does not contain cells.

[0089] “Conservative amino acid substitutions” refers to modifications that are physically, biologically, chemically or functionally similar to the corresponding reference amino acid, e.g., similar size, shape, electric charge, chemical properties, including the ability7to form covalent or hydrogen bonds, or the like. For example, conservative amino acid substitutions include those in which the amino acid residue is replaced with another amino acid residue from the same side chain family, e.g., serine may be substituted for threonine. Amino acid residues are usually divided into families based on common, similar side-chain properties, such as: 1. nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, methionine), 2. uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, ty rosine, proline, cysteine, tryptophan), 3. basic side chains (e.g., lysine, arginine, histidine, proline), 4. acidic side chains (e.g., aspartic acid, glutamic acid), 5. beta-branched side chains (e.g., threonine, valine, isoleucine), and 6. aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A conservative substitution may also involve the use of a non-natural amino acid.

[0090] ‘Copy number” refers to the number of identical copies of a vector, an expression cassette, an amplification unit, a gene or indeed any defined nucleotide sequence, that are present in a host cell at any time. For example, in some embodiments, a gene or another defined chromosomal nucleotide sequence may be present in one, two. or more copies on the chromosome. An autonomously replicating vector may be present in one, or several hundred copies per host cell.

[0091] “Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.

[0092] “Culturing” refers to the propagation of organisms on or in various kinds of media. For example, the term “culturing” can mean growing a population of cells under suitable conditions in a liquid or solid medium. In some embodiments, culturing refers to fermentative recombinant production of a heterologous polypeptide of interest and / or other desired end products (typically in a vessel or reactor).

[0093] “Cyclic” or “cyclized” refers to a molecule comprising a sequence of amino acid residues or analogues thereof without free amino and carboxy termini. In some embodiments, a cyclized peptide comprises a linkage between all amino acids in the peptide via amide (peptide) bonds, but other chemical linkers are also possible. In some25641601282Attorney Docket No. 277702-582798embodiments, an LN subunit and an Lc subunit can be fused via a peptide bond, thus forming a cyclic protein.

[0094] " Cysteine residue” refers to a cysteine amino acid.

[0095] '‘Cystine” refers to an oxidized cysteine-dimer. Cystines are sulfur-containing amino acids obtained via the oxidation of two cysteine molecules, and are linked with a disulfide bond.

[0096] “AAG” or “Delta Delta G” in the context of computational methods for determining the impact of point mutations on protein stability, refers to the change in the Gibbs free energy difference between a first protein (e.g., a wild-type protein), and a second protein (e.g., a mutant or variant of the first protein, such as a mutant comprising a point mutation). For example, in some embodiments, AAG=AGsecond-AGFirst. wherein AGsecond is the Gibbs free energy change associated with the stability of the second protein (e.g., a mutant or variant of the first protein, such as a mutant comprising a point mutation); and wherein AGrirst is the Gibbs free energy change associated with the stability of the first protein (e.g., the wild-type protein), and wherein AAG is calculated as the difference between the Gibbs free energy of the second protein and the first protein. Exemplary methods of calculating AAG are provide herein, and also provided in Kellog et al., Role of Conformational Sampling in Computing Mutation-Induced Changes in Protein Structure and Stability.” Proteins 79 (3): 830-38, the disclosure of which is incorporated herein by reference in its entirety.

[0097] '‘Defined medium” means a medium that is composed of known chemical components but does not contain crude proteinaceous extracts or by-products such as yeast extract or peptone.

[0098] “Degeneracy” or “codon degeneracy” refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide with a particular activity; such functionally equivalent variants are contemplated herein.

[0099] '‘Derived” or '‘derived from” refers to obtaining a peptide, polypeptide, protein or polynucleotide from a known and / or originating peptide, polypeptide, protein or polynucleotide. Thus, as used herein, the term “derived from” encompasses, without limitation: a protein or polynucleotide that is isolated or obtained directly from an originating source (e.g. an organism, such as a one or more species belonging to the Atracidae family); a 26641601282Attorney Docket No. 277702-582798synthetic or recombinantly generated protein or polynucleotide that is identical, substantially related to, or modified from, a protein or polynucleotide from an known / originating source; or protein or polynucleotide that is made from a protein or polynucleotide of anknow n / originating source or a fragment thereof. The term “substantially related”, as used herein, means that the protein may have been modified by chemical, physical or other means (e.g. sequence modification).

[0100] Accordingly, “derived” can refer to either directly or indirectly obtaining a protein or polynucleotide from a known and / or originating protein or polynucleotide. For example, in some embodiments, “derived” can refer to obtaining a protein or polynucleotide from a known and / or originating protein or polynucleotide by looking at the sequence of a known / originating protein or polynucleotide and preparing a protein or polynucleotide having a sequence similar, at least in part, to the sequence of the known and / or originating protein or polynucleotide. In yet other embodiments, “derived” can refer to obtaining a protein or polynucleotide from a known and / or originating protein or polynucleotide by isolating a protein or polynucleotide from an organism that is related to a known protein or polynucleotide. Other methods of "deriving” a protein or polynucleotide from aknown protein or polynucleotide are known to one of skill in the art.

[0101] In some embodiments, “derived” in the context of a protein (e.g., “a protein derived from an organism”) describes a condition wherein said protein was originally identified in an organism, and has been reproduced therefrom via isolation from the organism, or through synthetic or recombinant means.

[0102] “Disulfide bond” or “disulfide bridge” refers to a covalent bond betw een two cysteine residues derived by the coupling of two thiol groups on their side chains. In some embodiments, a disulfide bond occurs via the oxidative folding of two different thiol groups (-SH) present in a polypeptide. In some embodiments, a polypeptide can comprise four, six, or eight different thiol groups (i.e., four, six, or eight cysteine residues each containing a thiol group); thus, in some embodiments, a polypeptide can form two, three, or more intramolecular disulfide bonds.

[0103] “Double expression cassette” refers to two HVP expression cassettes contained on the same vector.

[0104] “Double transgene peptide expression vector” or “double transgene expression vector” means a yeast expression vector that contains tw o copies of the HVP expression cassette.27641601282Attorney Docket No. 277702-582798

[0105] “DNA” refers to deoxyribonucleic acid, comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T]. or cytosine [C]), which can be arranged in single-stranded or double-stranded form. For example, one or more nucleotides creates a polynucleotide.

[0106] “dNTPs” refers to the nucleoside triphosphates that compose DNA and RNA.

[0107] “Downstream” is context dependent, but generally refers to the spatial positioning along a polynucleotide or protein sequence. In the context of a polynucleotide, the term “downstream” refers to positions 3' of a location on the polynucleotide. Those having ordinary skill in the art are aware that transcription proceeds in a 5' to 3' manner along a DNA strand. This means that RNA is made by the sequential addition of ribonucleotide-5'-triphosphates to the 3' terminus of the growing chain (with a requisite elimination of the pyrophosphate). And, as it is well known, a polynucleotide sequence has a 5' end and a 3' end, so called for the carbons on the sugar (deoxyribose or ribose) ring of the nucleotide backbone. Hence, relative to the position on the polynucleotide sequence, the term downstream relates to the region towards the 3' end of the sequence, and the term upstream relates to the region towards the 5' end of the strand. In either a linear or circular nucleic acid molecule, discrete elements (e g., particular nucleotide sequences) may be referred to as being “downstream” or “3'” relative to a further element if they are bonded or would be bonded to the same nucleic acid in the 3' direction from that element.

[0108] In the context of a protein, the term “downstream” refers to positions toward the C-terminus of a location on the protein. As used herein, in the context of a protein, the term “downstream” and “C-terminal direction” and “C -terminally” are used interchangeably. The term “downstream” denotes a relative location within the primary amino acid sequence rather than placement at the absolute C-terminus, and does not exclude the possibility that an addition sequence can be located more downstream from a given location or component.

[0109] “Endogenous” refers to a polynucleotide, peptide, polypeptide, protein, or process that naturally occurs and / or exists in an organism, e.g., a molecule or activity that is already present in the host cell before a particular genetic manipulation.

[0110] “Enhancer element” refers to a DNA sequence operably linked to a promoter, which can exert increased transcription activity on the promoter relative to the transcription activity’ that results from the promoter in the absence of the enhancer element.

[0111] ‘ER” or “Endoplasmic reticulum” is a subcellular organelle common to all eukaryotes where some post translation modification processes occur.28641601282Attorney Docket No. 277702-582798

[0112] “ERSP” or “Endoplasmic reticulum signal peptide” is an N-terminus sequence of amino acids that — during protein translation of the mRNA molecule encoding an HVP — is recognized and bound by a host cell signal-recognition particle, which moves the protein translation ribosome / mRNA complex to the ER in the cytoplasm. The result is the protein translation is paused until it docks with the ER where it continues and the resulting protein is injected into the ER.

[0113] ‘"ersp” refers to a polynucleotide encoding the peptide, ERSP.

[0114] “ER trafficking” means transportation of a cell expressed protein into ER for post-translational modification, sorting and transportation.

[0115] “Excipient” refers to any agriculturally or pharmaceutically acceptable additive, carrier, surfactant, emulsifier, thickener, preservative, solvent, disintegrant, glidant. lubricant, diluent, filler, bulking agent, binder, emollient, stiffening agent, chelating agent, stabilizer, solubilizing agents, dispersing agent, suspending agent, antioxidant, antiseptic, wetting agent, humectant, fragrant, suspending agents, pigments, colorants, isotonic agents, viscosity enhancing agents, mucoadhesive agents, and / or any combination thereof, that can be added to an agricultural composition, preparation, and / or formulation, which may be useful in achieving a desired modification to the characteristics of the agricultural composition, preparation, and / or formulation. Such modifications include, but are not limited to, physical stability, chemical stability, pesticidal efficacy, and / or any combination thereof.

[0116] “Expression cassette” refers to (1) a DNA sequence of interest, e.g.. a polynucleotide operable to encode an HVP; and one or more of the following: (2) promoters, terminators, and / or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry' site (IRES); (5) introns; and / or (6) post-transcriptional regulatory elements. The combination (1) with at least one of (2)-(6) is called an “expression cassette.” In some embodiments, there can be numerous expression cassettes cloned into a vector. For example, in some embodiments, there can be a first expression cassette comprising a polynucleotide operable to encode an HVP. In alternative embodiments, there are two expression cassettes, each comprising a polynucleotide operable to encode an HVP (i.e., a double expression cassette). In other embodiments, there are three expression cassettes operable to encode an HVP (i.e., a triple expression cassette). In some embodiments, a double expression cassette can be generated by subcloning a second expression cassette into a vector containing a first expression cassette. In some embodiments, a triple expression cassette can be generated by subcloning a third expression cassette into a vector containing a first and a29641601282Attorney Docket No. 277702-582798second expression cassete. Methods concerning expression cassetes and cloning techniques are well-known in the art and described herein. See also HVP expression cassete.

[0117] “FECT” means a transient plant expression system using Foxtail mosaic virus with elimination of coating protein gene and triple gene block.

[0118] “Fermentation beer” refers to spent fermentation medium, i.e., fermentation medium supernatant after removal of organisms, that has been inoculated with and consumed by a transformed host cell (e.g., a yeast cell operable to express an HVP of the present disclosure). In some embodiments, fermentation beer refers to the solution that is recovered following the fermentation of the transformed host cell. The term “fermentation” refers broadly to the enzymatic and anaerobic or aerobic breakdown of organic substances (e g., a carbon substrate) nutrient substances by microorganisms under controlled conditions (e.g.. temperature, oxygen, pH, nutrients, and the like) to produce fermentation products (e.g., one or more peptides of the present disclosure). While fermentation typically describes processes that occur under anaerobic conditions, as used herein it is not intended that the term be solely limited to strict anaerobic conditions, as the term “fermentation” used herein may also occur processes that occur in the presence of oxygen.

[0119] “GFP” means a green fluorescent protein from the jellyfish, Aequorea victoria.

[0120] “Growth medium” refers to a nutrient medium used for growing cells in vitro.

[0121] “Gut” as used herein can refer to any organ, structure, tissue, cell, extracellular matrix, and / or space comprising the gut, for example: the foregut, e.g., mouth, pharynx, esophagus, crop, proventriculus, or crop; the midgut, e.g., midgut caecum, ventriculus; the hindgut, e.g., pylorum, ileum, rectum or anus; the peritrophic membrane; microvilli; the basement membrane; the muscle layer; Malpighian tubules; or rectal ampulla.

[0122] " Hexathelidae" refers to a family of mygalomorph spiders that previously contained the genera: Atracidae, Macrothelidae and Porrhothelidae, however, Atracidae, Macrothelidae and Porrhothelidae have since been classified as their own families. See Hedin et al., Phylogenomic reclassification of the world’s most venomous spiders (Mygalomorphae, Atracidae), with implications for venom evolution. Sci Rep. 2018; 8: 1636.

[0123] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function 30641601282Attorney Docket No. 277702-582798of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared xlOO. Thus, in some embodiments, the term ■‘homologous” refers to the sequence similarity between two polypeptide molecules, or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology.“Homology” or “homologous” or “analogous” refer to the overall relatedness between polymeric molecules, e.g., between two or more nucleic acid molecules (e.g., DNA molecules and / or RNA molecules), or between two or more polypeptide molecules.

[0124] Homologj' is a qualitative term that describes a relationship between polymeric molecules and can be based upon the quantitative similarity or identity’. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. Polymeric molecules (e.g., nucleic acid molecules such as DNA molecules and / or RNA molecules, or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are referred to herein as “homologous,” or “homologs.” Where a position in both of the two or more compared polymeric molecules is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine), the two or more compared sequences deemed to be homologous at that position. Thus, the homology' between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. Accordingly, in some embodiments, the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared xlOO. Generally, a comparison between two sequences is made when the two sequences are aligned to give maximum homology. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%. 45%. 50%. 55%. 60%. 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 31641601282Attorney Docket No. 277702-582798100% identical or similar. Accordingly, the term “homologous"’ necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0125] Identifying one or more nAChR [31 subunits having one or more homologous, analogous, or functionally equivalent amino acid residues to, e.g., one or more amino acid residues in a Drosophila melanogaster nAChR [31 subunit of SEQ ID NO: 1 (e.g., the amino acid residues: T153, 1154, D155, V156, T157. Y158, Q163. T165, 1167, K169, 1205. E206, V207, P208, Y210, N212, Y214, E223, Y229, 1231, T292, L294. V295, P503, H504, E507, Y508 and V509 of SEQ ID NO: 1), can be performed by aligning the mature amino sequence of the one or more nAChR [31 subunit amino acid sequences to the mature amino acid sequence of the Drosophila melanogaster nAChR [31 subunit (SEQ ID NO: 1). In the present disclosure, the Drosophila melanogaster nAChR [31 subunit has an immature amino acid sequence of:MESSCKSWLLCSILVLVAFSLVSASEDEERLVRDLFRGYNKLIRPVQNMTQKVGVRFGLAFV QLINVNEKNQVMKSNVWLRLVWYDYQLQWDEADYGGIGVLRLPPDKVWKPDIVLFNNADGNY EVRYKSNVLIYPTGEVLWVPPAIYQSSCTIDVTYFPFDQQTCIMKFGSWTFNGDQVSLALYN NKNFVDLSDYWKSGTWDIIEVPAYLNVYEGDSNHPTETDITFYIIIRRKTLFYTVNLILPTV LISFLCVLVFYLPAEAGEKVTLGISILLSLVVFLLLVSKILPPTSLVLPLIAKYLLFTFIMN TVSILVTVIIINWNFRGPRTHRMPMYIRSIFLHYLPAFLFMKRPRKTRLRWMMEMPGMSMPA HPHPSYGSPAELPKHISAIGGKQSKMEVMELSDLHHPNCKINRKVNSGGELGLGDGCRRESE SSDSILLSPEASKATEAVEFIAEHLRNEDLYIQTREDWKYVAMVIDRLQLYIFFIVTTAGTV G I LMD APH I FE YVDQDRI I E I YRGK(SEQ ID NO: 249)

[0126] As shown above, the immature amino acid sequence of SEQ ID NO: 1 contains a signal sequence consisting of amino acid residues 1-24 (underlined, as shown above): the mature amino acid sequence of SEQ ID NO: 1 consists of amino acid residues 25-521. Accordingly, by aligning one or more candidate nAChR [31 subunit mature amino acid sequences with the Drosophila melanogaster nAChR [31 subunit mature amino acid sequence of SEQ ID NO: 1, amino acid residues that are homologous, analogous, or functionally equivalent to amino acid residues in the Drosophila melanogaster nAChR pi subunit of SEQ ID NO: 1 (e.g., T153, 1154, D155, V156, T157, Y158, Q163, T165, 1167, KI 69, 1205, E206, V207, P208, Y210, N212, Y214, E223, Y229, 1231, T292, L294, V295, P503, H504, E507, Y508 and V509 of SEQ ID NO: 1) — can be identified in the one or more candidate nAChR pi subunit mature amino acid sequences. For clarify, and as used herein, residue site nomenclature, e.g., T153, 1154, D155, V156, T157, Y158, Q163, T165, 1167, K169, 1205, E206, V207, P208, Y210, N212, Y214, E223, Y229, 1231, T292, L294, V295, P503, H504,32641601282Attorney Docket No. 277702-582798E507, Y508 and V509, refers to the entirety of SEQ ID NO: 1, i.e., the immature amino acid sequence.

[0127] Typically, the term '‘homology’’ implies that the compared polymeric molecules diverged in evolution from a common origin, i.e., a first polymeric molecule is related to a second polymeric molecule by descent from a common ancestral polymeric molecule; however, as used herein, it is not intended that homologous polymeric molecules (homologs) necessarily be evolutionarily related. As used herein, the terms “homology” or “homologous” or “analogous” can refer to a first polymeric molecule (or portion thereof) that exhibits similar structural or functional characteristics to a second polymeric molecule (e.g., a reference sequence), wherein the first polymeric molecule and the second polymeric molecule may or may not necessarily share a direct evolutionary relationship. Thus, as used herein, the terms “homologous” or “analogous” or “homology” can refer to the relatedness between a first polymeric molecule and a second polymeric molecule (or reference polymeric molecule) — irrespective of the evolutionary relationship between the first polymeric molecule and the second polymeric molecule. For example, in some embodiments.

[0128] There may be partial homolog}', or complete homology and thus identical. “Sequence identity” refers to a measure of relatedness between two or more nucleic acid sequences or two or more polypeptide sequences, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues or amino acid residues that are identical and in the same relative positions in their respective larger sequences. See '‘Identity.”

[0129] “Homologous recombination” refers to the event of substitution of a segment of DNA by another one that possesses identical regions (homologous) or nearly so. For example, in some embodiments, “homologous recombination” refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Briefly, homologous recombination is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Although homologous recombination varies widely among different organisms and cell types, most forms involve the same basic steps: after a double-strand break occurs, sections of DNA around the 5' ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3' end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. After strand invasion, the further sequence of events may follow either of two main pathways, i.e., the doublestrand break repair pathway, or the synthesis -dependent strand annealing pathway.33641601282Attorney Docket No. 277702-582798Homologous recombination is conserved across all three domains of life as well as viruses, suggesting that it is a nearly universal biological mechanism. For example, in some embodiments, homologous recombination can occur using a site-specific integration (SSI) sequence, whereby there is a strand exchange crossover event between nucleic acid sequences substantially similar in nucleotide composition. These crossover events can take place between sequences contained in the targeting construct of the invention (i.e., the SSI sequence) and endogenous genomic nucleic acid sequences (e.g., the polynucleotide encoding the subunit). In addition, in some embodiments, it is possible that more than one site-specific homologous recombination event can occur, which would result in a replacement event in which nucleic acid sequences contained within the targeting construct have replaced specific sequences present within the endogenous genomic sequences.

[0130] “HVP” or “HXTX variant peptide’’ or “hexatoxin variant peptide” or “hexatoxin mutant” or “HXTX mutant peptide” or “peptides of the present disclosure” or “insecticidal peptides of the present disclosure” or “insecticidal peptides” refer to peptides having one or more mutations relative to a wild-type peptide isolated from spiders belonging to the Hexathelidae, Atracidae. Macrothelidae, and Porrhothelidae family of spiders. The Hexathelidae family of spiders formerly contained the Atracidae, Macrothelidae, and Porrhothelidae families of spiders; however, molecular phylogenetics revealed that Hexathelidae was not monophyletic, thus the genera Atracidae, Macrothelidae and Porrhothelidae were split off into new families. See Hedin et al., Phylogenomic reclassification of the world's most venomous spiders (Mygalomorphae, Atracidae), with implications for venom evolution. Set REP. 2018; 8: 1636. One such spider that produces wild-type venom toxins is known by its common name as the Australian Blue Mountains Funnel-web Spider, which includes three genera: Atrax. Hadronyche. and Illawarra, comprising 35 species, e.g., the species Hadronyche versuta and Atrax robustus. For the sake of brevity, as used herein, venom toxins isolated from the Hexathelidae, Atracidae, Macrothelidae, and Porrhothelidae families of spiders are all referred to herein as “ACTX” or “ACTX peptide” or “atracotoxin” or “hexatoxin” or “HXTX” or “HXTX peptide.” Examples of ACTX peptides isolated from Atracidae family species are the Omega- ACTX, Kappa-ACTX, and U-ACTX peptides.

[0131] In some embodiments, “HVP” or “HXTX variant peptide” or “hexatoxin variant peptide” or “hexatoxin mutant” or “HXTX mutant peptide” refers to peptides having one or more mutations relative to the mutant peptide, “Hybrid+2” (or “Spear” or “GS-CO / K-Hxtx-Hvla,” all used herein interchangeably), which has an amino acid sequence of:34641601282Attorney Docket No. 277702-582798“GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO:1). As used herein, the expression ‘“an HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1” means that all of the HVPs disclosed and claimed herein consists of or comprises an amino acid sequence that has at least one amino acid substitution (a replacement of one amino acid for another amino acid) relative to the amino acid sequence of SEQ ID NO: 1. HVPs may contain the same, or a greater, or a lesser number of amino acid residues relative to SEQ ID NO: I, but in all cases, the HVP will contain at least one amino acid substitution relative to the sequence of SEQ ID NO: 1. In some embodiments, an HVP can have an amino acid sequence according to Formula (I): Xi-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4 is I, T or V; X5is M or V; X6is G, H, N, Q or D: X7is Q or R,; X8is D, G, N or S; X9 is P, W. Y or L; X10 is H, K orN; X11 is C, P or T, or absent: X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or an agriculturally acceptable salt thereof. (SEQ ID NO: 131).

[0132] '‘HVP expression cassette” refers to one or more regulatory elements such as promoters; enhancer elements; mRNA stabilizing polyadenylation signal; an internal ribosome entry site (IRES); introns; post-transcriptional regulatory elements; and a polynucleotide operable to encode an HVP, e.g., an HVP ORF. For example, one example of an HVP expression cassette is one or more segments of DNA that contains a polynucleotide segment operable to express an HVP, a ADH1 promoter, a LAC4 terminator, and an alpha-MF secretory signal. An HVP expression cassette contains all of the nucleic acids necessary to encode an HVP or an HVP-insecticidal protein.

[0133] " HVP ORF” refers to a polynucleotide operable to encode an HVP, or an HVP-insecticidal protein.

[0134] “HVP ORF diagram” refers to the composition of one or more HVP ORFs, as written out in diagram or equation form. For example, a “HVP ORF diagram” can be written out as using acronyms or short-hand references to the DNA segments contained within the expression ORF. Accordingly, in one example, a “HVP ORF diagram” may describe the 35641601282Attorney Docket No. 277702-582798polynucleotide segments encoding the ERSP, LINKER. STA, and HVP, by diagramming in equation form the DNA segments as ‘ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “Zz er” or “£” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “std” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and “hvp” (i.e., the polynucleotide sequence encoding an HVP), respectively. An example of an HVP ORF diagram is “ersp-sta- inkeri-hvp^ ” or “ersp-(hvprlinken)N-sta’' and / or any combination of the DNA segments thereof.

[0135] '‘HVP-insecticidal protein” or “HVP-insecticidal polypeptide” or “insecticidal protein” or “insecticidal polypeptide” refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, comprising: (1) at least one HVP, or two or more HVPs; and (2) additional peptides, polypeptides, or proteins that are not an HVP. For example, in some embodiments, an HVP-insecticidal protein can be a fusion protein comprising at least one HVP, and at least one additional peptide or protein that is not a HVP, wherein at least one HVP is linked directly or indirectly to a non-HVP protein.

[0136] In some embodiments, an HVP-insecticidal protein can be a fusion protein comprising at least one HVP, and at least one additional peptide, polypeptide, or protein that is not a HVP, wherein at least one HVP is linked directly or indirectly to a non-HVP protein; wherein the at least one additional peptides, polypeptides, or proteins have the ability to increase the mortality and / or inhibit the growth of insects when the insects are exposed to an HVP-insecticidal protein, relative to an HVP alone; increase the expression of said HVP-insecticidal protein, e.g., in a host cell or an expression system; and / or affect the post-translational processing of the HVP-insecticidal protein.

[0137] In some embodiments, an HVP-insecticidal protein can be a polymer comprising two or more HVPs. In some embodiments, an HVP-insecticidal protein can be a polymer comprising two or more HVPs, wherein the HVPs are operably linked via a linker peptide, e.g., a cleavable and / or non-cleavable linker. In some embodiments, an HVP-insecticidal protein can refer to a one or more HVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and / or any other combination thereof. In some embodiments, an HVP-insecticidal protein can be a non-naturally occurring protein comprising (1) an HVP; and (2) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBL or a histidine tag or similar marker.

[0138] “HVP construct” refers to the three-dimensional arrangement / orientation of peptides, polypeptides, and / or motifs of operably linked polypeptide segments (e.g., an HVP- 36641601282Attorney Docket No. 277702-582798insecticidal protein). For example, an HVP ORF can include one or more of the following components or motifs: an HVP; an endoplasmic reticulum signal peptide (ERSP); a linker peptide (L); a translational stabilizing protein (STA); or any combination thereof. And, as used herein, the term “HVP construct” is used to describe the designation and / or orientation of the structural motif. In other words, the HVP construct describes the arrangement and orientation of the components or motifs contained w ithin a given HVP ORF. For example, in some embodiments, an HVP construct describes, without limitation, the orientation of one of the following HVP-insecticidal proteins: ERSP-HVP; ERSP-(HVP)N; ERSP-HVP-L; ERSP-(HVP)N-L; ERSP-(HVP-L)N; ERSP-L-HVP; ERSP-L-(HVP)N; ERSP-(L-HVP)N; ERSP-STA-HVP; ERSP-STA-(HVP)N; ERSP-HVP-STA; ERSP-(HVP)N-STA; ERSP-(STA-HVP)N; ERSP-(HVP-STA)N; ERSP-L-HVP-STA; ERSP-L-STA-HVP; ERSP-L-(HVP-STA)N; ERSP-L-(STA-HVP)N; ERSP-L-(HVP)N-STA; ERSP-(L-HVP)N-STA; ERSP-(L-STA-HVP)N; ERSP-(L-HVP-STA)N; ERSP-(L-STA)N-HVP; ERSP-(L-HVP)N-STA; ERSP-STA-L-HVP; ERSP-STA-HVP-L; ERSP-STA-L-(HVP)x; ERSP-(STA-L)N-HVP; ERSP-STA-(L-HVP)N; ERSP-(STA-L-HVP)N; ERSP-STA-(HVP)N-L; ERSP-STA-(HVP-L)N; ERSP-(STA-HVP)N-L; ERSP-(STA-HVP-L)N; ERSP-HVP-L-STA; ERSP-HVP-STA-L; ERSP-(HVP)N-STA-L ERSP-(HVP-L)N-STA; ERSP-(HVP-STA)N-L; ERSP-(HVP-L-STA)N; or ERSP-(HVP-STA-L)N; wherein N is an integer ranging from 1 to 200. See also “Structural motif.”

[0139] “HXTX” or “HXTX peptide” or “hexatoxin” or “ACTX” or “ACTX peptide” or “atracotoxin” (all used interchangeably) refer to venom toxins isolated from spiders belonging to the Hexathelidae, Atracidae, Macrothelidae, and Porrhothelidae family of spiders. The Hexathelidae family of spiders formerly contained the Atracidae, Macrothelidae, and Porrhothelidae families of spiders; however, molecular phylogenetics revealed that Hexathelidae was not monophyletic, thus the genera Atracidae, Macrothelidae and Porrhothelidae were split off into new families. See Hedin et al., Phylogenomic reclassification of the world’s most venomous spiders (Mygalomorphae, Atracidae), with implications for venom evolution. Sci Rep. 2018; 8: 1636. One such spider that produces these venom toxins is known by its common name as the Australian Blue Mountains Funnelweb Spider, which includes three genera: Atrax, Hadronyche, and Illawarra, comprising 35 species, e.g., the species Hadronyche versuta and Atrax robustus. For the sake of brevity, as used herein, venom toxins isolated from the Hexathelidae, Atracidae, Macrothelidae, and Porrhothelidae families of spiders are all referred to herein as “ACTX” or “ACTX peptide” or “atracotoxin” or “hexatoxin” or “HXTX” or “HXTX peptide.” Examples of ACTX 37641601282Attorney Docket No. 277702-582798peptides isolated from Atracidae family species are the Omega- ACTX, Kappa- ACTX, and U-ACTX peptides.

[0140] " Hybrid " or “Hybrid peptide,” aka “hybrid toxin,” aka “hybrid-ACTX-Hvla,” aka “native hybrid ACTX-Hvla,” as well as “U peptide,” aka “U toxin,” aka “native U,” aka “U-ACTX-Hvla,” aka “native U-ACTX-Hvla,” all refer to an ACTX peptide, which was discovered from a spider know n as the Australian Blue Mountains Funnel-web Spider, Hadronyche versuia. and is a positive allosteric modulators of the nicotinic acetylcholine receptor, and may also be a dual antagonist to insect voltage-gated Ca2+channels and voltage-gated K+channels. See Chambers et al., Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Lett. 2019 Jun;593(12): 1336-1350; and Windley et al.. Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors. Neuropharmacology. 2017 Dec; 127: 224-242, the disclosures of which are incorporated herein by reference in their entireties. An exemplary Hybrid peptide is provided herein, having the amino acid sequence:“QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO: 2)

[0141] “Hybrid+2” or “H+2” or “U+2 peptide” or “U+2 protein” or “U+2 toxin” or “U+2” or “U+2-ACTX-Hvla” or “Spear” or “GS-© / K-Hxtx-Hvla” all refer to a U-ACTX-Hvla having an additional dipeptide operably linked to the native peptide. The additional dipeptide that is operably linked to the U peptide is indicated by the “+2” or “plus 2” can be selected from among several peptides, any of which may result in a “U+2 peptide” with unique properties as discussed herein. In some preferred embodiments, the dipeptide is “GS”; an exemplary U+2-ACTX-HvTa peptide is set forth in SEQ ID NO: 1, having the amino acid sequence of “GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO: 1)

[0142] “Hybridize” refers to the annealing of one single-stranded polynucleotide to another polynucleotide based on the well-understood principle of sequence complementarity. In some embodiments, the other polynucleotide is a single-stranded polynucleotide. The propensity for hybridization between polynucleotides depends on the temperature and ionic strength of their milieu, the length of the polynucleotides, and the degree of complementarity. The effect of these parameters on hybridization are well known in the art.

[0143] “Hybridization” refers to any process by which a strand of polynucleotide binds with a complementary strand through base pairing. Two single-stranded polynucleotides “hybridize” when they form a double-stranded duplex. Thus, as used herein,38641601282Attorney Docket No. 277702-582798the term ‘‘hybridize” refers to the annealing of one single-stranded polynucleotide to another polynucleotide based on the well-understood principle of sequence complementarity. In some embodiments, the other polynucleotide is a single-stranded polynucleotide. The propensity for hybridization between polynucleotides depends on the temperature and ionic strength of their milieu, the length of the polynucleotides, and the degree of complementarity'. The effect of these parameters on hybridization are well known in the art. When two single-stranded polynucleotides hybridize and form a double-stranded duplex, the region of double-strandedness can include the full-length of one or both of the single-stranded polynucleotides, or all of one single stranded polynucleotide and a subsequence of the other single stranded polynucleotide, or the region of double-strandedness can include a subsequence of each polynucleotide. Hybridization also includes the formation of duplexes which contain certain mismatches, provided that the two strands are still forming a double stranded helix. See “Stringent hybridization conditions ” below.

[0144] TCso” or “IC50” refers to half-maximal inhibitory concentration, which is a measurement of how much of an agent is needed to inhibit a biological process by half, thus providing a measure of potency of said agent.

[0145] '‘ICK” or “Inhibitor cystine knot” or “ICK motif’ refers to a disulfide bond structural motif comprising three disulfide bonds. In some embodiments, a protein having an ICK motif has at least 6 motif-forming cysteine residues (i.e., 3 pairs of motif-forming cysteine residues), wherein the 3 pairs of motif-forming cysteine residues are operable to form the three disulfide bonds. Note: there may be other cysteine residues in a protein having an ICK motif, but the motif-forming cysteine residues are those residues that contribute to the disulfide bond structural motif (i.e., the ICK motif). The ICK motif occurs when two disulfide bonds and their connecting subunits form an internal ring structure, and that structure is then threaded by the third disulfide bond to form an interlocking and cross braced structure; i.e., an ICK comprises an embedded ring formed by two disulfide bonds and their connecting subunits, which is threaded by a third disulfide bond.

[0146] “Identity” refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing said sequences. The term “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity ” and “similarity” can be readily calculated by any one of the myriad methods known to those having ordinary skill in the art. including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University 39641601282Attorney Docket No. 277702-582798Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994:, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the disclosures of which are incorporated herein by reference in their entireties. Furthermore, methods to determine identity and similarity are codified in publicly available computer programs. For example, in some embodiments, methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990), the disclosures of which are incorporated herein by reference in their entireties.

[0147] "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0148] “Inactive” refers to a condition wherein something is not in a state of use, e.g., lying dormant and / or not working. For example, when used in the context of a gene or when referring to a gene, the term inactive means said gene is no longer actively synthesizing a gene product, having said gene product translated into a protein, or otherwise having the gene perform its normal function. For example, in some embodiments, the term inactive can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing: RNA splicing; or other post-transcriptional modifications); interference with noncoding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and / or inhibition and / or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.

[0149] “Inhibiting” or “inhibit” or “combating” or “combat” or “controlling” or “control,” or any variation of these terms, refers to making something (e.g., the number of pests, the functions and / or activities of the pest, and / or the deleterious effect of the pest on a plant or animal susceptible to attack thereof) less in size, amount, intensity, or degree. For example, in some embodiments, the application of a pesticidally effective amount of an HVP 40641601282Attorney Docket No. 277702-582798or agriculturally acceptable salt thereof, or an agricultural composition comprising an HVP or agriculturally acceptable salt thereof, to (i) the pest, a locus of the pest, a food supply of the pest, a habitat of the pest, or a breeding ground of the pest; (ii) a plant, a seed, a plant part, a locus of a plant, or an environment of a plant that is susceptible to an attack by the pest; (iii) an animal, a locus of an animal, or an environment of an animal susceptible to an attack by the pest; or (iv) a combination thereof, results in the following effect: a decrease in the number of pests, or inhibition of the pest’s activities (e.g., the pest dies stops or slows its movement; stops or slows its feeding; stops or slows its grow th: becomes confused, e g., with regard to navigation, locating food, sleeping behaviors, and / or mating; fails to pupate if applicable; interferes with reproduction of the pest; and / or precludes the pest from producing offspring and / or precludes the insect from producing fertile offspring) relative to the number of pests or activities thereof that had not been exposed to a pesticidally effective amount of an HVP or agriculturally acceptable salt thereof, or an agricultural composition comprising an HVP or agriculturally acceptable salt thereof.

[0150] In some embodiments, combating, controlling, or inhibiting a pest, includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, in the number of pests or the activities thereof treated with peptides and / or compositions of the present disclosure, compared to untreated pests. About as used herein means within ± 10%, preferably ± 5% of a given value.

[0151] Thus, in some embodiments, the terms “combating, controlling, or inhibiting a pest,’’ refers to a decrease in the number of pests, or an inhibition of the activities of the pests (e.g., movement; feeding; growth; level of awareness or alertness, e.g., with regard to navigation, locating food, sleeping behaviors, and / or mating; pupation if applicable; reproduction; ability to produce offspring and / or ability to produce fertile offspring) that have received a pesticidally effective amount of an HVP of the present disclosure, or an agricultural composition thereof, that is at least about 0.1%. at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 2.25%, at least about 2.5%, at least about 2.75%, at least about 3%, at least about 3.25%, at least about 3.5%, at least about 3.75%. at least about 4%, at least about 4.25%, at least about 4.5%. at least about 4.75%. at least about 5%, at least about 5.25%, at least about 5.5%, at least about 5.75%, at least about 41641601282Attorney Docket No. 277702-5827986%, at least about 6.25%, at least about 6.5%, at least about 6.75%, at least about 7%, at least about 7.25%. at least about 7.5%, at least about 7.75%, at least about 8%, at least about 8.25%, at least about 8.5%, at least about 8.75%, at least about 9%, at least about 9.25%, at least about 9.5%, at least about 9.75%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%. at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%. at least about 41%. at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%. at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%. at least about 100%, or a greater than a 100%, relative to the number of pests, or the inhibition of activities of the pests (e.g.. movement; feeding; growth; level of awareness or alertness, e.g., with regard to navigation, locating food, sleeping behaviors, and / or mating; pupation if applicable; reproduction; abi I i ty to produce offspring and / or ability to produce fertile offspring) that have not received a pesticidally effective amount of an HVP of the present disclosure, or an agricultural composition thereof.

[0152] “Inoperable” refers to the condition of a thing not functioning, malfunctioning, or no longer able to function. For example, when used in the context of a gene or when referring to a gene, the term inoperable means said gene is no longer able to operate as it normally would, either permanently or transiently. For example, “inoperable,” in some embodiments, means that a gene is no longer able to synthesize a gene product, having said gene product translated into a protein, or is otherwise unable to gene perform its normal function. For example, in some embodiments, the term inoperable can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA 42641601282Attorney Docket No. 277702-582798maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and / or inhibition and / or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.

[0153] “Insect” includes all organisms in the class ‘'Insecta.” The term “pre-adulf ' insects refers to any form of an organism prior to the adult stage, including, for example, eggs, larvae, and nymphs. As used herein, the term “insect refers to any arthropod and nematode, including acarids, and insects known to infest all crops, vegetables, and trees and includes insects that are considered pests in the fields of forestry', horticulture and agriculture. Examples of specific crops that might be protected with the methods disclosed herein are soybean, com, cotton, alfalfa and the vegetable crops. A list of specific crops and insects is enclosed herein.

[0154] “Insect gut environment” or “gut environment” means the specific pH and proteinase conditions found within the fore, mid or hind gut of an insect or insect larva.

[0155] “Insect hemolymph environment” means the specific pH and proteinase conditions of found within an insect or insect larva.

[0156] As used herein, the term “insecticidal” is generally used to refer to the ability of a polypeptide or protein used herein, to increase mortality or inhibit growth rate of insects. As used herein, the term “nematicidal” refers to the ability of a polypeptide or protein used herein, to increase mortality or inhibit the growth rate of nematodes. In general, the term “nematode” comprises eggs, larvae, juvenile and mature forms of said organism.

[0157] “Insecticidal activity” means that upon or after exposing the insect to compounds, agents, or peptides, the insect either dies stops or slows its movement; stops or slows its feeding; stops or slows its growth; becomes confused (e.g., with regard to navigation, locating food, sleeping behaviors, and / or mating); fails to pupate; interferes with reproduction; and / or precludes the insect from producing offspring and / or precluding the insect from producing fertile offspring.

[0158] “Integrative expression vector” or “integrative vector” means a yeast expression vector which can insert itself into a specific locus of the yeast cell genome and stably becomes a part of the yeast genome.

[0159] “Intervening linker” refers to a short peptide sequence in the protein separating different parts of the protein, or a short DNA sequence that is placed in the reading frame in the ORF to separate the upstream and downstream DNA sequences. For example, in 43641601282Attorney Docket No. 277702-582798some embodiments, an intervening linker may be used allowing proteins to achieve their independent secondary and tertiary’ structure formation during translation. In some embodiments, the intervening linker can be either resistant or susceptible to cleavage in plant cellular environments, in the insect and / or lepidopteran gut environment, and in the insect hemoly mph and lepidopteran hemolymph environment.

[0160] “Isolated” refers to separating a thing and / or a component from its natural environment, e.g., a toxin isolated from a given genus or species means that toxin is separated from its natural environment (e g., removed from the organism).

[0161] “kb” refers to kilobase, i.e., 1000 bases. As used herein, the term “kb” means a length of nucleic acid molecules. For example, 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long. A length of double-stranded DNA that is 1 kb long, contains two thousand nucleotides (i.e., one thousand on each strand). Alternatively, a length of singlestranded RNA that is 1 kb long, contains one thousand nucleotides.

[0162] “KD50” or " Knockdown dose 50” or “paralytic dose 50” or “PD50” refers to the median dose required to cause paralysis or cessation of movement in 50% of a population, for example, and without limitation, a population of Musca domestica (common housefly), or a population ol' v / es' aegypti (mosquito).

[0163] “kDa” refers to kilodalton, a unit equaling 1,000 daltons; a “Dalton” or “dalton” is a unit of molecular weight (MW).

[0164] “Knock in” or “knock-in” or “knocks-in” or “knocking-in” refers to the replacement of an endogenous gene w ith an exogenous or heterologous gene, or part thereof. For example, in some embodiments, the term “knock-in” refers to the introduction of a nucleic acid sequence encoding a desired protein to a target gene locus by homologous recombination, thereby causing the expression of the desired protein. In some embodiments, a “knock-in” mutation can modify a gene sequence to create a loss-of-function or gain-of-function mutation. The term “knock-in” can refer to the procedure by which a exogenous or heterologous polynucleotide sequence or fragment thereof is introduced into the genome, (e.g., “they performed a knock-in” or “they knocked-in the heterologous gene”), or the resulting cell and / or organism (e.g., “ the cell is a “knock-in” or “the animal is a “knock-in”).

[0165] “Knock out” or “knockout” or “knock-out” or “knocks-ouf ’ or “knocking-out” refers to a partial or complete suppression of the expression gene product (e.g., mRNA) of a protein encoded by an endogenous DNA sequence in a cell. In some embodiments, the “knock-out” can be effectuated by targeted deletion of a whole gene, or part of a gene encoding a peptide, polypeptide, or protein. As a result, the deletion may render a gene 44641601282Attorney Docket No. 277702-582798inactive, partially inactive, inoperable, partly inoperable, or otherwise reduce the expression of the gene or its products in any cell in the whole organism and / or cell in which it is normally expressed. The term "knock-out" can refer to the procedure by which an endogenous gene is made completely or partially inactive or inoperable (e.g., “they performed a knock-out” or “they knocked-out the endogenous gene”), or the resulting cell and / or organism (e.g., “ the cell is a “knock-out” or “the animal is a “knock-out”).

[0166] “ / ” or '"linker'' refers to a nucleotide encoding intervening linker peptide.

[0167] “L” in the proper context refers to an intervening linker peptide, which links a translational stabilizing protein (STA) with an additional polypeptide, e.g., an HVP, and / or multiple HVPs. When referring to amino acids, “L” can also mean leucine.

[0168] “LAC4 promoter” or “Lac4 promoter” or “pLac4” refers to a DNA segment comprised of the promoter sequence derived from the K. lactis [3-galactosidase gene. The LAC4 promoters is strong and inducible reporter that is used to drive expression of exogenous genes transformed into yeast.

[0169] “LAC4 terminator” or “Lac4 terminator” refers to a DNA segment comprised of the transcriptional terminator sequence derived from the K. lactis (3-galactosidase gene.

[0170] “LD20” refers to a dose required to kill 20% of a population.

[0171] “LD50” refers to lethal dose 50 which means the dose required to kill 50% of a population.

[0172] “Lepidopteran gut environment” means the specific pH and proteinase conditions of found within the fore, mid or hind gut of a lepidopteran insect or larva.

[0173] “Lepidopteran hemolymph environment” means the specific pH and proteinase conditions of found within lepidopteran insect or larva.

[0174] “Linker” or “LINKER” or “peptide linker” or “L” or “intervening linker” refers to a short peptide sequence operable to link two peptides together. Linker can also refer to a short DNA sequence that is placed in the reading frame of an ORF to separate an upstream and downstream DNA sequences. In some embodiments, a linker can be cleavable by an insect protease. In some embodiments, a linker may allow proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, the linker can be either resistant or susceptible to cleavage in plant cellular environments, in the insect and / or lepidopteran gut environment, and / or in the insect hemolymph and lepidopteran hemolymph environment. In some embodiments, a linker can be cleaved by a protease, e.g., in some embodiments, a linker can be cleaved by a plant protease (e.g., papain, bromelain, ficin, actinidin, zingibain, and / or cardosins), an insect 45641601282Attorney Docket No. 277702-582798protease, a fungal protease, a vertebrate protease, an invertebrate protease, a bacteria protease, a mammal protease, a reptile protease, or an avian protease. In some embodiments, a linker can be cleavable or non-cleavable. In some embodiments, a linker comprises a binary or tertiary region, wherein each region is cleavable by at least two types of proteases: one of which is an insect and / or nematode protease and the other one of which is a human protease. In some embodiments, a linker can have one of (at least) three roles: to cleave in the insect gut environment, to cleave in the plant cell, or to be designed not to intentionally cleave.

[0175] '‘Locus of a pest” refers to the habitat of a pest food supply of a pest; breeding ground of a pest; area traveled by or inhabited by a pest; material infested, eaten, used by a pest; and / or any environment in which a pest inhabits, uses, is present in, or is expected to be. In some embodiments, the locus of a pest includes, without limitation, a pest habitat; a pest food supply; a pest breeding ground; a pest area; a pest environment; any surface or location that may be frequented and / or infested by a pest; any plant or animal, or a locus of a plant or animal, susceptible to attack by a pest; and / or any surface or location where a pest may be found, may be expected to be found, or is likely to be attacked by a pest.

[0176] " Locus of a plant” refers to any place in which a plant is growing; any place where plant propagation materials of a plant are sown; any place where plant propagation materials of a plant will be placed into the soil; or any area where plants are stored, including without limitation, live plants and / or harvested plants, leaves, seeds, fruits, or parts thereof.

[0177] “Locus of an animal” refers to any place where animals live, eat, breed, sleep, or are otherw ise present.

[0178] “Medium” (plural “media”) refers to a nutritive solution for culturing cells in cell culture.

[0179] “MO A” refers to mechanism of action.

[0180] “Molecular w eight (MW)” refers to the mass or weight of a molecule, and is typically measured in “daltons (Da)” or kilodaltons (kDa). In some embodiments, MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, or light scattering. In some embodiments, the SDS-PAGE method is as follows: the sample of interest is separated on a gel with a set of molecular weight standards. The sample is run, and the gel is then processed with a desired stain, followed by destaining for about 2 to 14 hours. The next step is to determine the relative migration distance (Rf) of the standards and protein of interest. The migration distance can be determined using the following equation:46641601282Attorney Docket No. 277702-582798Migration distance of the proteinMigration distance of the dye frontFormula (a)

[0181] Next, the logarithm of the MW can be determined based on the values obtained for the bands in the standard; e.g., in some embodiments, the logarithm of the molecular weight of an SDS-denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. After plotting the graph, interpolating the value derived will provide the molecular weight of the unknown protein band.

[0182] " Motif ’ refers to dominant feature and / or distinct pattern in a molecule; e.g.. a distinct pattern of amino acids that operate in a function-specific protein sequence. In some embodiments, a motif is a polynucleotide or polypeptide sequence that is implicated in having some biological significance and / or exerts some effect or is involved in some biological process.

[0183] '‘Multiple cloning site” or “MCS” refers to a segment of DNA found on a vector that contains numerous restriction sites in which a DNA sequence of interest can be inserted.

[0184] “Mutant” refers to an organism, DNA sequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein, that has an alteration, variation, or modification (for example, in the nucleotide sequence or the amino acid sequence), which causes said organism and / or sequence to be different from the naturally occurring or wild-type organism, wild-type sequence, and / or reference sequence with which the mutant is being compared. In some embodiments, this alteration, variation, or modification can be one or more nucleotide and / or amino acid substitutions or modifications (e.g., deletion or addition). In some embodiments, the one or more amino acid substitutions or modifications can be conservative: here, such a conservative amino acid substitution and / or modification in a “mutant” does not substantially diminish the activity of the mutant in relation to its non-mutant form. For example, in some embodiments, a “mutant” possesses one or more conservative amino acid substitutions when compared to a peptide w ith a disclosed and / or claimed sequence, as indicated by a SEQ ID NO.

[0185] “N-terminus” or “N-terminal” refers to the free amine group (i.e., -NH2) that is positioned on beginning or start of a polypeptide.

[0186] '‘Nicotinic acetylcholine receptor (nAChR)” refers to a class of ligand-gated ion channels that mediate fast synaptic transmission in the nervous system (e.g., of an insect). These receptors are integral to the functioning of cholinergic synapses, where they facilitate 47641601282Attorney Docket No. 277702-582798the rapid actions of acetylcholine (ACh) by allowing the flow of cations through their central pore. The cholinergic pathway is a critical excitatory neurotransmission system in the insect nervous system. In insects, acetylcholine (ACh) acts through two classes of receptors: muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). mAChRs help regulate ACh release at presynaptic sites, whereas nAChRs mediate fast postsynaptic responses.

[0187] Nicotinic acetylcholine receptors (nAChRs) are prototypical members of the Cys-loop ligand-gated ion channel superfamily, which includes ionotropic GABA, glycine and serotonin receptors. nAChRs are ligand-gated ion channels that sen e as the foundation for rapid sy naptic communication in insects, and mediate the fast actions of acetylcholine (ACh) in the nervous system and at neuromuscular junctions. Briefly, the primary function of insect nAChRs is to mediate cholinergic synaptic transmission by responding to the binding of ACh. Upon ACh binding, the receptor undergoes a conformational change that opens the central pore, permitting an influx of cations. The resulting depolarization of the neuron propagates the synaptic signal, thereby enabling rapid communication within the nervous system.

[0188] Typically, nAChRs assemble as pentamers of homologous subunits arranged around a central ion channel. Each nAChR subunit has four transmembrane domains (TM1-4) and possesses an N-terminal extracellular domain containing the characteristic Cys-loop motif consisting of two disulfide bond-forming cysteines separated by 13 amino acid residues. S TF. F. VF. THANY, INSF. CT NICOTINIC ACETYL CHOLINE RECEPTORS (1st, 2010). The Cys-loop plays a role in nAChR assembly as well the kinetics of ion channel gating. nAChRs can exist as homomers of a subunits or as heteromers of either two kinds of a subunit, or, more commonly, of various combinations of a and non- a subunits. The ACh-binding site is located at the interface of two adjacent subunits and is formed by six distinct regions (loops A-F) in the N-terminal extracellular domain with loops A, B and C being contributed by an a subunit and loops D, E and F by either an a or non-a subunit. Subunits possessing two adjacent cysteine residues in loop C which are important for ACh binding are defined as a subunits while subunits lacking these vicinal cysteines are classified as non-a ([3, 5, s or y). ANDREW K. JONES A D DAVID B. SATTELLE, INSECT NICOTINIC ACETYLCHOLINE RECEPTORS (1st, 2010).

[0189] nAChR subunits can be divided into the following domains: a signal sequence; an amino-terminal extracellular ligand-binding domain; four transmembrane domains that form the transmembrane pore; and, between the third and fourth transmembrane domains, a 48641601282Attorney Docket No. 277702-582798large cytoplasmic loop. The second transmembrane domains TM2 of each subunit line the pore with a stripe of polar amino acids on the TM2 alpha helix facing the pore lumen. The TM2 domains are circumscribed by a ring of alternating TM1 and TM3 domains, with the TM4 domains deployed on the outside facing the lipid. The pore, starting at the lip of the extracellular domains, is funnel-shaped with the spout at the cytoplasmic end of the transmembrane domains. Id.

[0190] The ligand binding site sits at the interface between two subunits near the outer surface of the channel. A channel can have up to five ligand-binding sites depending on the how many adjacent subunit pairs bind ligand, ligand-binding pocket is a cage formed by aromatic side chains. The transmembrane domains form the channel gate and the ion selective filter. Rings of charged amino acids at the cytoplasmic side of the TM2 domain, where it links to the TM1 domain, determine the ion selectivity of the open pore. It is widely believed that agonists bind at subunit interfaces, generally at interfaces between an a and a non- a subunit. Some nAChRs subunits (always a subunits) are capable of forming functional homomeric receptors; the most extensively studied example being the vertebrate a7 subunit. However, more commonly, nAChRs are heteromeric complexes containing both a and non- a subunits.

[0191] A core group of nAChR subunits appears to be highly conserved in different insect species. Comparative genomics has allowed the characterization of complete nAChR gene families from disparate species such as Anopheles gambiae (malaria mosquito). Apis mellifera (honeybee), Tribolium castaneum (red flour beetle) and Bombyx mori (silkworm). See Holt et al. The genome sequence of the malaria mosquito Anopheles gambiae. Science.2002; 298: 129-149; Jones et al. The nicotinic acetylcholine receptor gene family of the malaria mosquito, Anopheles gambiae. Genomics. 2005; 85: 176-187; Jones et al. The nicotinic acetylcholine receptor gene family of the honeybee. Apis mellifera. Genome Res.2006; 16:1422-1430; Jones et al. The cys-loop ligand-gated ion channel gene superfamily of the red flour beetle, Tribolium castaneum. BMC Genomics. 2007; 8:327; Richards et al. The genome of the model beetle and pest Tribolium castaneum. Nature. 2008; 452:949-955; and Shao et al. The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori. BMC Genomics. 2007; 8:324. Each of the five insect nAChR gene families has seven core groups of subunits that are highly conserved between species. Thus, Anopheles, Apis, Bombyx and Tribolium have subunit equivalents of Dal -7, D 1 and D|32, suggesting that the core groups are common to insects. Immunohistochemistry and in situ hybridization techniques have revealed that several Drosophila nAChR subunits (Dal, Da2, Da3, Da4, Da7, Dpi and 49641601282Attorney Docket No. 277702-582798Df>2) have overlapping distributions in various regions of the nervous system. Likewise, mutation experiments have shown that Dot7 mediates the Drosophila escape response.

[0192] Expression of functional insect nAChRs in heterologous systems has so far proved difficult. NEIL S. MILLAR AND STUART J. LANDSELL, INSECT NICOTINIC ACETYLCHOLINE RECEPTORS (1st, 2010). Further confounding analysis of the insect nAChR is that insect species possess a distinct complement of divergent nAChR subunits (i.e., at least one divergent subunit having low homology to other known subunits), and are prone to RNA editing events. Id. For example, RNA A-to-I editing involves the modification of select adenosine (A) residues to inosine (I) in pre-mRNA transcripts by adenosine deaminases acting on RNA (ADARs). Because inosine is interpreted by cellular machineries as guanosine (G). A-to-I editing generates transcripts with a nucleotide composition different from that of the corresponding genomic DNA. RNA A-to-I editing has been observed in five D. melanogaster nAChR subunits which alter amino acid residues in functionally significant regions. Id.

[0193] “NCBF’ refers to the National Center for Biotechnology Information.

[0194] "nm” refers to nanometers.

[0195] '‘Non-Polar amino acid” is an amino acid that is weakly hydrophobic and includes glycine, alanine, proline, valine, leucine, isoleucine, phenylalanine and methionine. Glycine or gly is the most preferred non-polar amino acid for the dipeptides of this invention.

[0196] “Normalized peptide yield” means the peptide yield in the conditioned medium divided by the corresponding cell density at the point the peptide yield is measured. The peptide yield can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg / L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu.sec. The cell density can be represented by visible light absorbance of the culture at wavelength of 600 nm (OD600).

[0197] “OD” refers to optical density. Typically, OD is measured using a spectrophotometer. When measuring grow th over time of a cell population, OD600 is preferable to UV spectroscopy; this is because at a 600 nm wavelength, the cells will not be harmed as they would under too much UV light.

[0198] “OD660nm” or “ODeeonm” refers to optical densities of a liquid sample measured (for example, yeast cell culture) when measured in a spectrophotometer at 660 nanometers (nm).

[0199] “One letter code” means the peptide sequence which is listed in its one letter code to distinguish the various amino acids in the primary structure of a protein: alanine=A,50641601282Attorney Docket No. 277702-582798arginine=R, asparagine=N, aspartic acid=D, asparagine or aspartic acid=B, cysteine=C, glutamic acid=E. glutamine=Q, glutamine or glutamic acid=Z, glycine=G, histidine=H, isoleucine=I, leucine=L, lysine=K, methionine=M, phenylalanine=F, proline=P, serine=S, threonine=T, tryptophan=W, tyrosine=Y, and valine=V.

[0200] “Open reading frame” or “ORF” refers to a length of RNA or DNA sequence, between a translation start signal (e.g.. AUG or ATG, respectively) and any one or more of the known termination codons, which encodes one or more polypeptide sequences. Put another way, the ORF describes the frame of reference as seen from the point of view of a ribosome translating the RNA code, insofar that the ribosome is able to keep reading (i.e., adding amino acids to the nascent protein) because it has not encountered a stop codon. Thus, “open reading frame” or “ORF” refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. Here, the terms “initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides (i.e., a codon) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

[0201] In some embodiments, an ORF is a continuous stretch of codons that begins with a start codon (usually ATG for DNA, and AUG for RNA) and ends at a stop codon (usually UAA, UAG or UGA). In other embodiments, an ORF can be length of RNA or DNA sequence, between a translation start signal (e g., AUG or ATG) and any one or more of the known termination codons, wherein said length of RNA or DNA sequence encodes one or more polypeptide sequences. In some other embodiments, an ORF can be a DNA sequence encoding a protein which begins with an ATG start codon and ends with a TGA, TAA or TAG stop codon. ORF can also mean the translated protein that the DNA encodes. Generally, those having ordinary skill in the art distinguish the terms “open reading frame” and “ORF,” from the term “coding sequence,” based upon the fact that the broadest definition of “open reading frame” simply contemplates a series of codons that does not contain a stop codon. Accordingly, while an ORF may contain introns, the coding sequence is distinguished by referring to those nucleotides (e.g., concatenated exons) that can be divided into codons that are actually translated into amino acids by the ribosomal translation machinery (i.e., a coding sequence does not contain introns); however, as used herein, the terms “coding sequence”; “CDS”; “open reading frame”; and “ORF,’ are used interchangeably.

[0202] “Operable” refers to the ability to be used, the ability to do something, and / or the accomplishing or achieving some function or result. For example, in some embodiments, “operable” refers to the ability of a pair of cysteine residues to form a disulfide bond. In other 51641601282Attorney Docket No. 277702-582798embodiments, operable refers to the ability- of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to encode a peptide, polypeptide, and / or protein. For example, in some embodiments, a polynucleotide may be operable to encode a protein, which means that the polynucleotide contains information that imbues it with the ability- to create a protein (e.g., by transcribing mRNA, which is in turn translated to protein).

[0203] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, in some embodiments, operably linked can refer to two or more DNA, peptide, or polypeptide sequences. In other embodiments, operably linked can mean that the two adjacent DNA sequences are placed together such that the transcriptional activation of one DNA sequence can act on the other DNA sequence. In yet other embodiments, the term “operably linked” can refer to two or more peptides and / or polypeptides, wherein said two or more peptides and / or polypeptides are connected in such a way as to yield a single polypeptide chain; alternatively, the term operably linked can refer to two or more peptides that are connected in such a way that one peptide exerts some effect on the other. In yet other embodiments, operably linked can refer to two adjacent DNA sequences are placed together such that the transcriptional activation of one can act on the other.

[0204] “Out-recombined” or “out-recombination” refers to the removal of a gene and / or polynucleotide sequence (e g., an endogenous gene) that is flanked by two sitespecific recombination sites (e.g., the 5’- and 3’- nucleotide sequence of a target gene that is homologous to the homology arms of a target vector) during in vivo homologous recombination. See “knockout.”

[0205] “Peptide yield” means the insecticidal peptide concentration in the conditioned medium which is produced from the cells of a peptide expression yeast strain. It can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg / L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu.sec.

[0206] “Pest” refers to any organism (e.g.. including, but is not limited to: insects, fungi, bacteria, nematodes, mites, ticks, snails, and the like) that is annoying, troublesome, detrimental, harmful, a nuisance, attacks, is directly or indirectly harmful and / or causes damage or destruction, for example, to people, plants, crops, pets, and / or livestock.

[0207] In some embodiments, a pest can be an organism (e.g., an insect) that directly or indirectly harms the plant. In some embodiments, a pest can have a harmful direct effect52641601282Attorney Docket No. 277702-582798on plant. For example, in some embodiments, a pest can be insect (e.g., a phytophagous insect pest) that causes a harmful direct effect on a plant by feeding on the plant leaves.

[0208] In yet other embodiments, a pest can be an insect (e.g., a phytopathogenic insect) that causes a harmful indirect effect on a plant, e.g., via transmission of a disease agent (e.g., a virus, bacteria, etc.) from the insect to the plant. Accordingly, in some embodiments, the pest serves as a vector for pathogen transmission.

[0209] In some embodiments, a pest can be a phytophagous insect that consumes, eats, or otherwise injures a seed or plant grown therefrom, or any part of a plant, e.g., plant tissues, plant cells, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, propagules, embryos and progeny of the same.

[0210] “Pesticidal’' is generally used to refer to the ability of a peptide of the present disclosure, or a composition comprising the same, to increase mortality, inhibit the growth rate, or interfere with one or more activities, of a pest, e.g., a deleterious arthropod insect. As used herein, “insecticidal” is generally used to refer to the ability of a peptide of the present disclosure, or a composition comprising the same, to increase mortality, inhibit the growth rate, or interfere with one or more activities, of an insect.

[0211] “Pesticidal activity” or “insecticidal activity” (e.g., in the context of insect pests) means that upon or after exposing the pest to a peptide of the present disclosure or a composition comprising a peptide of the present disclosure and an excipient, the pest (e.g., a deleterious insect) either dies, stops or slows its movement; stops or slows its feeding; stops or slows its growth; becomes confused (e.g., with regard to navigation, locating food, sleeping behaviors, and / or mating); fails to pupate; interferes with reproduction; and / or precludes the insect from producing offspring and / or precludes the insect from producing fertile offspring, or any combination of the foregoing.

[0212] “Pesticidal activity assay” or “insecticidal activity assay” (e.g., in the context of insect pests) refers to an assay wherein one or more pests (e.g., one or more deleterious insects) are treated with either an agent (e.g., a peptide of the present disclosure), or a comparator or control, and wherein the insecticidal activity of the agent or the comparator or control is then determined 12-, 18-, or 24-hours post-treatment based on the dose (ng / pL) of the agent, or the dose (ng / pL) of the comparator or control, w hich is required to achieve 100% insect mortality, i.e., 100% of insects are dead, unmoving, or unable to hold on to the side of a container (or host in the context of a parasite) when said container (or host) is upended.53641601282Attorney Docket No. 277702-582798

[0213] In some embodiments, the pesticidal activity of the agent relative to the pesticidal activity the comparator or control can be compared, wherein (1) at least a first group of one or more pests, e.g., an insect pest, is treated with the agent (e.g., a peptide of the present disclosure); and (2) at least a second group of one or more pests is treated with the comparator or control; and wherein the pesticidal activity of the agent relative to the comparator or control is then determined, e.g., 24-hours post-treatment, based on the dose (ng / pL) of the agent, relative to the dose (ng / pL) of the comparator or control, required to achieve 100% insect mortality (i.e., 100% of the insects are dead, unmoving, or unable to hold on to the side of a container or host, when said container or host is up-ended).

[0214] Accordingly, where an agent such as a peptide of the present disclosure has an pesticidal activity that is greater than a comparator or control, e.g., a comparator such as a vehicle, water, etc. — as measured via an pesticidal activity assay as described herein — the dose (ng / pL) of the agent required to achieve 100% pest mortality (i.e., 100% of the one or more insects are dead, unmoving, or unable to hold on to the side of a container or host when said container or host is up-ended) will be less than the dose (ng / pL) of the comparator or control required to achieve 100% pest mortality.

[0215] For example, in some embodiments, a pesticidal activity assay can be performed as follows: one or more insects can be immobilized via CO2 for 10 minutes, and then transferred to a CO2 pad to keep them immobilized. Insects of a predetermined size (e.g., weight in mg) can be picked for topical application of the agent or control. A dose concentration of an agent (e.g., a peptide of the present disclosure) and a comparator (e.g., water, vehicle, or a untreated control) can be estimated by peak area using reverse phase HPLC. Next, the treatment can be applied, e.g., 0.5 pL of a solution containing the agent, or a control solution containing water, can be topically applied to each insect, or the environment (e.g., host environment). Here, the solution containing the agent is topically applied to insects in a first group, and the control solution containing water is topically applied to insects in a second group. The treated insects and untreated insects can then each be transferred to their own container, which may optionally contain a host organism (e.g.. to determine whether the agent has an effect on the host). 'Insect mortality / ’ i.e., insect knock-down and insect death, can then be assessed at 24-hours post-treatment of the single dose; here, ‘‘insect mortality” is the dose (ng / pL) required for 100% of the insects to be either dead, unmoving, or unable to hold on to the side of a container or host when said container or host is up-ended.

[0216] “Pesticidal effect” or “insecticidal effect” (e.g., in the context of an insect pest) refers to the removal or the reduction of harm of pests (e.g., deleterious insects). The 54641601282Attorney Docket No. 277702-582798concept of “insecticidal effect'’ includes reducing of the target insect, killing of insects (extermination), insect proliferation inhibition, insect development inhibition, insect growth inhibition, repelling of insects (repellence), reducing of the survival rate of the target insect and the removal or the reduction of harm of insects (for example, inhibition of ingestion capacity of agricultural insects). “Insecticidal effect” includes killing of any individual or group of insects.

[0217] “Pesticidally-effective amount” or “insecticidally-effective amount” or refers to an amount of (1) a peptide of the present disclosure, or an agriculturally acceptable salt thereof; and / or (2) a pesticidal composition comprising: a peptide of the present disclosure, or an agriculturally acceptable salt thereof, and an excipient; that is sufficient to: partially or completely inhibit an pest (e.g., a deleterious arthropod insect); bring about the death of at least one pest; partially or completely reduce or decrease insect growth, feeding, or normal physiological development; partially or completely inhibit or decrease the normal insect cellular processes, including maintenance and growth; and / or attenuate or decrease the severity of an insect infestation. This amount will vary depending on such factors including but not limited to: the specific target insect to be controlled; the specific environment, location, plant, crop, or agricultural site to be treated; the environmental conditions, method, rate, concentration, stability, and quantity applied. Further, those having ordinary’ skill in the art will recognize that the insecticidally-effective amount may also vary with respect to climatic conditions, environmental considerations, and / or frequency of application and / or severity of insect infestation. In some embodiments, insecticidally-effective amounts can be measured by use of assays that measure the reduction in growth or decline in insect populations. One measure of reduction can be to express the decrease in population in logarithmic scale typical of a specific microbial species. That is, a 1 log reduction is equivalent to a 90% reduction versus a control, a 2 log reduction is a 99% reduction, etc.

[0218] “Pharmaceutically acceptable salt” is synonymous with agriculturally7acceptable salt, and as used herein refers to a compound that is modified by making acid or base salts thereof.

[0219] “Plant” shall mean whole plants, plant tissues, plant cells, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).55641601282Attorney Docket No. 277702-582798

[0220] “Plant transgenic protein” means a protein from a heterologous species that is expressed in a plant after the DNA or RNA encoding it was delivered into one or more of the plant cells.

[0221] “Plant-incorporated protectant” or “PIP” means an insecticidal protein produced by transgenic plants, and the genetic material necessary for the plant to produce the protein.

[0222] “Plant cleavable linker” means a cleavable linker peptide, or a nucleotide encoding a cleavable linker peptide, which contains a plant protease recognition site and can be cleaved during the protein expression process in the plant cell.

[0223] “Plant regeneration media” means any media that contains the necessary' elements and vitamins for plant growth and plant hormones necessary to promote regeneration of a cell into an embryo which can germinate and generate a plantlet derived from tissue culture. Often the media contains a selectable agent to which the transgenic cells express a selection gene that confers resistance to the agent.

[0224] “Plasmid” refers to a DNA segment that acts as a carrier for a gene of interest, and, when transformed or transfected into an organism, can replicate and express the DNA sequence contained within the plasmid independently of the host organism. Plasmids are a type of vector, and can be “cloning vectors” (i.e., simple plasmids used to clone a DNA fragment and / or select a host population carrying the plasmid via some selection indicator) or “expression plasmids” (i.e., plasmids used to produce large amounts of polynucleotides and / or polypeptides).

[0225] “Polar amino acid” is an amino acid that is polar and includes serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan and tyrosine; preferred polar amino acids are serine, threonine, cysteine, asparagine and glutamine; with serine being most highly preferred.

[0226] “Polynucleotide” refers to a polymeric-form of nucleotides (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof) of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides. As used herein, the term “polynucleotide” includes double- and single-stranded DNA, as well as double- and singlestranded RNA; it also includes modified and unmodified forms of a polynucleotide (modifications to and of a polynucleotide, for example, can include methylation, phosphorylation, and / or capping). In some embodiments, a polynucleotide can be one of the following: a gene or gene fragment (for example, a probe, primer, EST, or SAGE tag); genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer 56641601282Attorney Docket No. 277702-582798RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probe; primer or amplified copy of any of the foregoing.

[0227] In yet other embodiments, a polynucleotide can refer to a polymeric-form of nucleotides operable to encode the open reading frame of a gene.

[0228] In some embodiments, a polynucleotide can refer to cDNA.

[0229] In some embodiments, polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The structure of a polynucleotide can also be referenced to by its 5’- or 3’- end or terminus, which indicates the directionality of the polynucleotide. Adjacent nucleotides in a single-strand of polynucleotides are typically joined by a phosphodiester bond between their 3’ and 5' carbons. However, different intemucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc. This means that the respective 5’ and 3’ carbons can be exposed at either end of the polynucleotide, which may be called the 5' and 3’ ends or termini. The 5’ and 3’ ends can also be called the phosphoryl (PO4) and hydroxyl (OH) ends, respectively, because of the chemical groups attached to those ends. The term polynucleotide also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment that makes or uses a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0230] In some embodiments, a polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with nonnatural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.

[0231] In some embodiments, a polynucleotide can also be further modified after polymerization, such as by conjugation with a labeling component. Additionally, the sequence of nucleotides in a polynucleotide can be interrupted by non-nucleotide components. One or more ends of the polynucleotide can be protected or otherwise modified to prevent that end from interacting in a particular way (e g. forming a covalent bond) with other polynucleotides.

[0232] In some embodiments, a polynucleotide can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) can also be present, for example, as a natural replacement for thymine when the 57641601282Attorney Docket No. 277702-582798polynucleotide is RNA. Uracil can also be used in DNA. Thus, the term “sequence” refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and non-natural bases.

[0233] The term “RNA molecule” or ribonucleic acid molecule refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and ty pically uracil rather than thymine as one of the pyrimidine bases. An RNA molecule of the invention is generally single-stranded, but can also be double-stranded. In the context of an RNA molecule from an RNA sample, the RNA molecule can include the single-stranded molecules transcribed from DNA in the cell nucleus, mitochondrion or chloroplast, which have a linear sequence of nucleotide bases that is complementary to the DNA strand from which it is transcribed.

[0234] In some embodiments, a polynucleotide can further comprise one or more heterologous regulatory elements. For example, in some embodiments, the regulatory element is one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; or combinations thereof.

[0235] “Post-transcriptional regulatory- elements” are DNA segments and / or mechanisms that affect mRNA after it has been transcribed. Mechanisms of post-transcriptional mechanisms include splicing events; capping, splicing, and addition of a Poly (A) tail, and other mechanisms known to those having ordinary skill in the art.

[0236] “Promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

[0237] “Protein” and “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in yvhich one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms “protein” and “polypeptide” and “peptide” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0238] “Ratio” refers to the quantitative relation between two amounts showing the number of times one value contains or is contained yvithin the other.

[0239] “Reading frame” refers to one of the six possible reading frames, three in each direction, of the double stranded DNA molecule. The reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA58641601282Attorney Docket No. 277702-582798molecule. In some embodiments, a reading frame is a way of dividing the sequence of nucleotides in a polynucleotide and / or nucleic acid (e.g., DNA or RNA) into a set of consecutive, non-overlapping triplets.

[0240] ■‘Recombinant DNA” or “rDNA” refers to DNA that is comprised of two or more different DNA segments.

[0241] ‘Recombinant vector” means a DNA plasmid vector into which foreign DNA has been inserted.

[0242] '‘Regulatory elements” refers to a genetic element that controls some aspect of the expression and / or processing of nucleic acid sequences. For example, in some embodiments, a regulatory element can be found at the transcriptional and post-transcriptional level. Regulatory elements can be cis-regulatory elements (CREs), or trans-regulatory elements (TREs). In some embodiments, a regulatory element can be one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; and / or other elements that influence gene expression, for example, in a tissuespecific manner; temporal-dependent manner; to increase or decrease expression; and / or to cause constitutive expression.

[0243] “Restriction enzy me” or “restriction endonuclease” refers to an enzyme that cleaves DNA at a specified restriction site. For example, a restriction enzyme can cleave a plasmid at an EcoRI. SacII or BstXI restriction site allowing the plasmid to be linearized, and the DNA of interest to be ligated.

[0244] “Restriction site” refers to a location on DNA comprising a sequence of 4 to 8 nucleotides, and whose sequence is recognized by a particular restriction enzy me.

[0245] “Selection gene” means a gene which confers an advantage for a genetically modified organism to grow under the selective pressure.

[0246] “Serovar” or “serotype” refers to a group of closely related microorganisms distinguished by a characteristic set of antigens. In some embodiments, a serovar is an antigenically and serologically distinct variety of microorganism.

[0247] “577.” refers to species.

[0248] '‘ssp ” or “su-bsp ” refers to subspecies.

[0249] “Subcloning” or “subcloned” refers to the process of transferring DNA from one vector to another, usually advantageous vector. For example, polynucleotide encoding an HVP can be subcloned into a pLB102 plasmid subsequent to selection of yeast colonies transformed with pKLACl plasmids.59641601282Attorney Docket No. 277702-582798

[0250] “SSI” is an acronym that is context dependent. In some contexts, it can refer to “site-specific integration,” which is used to refer to a sequence that will permit in vivo homologous recombination to occur at a specific site within a host organism’s genome. Thus, in some embodiments, the term “site-specific integration” refers to the process directing a transgene to a target site in a host-organism's genome, allowing the integration of genes of interest into pre-selected genome locations of a host-organism. However, in other contexts, SSI can refer to “surface spraying indoors,” which is a technique of applying a variable volume sprayable volume of an insecticide onto surfaces where vectors rest, such as on walls, windows, floors and ceilings.

[0251] “STA” or “Translational stabilizing protein” or “stabilizing domain” or “stabilizing protein” (used interchangeably herein) means a peptide or protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation. The protein can be between 5 and 50 amino acids long. The translational stabilizing protein is coded by a DNA sequence for a protein that is operably linked with a sequence encoding an insecticidal protein or an HVP in the ORF. The operably-linked STA can either be upstream or downstream of the HVP and can have any intervening sequence between the tw o sequences (STA and HVP) as long as the intervening sequence does not result in a frame shift of either DNA sequence. The translational stabilizing protein can also have an activity which increases delivery of the HVP across the gut wall and into the hemolymph of the insect.

[0252] '‘sta” means a nucleotide encoding a translational stabilizing protein.

[0253] “Stability ” or “increased stability ” or “stable mutant” as used herein, refers to the ability' of a heterologous peptide (e.g., an HVP as described herein) to produce a single homogeneous species when said heterologous peptide is expressed in a recombinant system using standard fermentation techniques (e.g., such as those described herein). For example, in some embodiments, standard fermentation techniques such as those described herein (e.g., involving the application of temperature, pH, and the like), can cause heterologous peptides to incur post- translational modifications during the fermentation process. Examples of such post-translational modifications are well known in the art, and include, e.g., phosphorylation, dephosphorylation, glycosylation, deamidation, and the like. Amino acids that are susceptible to post-translational modifications include, without limitation, amino acid residues having an -OH or -NH2 group on their side chains, e.g., Serine (S), Threonine (T), Tyrosine (Y), Asparagine (N), Glutamine (Q), positively charged Lysine (K) and Arginine (R) residues, and negatively charged Aspartic acid (D) and Glutamic acid (E) residues (as they can lose a “-H”60641601282Attorney Docket No. 277702-582798or “-OH” group). Post-translational modifications implicated in leading to the instability of a peptide may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), clipping / hydrolysis / fragmentation, succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, and the like.

[0254] In some embodiments, post-translational modifications can result in the production of variants of a heterologous peptide, i.e., one or more heterogeneous species of the heterologous peptide. In some embodiments, the one or more heterogeneous species of the heterologous peptide can be observed, e.g., as separate peaks using HPLC.

[0255] Accordingly, the present disclosure contemplates heterologous peptides (i.e., HVPs or HVP-insecticidal proteins) that are resistant to post-translation modifications, and whose expression results in the production of a single, homogenous species of the heterologous peptide, when expressed in a recombinant system using standard fermentation techniques as described herein.

[0256] As used herein, the term "‘stable composition" refers to a composition or formulation that maintains the chemical and / or physical stability of the active ingredient (e.g., an HVP or HVP-insecticidal protein) to within acceptable limits after an extended period of storage at the intended storage conditions of the product. In some embodiments, a stable composition has less than 10% degradation over two years or less than 5% degradation over two years.

[0257] '‘Stringent hybridization” or ‘'stringent hybridization conditions” refers to conditions under which a polynucleotide (e.g., a nucleic acid probe, primer or oligonucleotide) will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not to other sequences. In some embodiments, the term ‘'stringent hybridization” or “stringent hybridization conditions” refers to the conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold, 5-fold, or 10-fold over background).

[0258] Stringent hybridization conditions are sequence- and length-dependent, and will be different in different circumstances. Similarly, stringent hybridization conditions depend on % (percent)-identity (or %-mismatch) over a certain length of nucleotide residues. Generally, longer sequences hybridize specifically at higher temperatures than shorter sequences. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in 61641601282Attorney Docket No. 277702-582798sequences so that lower degrees of similarity' are detected (heterologous probing). Generally, a probe is less than about 1000 or 500 nucleotides in length

[0259] For example, in some embodiments, stringent hybridization conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e g., 10 to 50 nucleotides), and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). In some embodiments, stringent hybridization conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0260] In some embodiments, low stringency hybridization conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C. and a wash in lx to 2xSSC (20xSSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50-55°C. In some embodiments, moderate stringency hybridization conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5x to IxSSC at 55-60°C. In some embodiments, high stringency hybridization conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C.. and a final wash in 0.1 xSSC at 60 to 65°C. for at least about 20 minutes. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

[0261] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm(thermal melting point) can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500 / L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, ”% form” is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tmis the temperature (under defined ionic strength and pH) at which 50% of a complementary' target sequence hybridizes to a perfectly matched probe. Washes are ty pically performed at least until equilibrium is reached and a low background level of hybridization is achieved, such as for 2 hours, 1 hour, or 30 minutes.

[0262] In some embodiments, the Tmis reduced by about 1°C for each 1% of mismatching; thus, Tm, hybridization, and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity' are sought, the Tmcan be decreased 10°C.62641601282Attorney Docket No. 277702-582798

[0263] In some embodiments, stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, in some embodiments, stringent hybridization conditions can utilize a hybridization and / or wash temperature that is about 1°C, 2°C, 3°C, or 4°C lower than the thermal melting point (Tm).

[0264] Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. An exemplary description of the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes. Part I, Chapter 2 (Elsevier, New Y ork); and Ausubel et al., eds. ( 1995) Current Protocols in Molecular Biology. Chapter 2 (Greene Publishing and Wiley -Interscience, New York); the disclosures of which are incorporated herein by reference in their entireties.

[0265] In some embodiments, a polynucleotide of the present disclosure can stringently hybridize to a polynucleotide encoding an HVP. or a complementary nucleotide sequence thereof.

[0266] '‘Susceptible to attack by a pest(s)." refer to plants, or human or animal patients or subjects, susceptible to a pest or a pest infections.

[0267] “Toxin"’ refers to a venom and / or a poison, especially a protein or conjugated protein produced by certain animals, higher plants, and pathogenic bacteria. Generally, the term '‘toxin” is reserved natural products, e.g., molecules and peptides found in scorpions, spiders, snakes, poisonous mushrooms, etc., whereas the term “toxicant” is reserved for manmade products and / or artificial products e.g., man-made chemical pesticides. However, as used herein, the terms “toxin” and “toxicant” are used synonymously

[0268] “Transfection” and '‘transformation” both refer to the process of introducing exogenous and / or heterologous DNA or RNA (e.g., a vector containing a polynucleotide that encodes an HVP) into a host organism (e.g., a prokary ote or a eukaryote). Generally, those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and / or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and / or heterologous DNA or RNA into eukary otic cells. However, as used herein, the term “transformation” and “transfection"’ are used synonymously, regardless of whether a process describes the introduction exogenous and / or heterologous DNA or RNA into a prokary ote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).63641601282Attorney Docket No. 277702-582798

[0269] “Transgene” means a heterologous and / or exogenous DNA sequence encoding a protein which is transformed into a plant.

[0270] “Transgenic host cell” or “host cell” means a cell which is transformed with a gene and has been selected for its transgenic status via an additional selection gene.

[0271] “Transgenic plant” means a plant that has been derived from a single cell that was transformed with foreign DNA such that every cell in the plant contains that transgene.

[0272] “Transient expression system” means an Agrobacterium tumefaciens-based system which delivers DNA encoding a disarmed plant virus into a plant cell where it is expressed. The plant virus has been engineered to express a protein of interest at high concentrations, up to 40% of the TSP.

[0273] “Triple expression cassette refers to three HVP expression cassettes contained on the same vector.

[0274] “TRBO” means a transient plant expression system using Tobacco mosaic virus with removal of the viral coating protein gene.

[0275] “Trypsin cleavage” means an in vitro assay that uses the protease enzyme trypsin (which recognizes exposed lysine and arginine amino acid residues) to separate a cleavable linker at that cleavage site. It also means the act of the trypsin enzyme cleaving that site.

[0276] “TSP” or “total soluble protein” means the total amount of protein that can be extracted from a plant tissue sample and solubilized into the extraction buffer.

[0277] “UBT” refers to ubiquitin. For example, in some embodiments, UBI can refer to a ubiquitin monomer isolated from Zea mays.

[0278] “var.” refers to varietas or variety'. The term “var.” is used to indicate a taxonomic category that ranks below the species level and / or subspecies (where present). In some embodiments, the term “var.” represents members differing from others of the same subspecies or species in minor but permanent or heritable characteristics.

[0279] “Vector” refers to the DNA segment that accepts a foreign gene of interest. The gene of interest is known as an “insert” or “transgene.”

[0280] “Wild type” or “WT” or “wild-type” or “wildtype” refer to the phenotype and / or genotype (i.e., the appearance or sequence) of an organism, polynucleotide sequence, and / or polypeptide sequence, as it is found and / or observed in its naturally occurring state or condition.

[0281] “Yield” refers to the production of a peptide, and increased yields can mean increased amounts of production, increased rates of production, and an increased average or 64641601282Attorney Docket No. 277702-582798median yield and increased frequency at higher yields. The term “yield” when used in reference to plant crop growth and / or production, as in “yield of the plant” refers to the quality and / or quantity of biomass produced by the plant.

[0282] The terms “first,” “second,” and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another, and the terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. All ranges disclosed herein are inclusive and combinable.

[0283] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0284] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, solid phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid phase peptide synthesis, immunology, cell culture, and formulation. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York. Second Edition (1989), whole of Vols I. II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc ), whole of series; J. F. Ramalho Ortigao, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R. B. (1963). J. Am. Chem. Soc.85, 2149-2154: Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, 3. eds.). vol. 2, pp. 1-284, Academic Press. New York. 12. Wiinsch. E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler,65641601282Attorney Docket No. 277702-582798E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis. Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach. Third Edition (John R. W. Masters, ed., 2000); each of these references are incorporated herein by reference in their entireties.

[0285] Although the disclosure of the invention has been described in detail for purposes of clarity and understanding, it will be obvious to those with skill in the art that certain modifications can be practiced within the scope of the appended claims. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually denoted.

[0286] Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0287] All patent applications, patents, and printed publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. And, all patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers, or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.

[0288] Hexatoxin variant peptides (HVPs)

[0289] The Hexcithelidae family of spiders formerly contained the Atracidae, Macrothelidae, and Porrhothelidae families of spiders; however, molecular phylogenetics revealed that Hexcithelidae was not monophyletic, thus the genera Atracidae, Macrothelidae and Porrhothelidae were split off into new families. See Hedin et al., Phylogenomic reclassification of the world’s most venomous spiders (Mygalomorphae, Atracidae), with implications for venom evolution. Sci Rep. 2018; 8: 1636. One such spider that produces these venom toxins is known by its common name as the Australian Blue Mountains Funnelweb Spider, which includes three genera: Atrax. Hadronyche. and Illawarra, comprising 35 species, e.g., the species Hadronyche versuta and Atrax robustus. For the sake of brevity, as 66641601282Attorney Docket No. 277702-582798used herein, venom toxins isolated from the Hexathelidae, Atracidae, Macrothelidae, and Porrhothelidae families of spiders are all referred to herein as “ACTX” or “ACTX peptide” or “atracotoxin” or “hexatoxin” or “HXTX” or “HXTX peptide.” Examples of ACTX peptides isolated from Atracidae family species are the Omega- ACTX, Kappa- ACTX, and U-ACTX peptides.

[0290] In some embodiments, an HVP comprises a DNA sequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein, that has an alteration, variation, or modification (for example, in the nucleotide sequence or the amino acid sequence), relative to a wild-ty pe or originating DNA sequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein from which the HVP was derived.

[0291] In some embodiments, an HVP comprise an alteration, variation, or modification that can be one or more nucleotide and / or amino acid substitutions or modifications (e.g., deletion or addition), in a DNA sequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein, relative to the wild-ty pe or originating DNA sequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein from which the HVP was derived.

[0292] An exemplary originating protein from which HVPs can be derived (i.e., an originating protein that is mutated, wherein one or more amino acid substitutions, deletions, or additions to the originating protein results) is the protein “Hybrid,” which has an amino acid sequence: “QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO: 2).

[0293] Another exemplary7originating protein from which HVPs can be derived (i.e., an originating protein that is mutated, wherein one or more amino acid substitutions, deletions, or additions to the originating protein results) is the protein “Hybrid+2,” which has an ammo acid sequence of “GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO: 1).

[0294] Exemplary HVPs

[0295] In some embodiments, a hexatoxin variant peptide (HVP) can be a mutant or variant that has a different amino acid sequence from wild type Hybrid protein (SEQ ID NO: 2), e.g., in some embodiments, this variance can be an amino acid substitution, amino acid deletion / insertion, or a change to the polynucleotide encoding the HVP. The result of this variation is a non-naturally occurring polypeptide and / or polynucleotide sequence encoding the same, relative to WT Hybrid, that possesses insecticidal activity against one or more insect species.67641601282Attorney Docket No. 277702-582798

[0296] In some embodiments, a hexatoxin variant peptide (HVP) can be a mutant or variant that has a different amino acid sequence from a Hybrid+2 protein (SEQ ID NO: 1). e.g., in some embodiments, this variance can be an amino acid substitution, amino acid deletion / insertion, or a change to the polynucleotide encoding the HVP. The result of this variation is a non-naturally occurring polypeptide and / or polynucleotide sequence encoding the same, relative to Hybrid+2, that possesses insecticidal activity against one or more insect species. HVPs of the present disclosure do not comprise the entire amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and are therefore, novel and engineered insecticidal peptides relative to the insecticidal peptides of SEQ ID NOs: 1 and 2.

[0297] The HVP of the present disclosure does not consist of or have the same or identical amino acid sequence in its entire length as the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2is L, Y or S; X3is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E. or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or an agriculturally acceptable salt thereof.68641601282Attorney Docket No. 277702-582798

[0298] In some embodiments, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, comprises, consists essentially of, or consists of, an ammo acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (II): Xi-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-X8-R-N-X9-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 132); wherein Xi is G, L, N, A, or E; X2is S, L, or A; X3 is N or D; X4 is D, S, K, or E; X5 is D or P; Xe is A or N; X7 is T, V, or F; Xs is E, A, or K; and X9 is E, A. or K: and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0299] In some embodiments, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, comprises, consists essentially of, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (III): G-S-Q-Y-C-V-P-V-X1-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-X2-R-N-X3-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 133); wherein Xi is N or D; X2 is E, A, or K; and X3 is E, A, or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0300] In some embodiments, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, comprises, consists essentially of, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%. 99% or 100% identical to an amino acid sequence according to Formula (IV): X1-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-E-R-N-E-N-G-H-T-V-Y-Y- 69641601282Attorney Docket No. 277702-582798C-R-A (SEQ ID NO: 134); wherein Xi is G, L, N, A, or E; X2is S. L, or A; X3is N or D; X is S, K. or E; Xs is D or P; Xe is A or N; X7 is T, V, or F; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0301] In some embodiments, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, comprises, consists essentially of, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (V): G-S-Q-Y-C-V-P-V-D-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-E-R-N-Xi-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 135); wherein Xi is A or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0302] In some embodiments, where sequences differ in conservative amino acid substitutions, the percent sequence identity7or homology7may be adjusted upwards to correct for the conservative nature of the substitution.

[0303] A summary of the HVPs evaluated here possessing mutations that confer novel and unexpected properties are provided in the table 1 below. A listing of illustrative mutants evaluated is provided at the end of the application.

[0304] Table 1. Summary7of HVPs possessing mutations that confer novel and unexpected properties relative to the insecticidal peptide Hybrid+2 (SEQ ID NO: 1). Table 1 provides a summary of HVPs having at least one mutation that confers at least one novel property to the HVP relative to Hybrid+2. The properties include: (1) increased expression; or (2) increased activity — relative to SEQ ID NO: 1.SEQMutation Amino Acid Sequence ID NO.D9N GSQYCVPVNQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 13 D21S D22G GSQYCVPVDQPCSLNTQPCCSGATCTQERNENGHTVYYCRA 137 D9H GSQYCVPVHQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 14 V6I S13G L14PN15K D21E E28D GSQYCIPVDQPCGPKTQPCCEDATCTQDRNENGHTVYYCRA 141 D21L GSQYCVPVDQPCSLNTQPCCLDATCTQERNENGHTVYYCRA 148D21T GSQYCVPVDQPCSLNTQPCCTDATCTQERNENGHTVYYCRA 15470641601282Attorney Docket No. 277702-582798SEQMutation Amino Acid Sequence ID NO.D9G GSQYCVPVGQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 16 D22Q GSQYCVPVDQPCSLNTQPCCDQATCTQERNENGHTVYYCRA 167 G1Q D21K T24V qSQYCVPVDQPCSLNTQPCCkDAVCTQERNENGHTVYYCRA 174 G1E S2L D21K T24V ELQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA 179 D9N L14YD21S GSQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA 190 G1L D9ND21S LSQYCVPVNQPCSLNTQPCCSDATCTQERNENGHTVYYCRA 192 D21S T24V GSQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA 193 G1Q D21S T24V qSQYCVPVDQPCSLNTQPCCsDAVCTQERNENGHTVYYCRA 194 G1L D21S T24V LSQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA 195 G1N D9NL14YD21S NSQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA 196 G1N D21S T24V NSQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA 197 GIF. S2I. D9NL14Y D21S ELQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA 198 GIA S2L D9N L14Y D21 S ALQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA 199 S13N GSQYCVPVDQPCNLNTQPCCDDATCTQERNENGHTVYYCRA 20 G1A S2L D21S T24V ALQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA 200 G1E S2AD9NL14YD21S EAQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA 201 G1E S2AD21S T24V EAQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA 202 D9N D21S T24V G33A GSQYCVPVNQPCSLNTQPCCSDAVCTQERNENAHTVYYCRA 203 GID S2A D9N T24V G33A DAQYCVPVNQPCSLNTQPCCDDAVCTQERNENAHTVYYCRA 206 G1L D9NL14Y D21K LSQYCVPVNQPCSYNTQPCCKDATCTQERNENGHTVYYCRA 209 S13D GSQYCVPVDQPCDLNTQPCCDDATCTQERNENGHTVYYCRA 21 D21K T24V GSQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA 211 L14W GSQYCVPVDQPCSWNTQPCCDDATCTQERNENGHTVYYCRA 22 N15H GSQYCVPVDQPCSLHTQPCCDDATCTQERNENGHTVYYCRA 24 D21E D22P A23N T24F GSQYCVPVDQPCSLNTQPCCEPNFCTQERNENGHTVYYCRA 244 GIT S2Y D21G D22N A23T T24H TYQYCVPVDQPCSLNTQPCCGNTHCTQERNENGHTVYYCRA 245 T16P GSQYCVPVDQPCSLNPQPCCDDATCTQERNENGHTVYYCRA 25 A23W GSQYCVPVDQPCSLNTQPCCDDWTCTQERNENGHTVYYCRA 27 GIN NSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 3 A23L GSQYCVPVDQPCSLNTQPCCDDLTCTQERNENGHTVYYCRA 32 T24Q GSQYCVPVDQPCSLNTQPCCDDAQCTQERNENGHTVYYCRA 33 T24I GSQYCVPVDQPCSLNTQPCCDDAICTQERNENGHTVYYCRA 34 T24V GSQYCVPVDQPCSLNTQPCCDDAVCTQERNENGHTVYYCRA 35 E28A GSQYCVPVDQPCSLNTQPCCDDATCTQARNENGHTVYYCRA 38 E28K GSQYCVPVDQPCSLNTQPCCDDATCTQKRNENGHTVYYCRA 39 GIF ESQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 4 R29L GSQYCVPVDQPCSLNTQPCCDDATCTQELNENGHTVYYCRA 41 R29F GSQYCVPVDQPCSLNTQPCCDDATCTQEFNENGHTVYYCRA 42 R29M GSQYCVPVDQPCSLNTQPCCDDATCTQEMNENGHTVYYCRA 45 R29Q GSQYCVPVDQPCSLNTQPCCDDATCTQEQNENGHTVYYCRA 46 N30D GSQYCVPVDQPCSLNTQPCCDDATCTQERDENGHTVYYCRA 47 E31A GSQYCVPVDQPCSLNTQPCCDDATCTQERNANGHTVYYCRA 48 E31K GSQYCVPVDQPCSLNTQPCCDDATCTQERNKNGHTVYYCRA 50 G33A GSqyCVPVDQPCSLNTQPCCDDATCTQERNENAHTVYYCRA 51 G33D GSqyCVPVDQPCSLNTQPCCDDATCTQERNENDHTVYYCRA 52 H34L GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGLTVYYCRA 55 H34N GSqyCVPVDQPCSLNTQPCCDDATCTQERNENGNTVYYCRA 56 T35L GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHLVYYCRA 58 T35P GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHPVYYCRA 59 GIL LSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 6 T35W GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHWVYYCRA 60 T35V GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHWYYCRA 61 Y37K GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVKYCRA 65V8M D9Q GSQYCVPMQQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 6871641601282Attorney Docket No. 277702-582798SEQMutation Amino Acid Sequence ID NO.D9N L14W GSQYCVPVnQPCSwNTQPCCDDATCTQERNENGHTVYYCRA 71 L14WR29H GSQYCVPVDQPCSwNTQPCCDDATCTQEhNENGHTVYYCRA 74 D9N L14Y GSQYCVPVnQPCSyNTQPCCDDATCTQERNENGHTVYYCRA 76 G1S SSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 8 D9N R29M GSQYCVPVnQPCSLNTQPCCDDATCTQEmNENGHTVYYCRA 82 T24V G33A GSQYCVPVDQPCSLNTQPCCDDAvCTQERNENaHTVYYCRA 84 T16C Q17C GSqyCVPVDQPCSLNCCPCCDDATCTQERNENGHTVYYCRA 86 D22G E31A GSQYCVPVDQPCSLNTQPCCDGATCTQERNANGHTVYYCRA 87 E28K E31A GSQYCVPVDQPCSLNTQPCCDDATCTQKRNANGHTVYYCRA 88 GIT TSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 9 D9N L14W R29F GSQYCVPVnQPCSwNTQPCCDDATCTQEf NENGHTVYYCRA 90 D9N T24V G33A GSQYCVPVnQPCSLNTQPCCDDAvCTQERNENaHTVYYCRA 93 D22G E28K E31A GSQYCVPVDQPCSLNTQPCCDGATCTQKRNANGHTVYYCRA 94 D9G D21 S D22G E28A E31 A GSQYCVPVGQPCSLNTQPCCSGATCTQARNANGHTVYYCRA 97D9G D21S D22G E28K E31 A GSQYCVPVGQPCSLNTQPCCSGATCTQKRNANGHTVYYCRA 99

[0305] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, said HVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to any one of the amino acid sequences provided in the foregoing Table 1; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0306] Table 2. Exemplary HVPs that have neither improved activity nor improved expression in the same yeast recombinant strain produced relative to the hexatoxin Hybrid+2 of SEQ ID NO: 1.SEQMutation Amino Acid Sequence IDNO.Q3A GSAYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 1072641601282Attorney Docket No. 277702-582798SEQMutation Amino Acid Sequence ID NO.D9H D21H D22H E28H GSQYCVPVHQPCSLNTQPCCHHATCTQHRNHNGHTVYYCRA too E31HY4F GSQFCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 11 V8M GSQYCVPMDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 12 Y4A V6T V8A D21SD22GT24S Q27E E28Q GSQACTPADQPCSLNTQPCCSGASCTEQLNENGHTVYYCRA 138 R29LP18W GSQYCVPVDQPCSLNTQWCCDDATCTQERNENGHTVYYCRA 139 V6I S13Q L14T T16L GSQYCIPVDQPCQTNLSPCCEDATCTQDRNENGHTVYYCRA Q17S D21E E28D 140 D21A GSQYCVPVDQPCSLNTQPCCADATCTQERNENGHTVYYCRA 142 D21E GSQYCVPVDQPCSLNTQPCCEDATCTQERNENGHTVYYCRA 143 D21F GSQYCVPVDQPCSLNTQPCCFDATCTQERNENGHTVYYCRA 144 D21G GSQYCVPVDQPCSLNTQPCCGDATCTQERNENGHTVYYCRA 145 D21H GSQYCVPVDQPCSLNTQPCCHDATCTQERNENGHTVYYCRA 146 D21I GSQYCVPVDQPCSLNTQPCCIDATCTQERNENGHTVYYCRA 147 D21M GSQYCVPVDQPCSLNTQPCCMDATCTQERNENGHTVYYCRA 149 D9Q GSQYCVPVQQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 15 D21N GSQYCVPVDQPCSLNTQPCCNDATCTQERNENGHTVYYCRA 150 D21P GSQYCVPVDQPCSLNTQPCCPDATCTQERNENGHTVYYCRA 151 D21Q GSQYCVPVDQPCSLNTQPCCQDATCTQERNENGHTVYYCRA 152 D21R GSQYCVPVDQPCSLNTQPCCRDATCTQERNENGHTVYYCRA 153 D21V GSQYCVPVDQPCSLNTQPCCVDATCTQERNENGHTVYYCRA 155 D21W GSQYCVPVDQPCSLNTQPCCWDATCTQERNENGHTVYYCRA 156 D21Y GSQYCVPVDQPCSLNTQPCCYDATCTQERNENGHTVYYCRA 157 D22A GSQYCVPVDQPCSLNTQPCCDAATCTQERNENGHTVYYCRA 158 D22E GSQYCVPVDQPCSLNTQPCCDEATCTQERNENGHTVYYCRA 159 D22F GSQYCVPVDQPCSLNTQPCCDFATCTQERNENGHTVYYCRA 160 D22H GSQYCVPVDQPCSLNTQPCCDHATCTQERNENGHTVYYCRA 161 D22I GSQYCVPVDQPCSLNTQPCCDIATCTQERNENGHTVYYCRA 162 D22L GSQYCVPVDQPCSLNTQPCCDLATCTQERNENGHTVYYCRA 163 D22M GSQYCVPVDQPCSLNTQPCCDMATCTQERNENGHTVYYCRA 164 D22N GSQYCVPVDQPCSLNTQPCCDNATCTQERNENGHTVYYCRA 165 D22P GSQYCVPVDQPCSLNTQPCCDPATCTQERNENGHTVYYCRA 166 D22R GSQYCVPVDQPCSLNTQPCCDRATCTQERNENGHTVYYCRA 168 D22S GSQYCVPVDQPCSLNTQPCCDSATCTQERNENGHTVYYCRA 169 D9K GSQYCVPVKQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 17 D22T GSQYCVPVDQPCSLNTQPCCDTATCTQERNENGHTVYYCRA 170 D22V GSQYCVPVDQPCSLNTQPCCDVATCTQERNENGHTVYYCRA 171 D22W GSQYCVPVDQPCSLNTQPCCDWATCTQERNENGHTVYYCRA 172 D22Y GSQYCVPVDQPCSLNTQPCCDYATCTQERNENGHTVYYCRA 173 G1L D21K T24V LSQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA 175 GIN D9N L14Y D21K NSQYCVPVNQPCSYNTQPCCKDATCTQERNENGHTVYYCRA 176 GIN D21KT24V NSQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA 177 G1E S2L D9N L14Y ELQYCVPVNQPCSYNTQPCCKDATCTQERNENGHTVYYCRA D21K 178 Q10P GSQYCVPVDPPCSLNTQPCCDDATCTQERNENGHTVYYCRA 18 GIA S2L D9N L14Y ALQYCVPVNQPCSYNTQPCCKDATCTQERNENGHTVYYCRA 180 D21KG1A S2L D21K T24V ALQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA 18173641601282Attorney Docket No. 277702-582798SEQMutation Amino Acid Sequence ID NO.G1E S2A D9N L14Y EAQYCVPVNQPCSYNTQPCCKDATCTQERNENGHTVYYCRA 182 D21KG1E S2A D21K T24V EAQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA 183 D9N D21K T24V G33A GSQYCVPVNQPCSLNTQPCCKDAVCTQERNENAHTVYYCRA 184 G1L D9N D21KT24V LSQYCVPVNQPCSLNTQPCCKDAVCTQERNENAHTVYYCRA 185 G33AGID S2A D9N D21K DAQYCVPVNQPCSLNTQPCCKDAVCTQERNENAHTVYYCRA 186 T24V G33AY37K Y38R R40D GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVKRCDA 187 Q10R T35GY37S GSQYCVPVDRPCSLNTQPCCDDATCTQERNENGHGVSYCRA 188 L14Y GSQYCVPVDQPCSYNTQPCCDDATCTQERNENGHTVYYCRA 189 Q10R GSQYCVPVDRPCSLNTQPCCDDATCTQERNENGHTVYYCRA 19 G1L D9N L14Y D21S LSQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA 191 G1L D9N D21S T24V LSQYCVPVNQPCSLNTQPCCSDAVCTQERNENAHTVYYCRA G33A 204 GID S2A D9N D21S DAQYCVPVNQPCSLNTQPCCSDAVCTQERNENAHTVYYCRA 205 T24V G33AGIL D9N T24V G33A LSQYCVPVNQPCSLNTQPCCDDAVCTQERNENAHTVYYCRA 207 D9N L14Y D21K GSQYCVPVNQPCSYNTQPCCKDATCTQERNENGHTVYYCRA 208 G1L D9N D21K LSQYCVPVNQPCSLNTQPCCKDATCTQERNENGHTVYYCRA 210 T16- GSQYCVPVDQPCSLN-QPCCDDATCTQERNENGHTVYYCRA 212 T16A GSQYCVPVDQPCSLNAQPCCDDATCTQERNENGHTVYYCRA 213 S13A L14AN15AT16A GSQYCVPVDQPCAAAAAPCCDDATCTQERNENGHTVYYCRA 214 Q17AD21E D22HA23E T24Q GSQYCVPVDQPCSLNTQPCCEHEQCTQERNENGHTVYYCRA 215 S2V D21GD22S A23P GVQYCVPVDQPCSLNTQPCCGSPHCTQERNENGHTVYYCRA 216 T24HG1M S2T D21N D22T MTQYCVPVDQPCSLNTQPCCNTVQCTQERNENGHTVYYCRA 217 A23V T24QG1I S2D D21A D22A IDQYCVPVDQPCSLNTQPCCAASGCTQERNENGHTVYYCRA 218 A23S T24GG1V S2D D21F D22E VDQYCVPVDQPCSLNTQPCCFETDCTQERNENGHTVYYCRA A23T T24D 219 G1D D21R D22GA23D DSQYCVPVDQPCSLNTQPCCRGDRCTQERNENGHTVYYCRA 220 T24RD21R D22N A23K T24S GSQYCVPVDQPCSLNTQPCCRNKSCTQERNENGHTVYYCRA 221 G1M S2T D21AD22F MTQYCVPVDQPCSLNTQPCCAFHHCTQERNENGHTVYYCRA 222 A23H T24HG1M S2L D21F D22Q MLQYCVPVDQPCSLNTQPCCFQKNCTQERNENGHTVYYCRA 223 A23K T24NS2I D21P D22I A23M GIQYCVPVDQPCSLNTQPCCPIMPCTQERNENGHTVYYCRA T24P 224 S2A D21P D22P A23G GAQYCVPVDQPCSLNTQPCCPPGFCTQERNENGHTVYYCRA 225 T24FG1S D21Y D22GA23H SSQYCVPVDQPCSLNTQPCCYGHQCTQERNENGHTVYYCRA 226 T24QS2I D21E D22S A23Q GIQYCVPVDQPCSLNTQPCCESQTCTQERNENGHTVYYCRA in S2F D21N D22K A23X GFQYCVPVDQPCSLNTQPCCNKXQCTQERNENGHTVYYCRA 228T24Q74641601282Attorney Docket No. 277702-582798SEQMutation Amino Acid Sequence ID NO.G1K D21H D22L A23G KSQYCVPVDQPCSLNTQPCCHLGMCTQERNENGHTVYYCRA 229 T24ML14F GSQYCVPVDQPCSFNTQPCCDDATCTQERNENGHTVYYCRA 23 S2L D21GD22A A23Y GLQYCVPVDQPCSLNTQPCCGAYSCTQERNENGHTVYYCRA 230 T24SS2K D21GD22Q A23G GKQYCVPVDQPCSLNTQPCCGQGNCTQERNENGHTVYYCRA 231 T24NG1S D21AD22P A23F SSQYCVPVDQPCSLNTQPCCAPFDCTQERNENGHTVYYCRA 232 T24DG1Q S2M D22KA23M QMQYCVPVDQPCSLNTQPCCDKMDCTQERNENGHTVYYCRA 233 T24DS2K D21P D22R T24S GKQYCVPVDQPCSLNTQPCCPRASCTQERNENGHTVYYCRA 234 G1M D21H A23F T24I MSQYCVPVDQPCSLNTQPCCHDFICTQERNENGHTVYYCRA 235 D21V D22H A23S T24S GSQYCVPVDQPCSLNTQPCCVHSSCTQERNENGHTVYYCRA 236 GIF S2M D21H D22G FMQYCVPVDQPCSLNTQPCCHGGLCTQERNENGHTVYYCRA 237 A23GT24LG1K D21H D22P A23T KSQYCVPVDQPCSLNTQPCCHPTHCTQERNENGHTVYYCRA 238 T24HG1D D21K D22Q DSQYCVPVDQPCSLNTQPCCKQATCTQERNENGHTVYYCRA 239 S2R D21H D22G A23G GRQYCVPVDQPCSLNTQPCCHGGLCTQERNENGHTVYYCRA 240 T24LS2T D21S D22GA23T GTQYCVPVDQPCSLNTQPCCSGTVCTQERNENGHTVYYCRA 241 T24VS2D D21K A23T T24S GDQYCVPVDQPCSLNTQPCCKDTSCTQERNENGHTVYYCRA 242 D21G A23RT24V GSQYCVPVDQPCSLNTQPCCGDRVCTQERNENGHTVYYCRA 243 T16K GSQYCVPVDQPCSLNKQPCCDDATCTQERNENGHTVYYCRA 26 D21K GSQYCVPVDQPCSLNTQPCCKDATCTQERNENGHTVYYCRA 28 D21S GSQYCVPVDQPCSLNTQPCCSDATCTQERNENGHTVYYCRA 29 D22G GSQYCVPVDQPCSLNTQPCCDGATCTQERNENGHTVYYCRA 30 D22K GSQYCVPVDQPCSLNTQPCCDKATCTQERNENGHTVYYCRA 31 T26Q GSQYCVPVDQPCSLNTQPCCDDATCQQERNENGHTVYYCRA 36 T26V GSQYCVPVDQPCSLNTQPCCDDATCVQERNENGHTVYYCRA 37 R29H GSQYCVPVDQPCSLNTQPCCDDATCTQEHNENGHTVYYCRA 40 R29Y GSQYCVPVDQPCSLNTQPCCDDATCTQEYNENGHTVYYCRA 43 R29K GSQYCVPVDQPCSLNTQPCCDDATCTQEKNENGHTVYYCRA 44 E31D GSQYCVPVDQPCSLNTQPCCDDATCTQERNDNGHTVYYCRA 49 G1Q QSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 5 G33V GSQYCVPVDQPCSLNTQPCCDDATCTQERNENVHTVYYCRA 53 H34E GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGETVYYCRA 54 T35I GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHIVYYCRA 57 T35G GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHGVYYCRA 62 T35S GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHSVYYCRA 63 Y37S GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVSYCRA 64 Y38R GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYRCRA 66 R40D GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCDA 67 N15H T16K GSQYCVPVDQPCSLHKQPCCDDATCTQERNENGHTVYYCRA 69 G1P PSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA 7 D9N R29F GSQYCVPVnQPCSLNTQPCCDDATCTQEfNENGHTVYYCRA 70 D9H R29H GSQYCVPVhQPCSLNTQPCCDDATCTQEhNENGHTVYYCRA 72N15H A23L GSQYCVPVDQPCSLhTQPCCDDITCTQERNENGHTVYYCRA 7375641601282Attorney Docket No. 277702-582798SEQMutation Amino Acid Sequence ID NO.D9N L14F GSQYCVPVnQPCSf NTQPCCDDATCTQERNENGHTVYYCRA 75 D9K R29F GSQYCVPVkQPCSLNTQPCCDDATCTQEfNENGHTVYYCRA 77 L14F R29Q GSQYCVPVDQPCSf NTQPCCDDATCTQEqNENGHTVYYCRA 78 L14Y R29M GSQYCVPVDQPCSyNTQPCCDDATCTQEmNENGHTVYYCRA 79 D9N G33A GSQYCVPVnQPCSLNTQPCCDDATCTQERNENaHTVYYCRA 80 R29F G33A GSQYCVPVDQPCSLNTQPCCDDATCTQEfNENaHTVYYCRA 81 D9N T24V GSQYCVPVnQPCSLNTQPCCDDAvCTQERNENGHTVYYCRA 83 T24V R29M GSQYCVPVDQPCSLNTQPCCDDAvCTQEmNENGHTVYYCRA 85 D9H L14W R29F GSQYCVPVhQPCSwNTQPCCDDATCTQEfNENGHTVYYCRA 89 D9N L14W G33A GSQYCVPVnQPCSwNTQPCCDDATCTQERNENaHTVYYCRA 91 D9N R29M G33A GSQYCVPVnQPCSLNTQPCCDDATCTQEmNENaHTVYYCRA 92 D9G D21K D22GE31A GSQYCVPVGQPCSLNTQPCCKGATCTQERNANGHTVYYCRA 95 D9K D21K D22K E28K GSQYCVPVKQPCSLNTQPCCKKATCTQKRNKNGHTVYYCRA 96 E31KD9G D21K D22GE28A GSQYCVPVGQPCSLNTQPCCKGATCTQARNANGHTVYYCRA 98E31A

[0307] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, said HVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to any one of the amino acid sequences provided in the foregoing Table 1; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0308] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, said HVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82%76641601282Attorney Docket No. 277702-582798identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to any one of the amino acid sequences provided in the foregoing Table 1; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0309] In some embodiments, the HVP comprises, consists essentially of, or consists of, an amino sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244 or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0310] In some embodiments, the HVP comprises, consists essentially of, or consists of, an amino sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5%77641601282Attorney Docket No. 277702-582798identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0311] In some embodiments, the HVP comprises, consists essentially of, or consists of, an amino sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0312] In some embodiments, the HVP consists essentially of an amino sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0313] In some embodiments, the HVP consists essentially of an amino sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof: and wherein the HVP further comprises 0, 1, 2, 3. 4, or 5 amino acid substitutions that are conservative amino acid substitutions.78641601282Attorney Docket No. 277702-582798

[0314] In some embodiments, the HVP consists of an amino sequence as set forth in any one of SEQ ID NOs: 13. 38. 39.48, 50, 190, 192. 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0315] In some embodiments, the HVP consists of an amino sequence as set forth in any one of SEQ ID NOs 3. 4, 6, 8-9. 13. 14. 16. 20-22, 24-25, 27. 32-35. 38-39, 41-42. 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0316] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “GSQYCVPVNQPCSLNTQPCCDDATCTQERNENGHTVYYCRA " (SEQ ID NO: 13); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.

[0317] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 79641601282Attorney Docket No. 277702-58279887% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “GSQYCVPVDQPCSLNTQPCCDDATCTQARNENGHTVYYCRA" (SEQ ID NO: 38); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0318] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “GSQYCVPVDQPCSLNTQPCCDDATCTQKRNENGHTVYYCRA" (SEQ ID NO: 39); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0319] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 80641601282Attorney Docket No. 277702-58279895% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “GSQYCVPVDQPCSLNTQPCCDDATCTQERNANGHTVYYCRA" (SEQ ID NO: 48); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0320] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: 'GSQYC VPVDQPCSLNTQPCCDDATCTQERNKNGHTVYYCRA" (SEQ ID NO: 50); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.

[0321] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at 81641601282Attorney Docket No. 277702-582798least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “GSQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA" (SEQ ID NO: 190); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.

[0322] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “LSQYCVPVNQPCSLNTQPCCSDATCTQERNENGHTVYYCRA" (SEQ ID NO: 192); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0323] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “LSQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 195);82641601282Attorney Docket No. 277702-582798wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0324] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: ‘NSQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 197); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0. 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.

[0325] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “ALQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 200); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the 83641601282Attorney Docket No. 277702-582798entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.

[0326] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “EAQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 202); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0327] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “GSQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 211); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and84641601282Attorney Docket No. 277702-582798wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0328] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “GSQYCVPVDQPCSLNTQPCCEPNFCTQERNENGHTVYYCRA" (SEQ ID NO: 244); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0329] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, a homopolymer or heteropolymer of two or more HVPs. wherein the amino acid sequence of each HVP is the same or different.

[0330] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an HVP that is a fused protein comprising two or more HVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each HVP may be the same or different.

[0331] In some embodiments, the linker is a cleavable linker.

[0332] In some embodiments, the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 102-111.

[0333] In some embodiments, the linker is cleavable inside at least one of (i) the gut or hemolymph of an insect, and (ii) cleavable inside the gut of a mammal.

[0334] Detailed methods concerning linkers are described below.

[0335] Polynucleotides encoding HVPs

[0336] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode a hexatoxin variant peptide (HVP).85641601282Attorney Docket No. 277702-582798

[0337] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S. T or G; X2is L, Y or S; X3 is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K orN; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I. Q, S. V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M. Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or a complementary polynucleotide sequence thereof.

[0338] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24- 86641601282Attorney Docket No. 277702-582798X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; Xs is Y, A, or F; X4is I, T or V; Xs is M or V; X6is G, H, N, Q or D; X7is Q or R,; Xs is D, G, N or S; X9is P, W, Y or L; Xio is H, K or N; Xu is C, P or T, or absent: X12 is Q or C; X13 is P or W; Xi4is E, G, H. I, K, L, N, P, Q, R, S, T. V or D; Xis is G. N, P, Q or D; Xi6 is L, N, R. T, W or A; X17 is F, H. I. Q, S. V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions, or a complementary polynucleotide sequence thereof.

[0339] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D. E, L, N, Q. S, T or G; X2is L, Y or S; X3 is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7is Q or R,; X8is D, G, N or S; X9 is P, W, Y or L; Xio is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I. Q, S, V or T; Xis is Q or E; X19 is D, K. Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or a complementary polynucleotide sequence thereof.

[0340] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (II): XI-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-X8-R-N-X9-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 132); wherein Xi is G, L, N, A, or E; X2 is S, L, or A; X3 is N or D; X4is D, S, K, or E; X5 is D or P; Xe is A or N; X7 is T, V, or F; X8is E, A. or K; and X9is E, A, or K; and wherein the HVP comprises at least one amino acid substitution 87641601282Attorney Docket No. 277702-582798relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0341] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (III): G-S-Q-Y-C-V-P-V-X1-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-X2-R-N-X3-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 133); wherein Xi is N or D; X2 is E, A, or K; and X3 is E, A. or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0342] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (IV): X1-X2-Q-Y-C-V-P-V-X3-Q-P-C-S-L-N-T-Q-P-C-C-X4-X5-X6-X7-C-T-Q-E-R-N-E-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 134); wherein Xi is G, L, N, A, or E; X2is S, L, or A; X3 is N or D; X4 is S, K, or E; X5 is D or P; Xe is A or N; X7 is T, V, or F; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0343] In addition, the present disclosure describes a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (V): G-S-Q-Y-C-V-P-V-D-Q-P-C-S-L-N-T-Q-P-C-C-D-D-A-T-C-T-Q-E-R-N-Xi-N-G-H-T-V-Y-Y-C-R-A (SEQ ID NO: 135); wherein Xi is A or K; and wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID 88641601282Attorney Docket No. 277702-582798NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0344] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192. 195, 197, 200, 202, 211. and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0345] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid89641601282Attorney Docket No. 277702-582798sequence set forth in SEQ ID NO: 1. with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0346] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0347] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192. 195, 197, 200, 202, 211, and 244. or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0348] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of. an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65%90641601282Attorney Docket No. 277702-582798identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0349] Nucleotide sequence homologs, e.g., HVPs encoded by polynucleotides that hybridize to each or any of the sequences disclosed in this application under stringent hybridization conditions, are also an embodiment of the present disclosure. The present disclosure also provides a method for detecting a first polynucleotide that hybridizes to a second polynucleotide, wherein the first polynucleotide (or its reverse complement sequence) encodes an HVP or fragment thereof, and hybridizes to the second polynucleotide. In such case, the second polynucleotide can be any of the polynucleotides operable to encode an HVP of the present disclosure, under stringent hybridization conditions.

[0350] In some embodiments, a polynucleotide of the present disclosure is operable to encode a hexatoxin variant peptide (HVP) having insecticidal activity’ against one or more insect species, said HVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to any one of the amino acid sequences provided in the foregoing Table 1; wherein the HVP comprises at least one 91641601282Attorney Docket No. 277702-582798amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1.

[0351] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195. 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof, or a complementary nucleotide sequence thereof.

[0352] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises an amino sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197. 200, 202. 211, and 244, or a complementary nucleotide sequence thereof.

[0353] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that consists essentially of an amino sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195. 197, 200, 202, 211, and 244, or a complementary nucleotide sequence thereof.

[0354] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that consists of an amino sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202. 211, and 244, or a complementary' nucleotide sequence thereof.

[0355] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“NSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO: 3): wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the 92641601282Attorney Docket No. 277702-582798entire amino acid sequence of SEQ ID NO: 1; or a complementary nucleotide sequence thereof. In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“GSQYCVPVNQPCSLNTQPCCDDATCTQERNENGHTVYYCRA " (SEQ ID NO: 13); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0356] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:■ GSQYC VPVDQPCSLNTQPCCDDATCTQARNENGHTVYYCRA" (SEQ ID NO: 38); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.93641601282Attorney Docket No. 277702-582798

[0357] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“GSQYCVPVDQPCSLNTQPCCDDATCTQKRNENGHTVYYCRA" (SEQ ID NO: 39); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0358] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“GSQYCVPVDQPCSLNTQPCCDDATCTQERNANGHTVYYCRA" (SEQ ID NO: 48); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0359] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence 94641601282Attorney Docket No. 277702-582798that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“GSQYCVPVDQPCSLNTQPCCDDATCTQERNKNGHTVYYCRA" (SEQ ID NO: 50); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0360] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an ammo acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“GSQYCVPVNQPCSYNTQPCCSDATCTQERNENGHTVYYCRA" (SEQ ID NO: 190); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0361] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81%95641601282Attorney Docket No. 277702-582798identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“LSQYCVPVNQPCSLNTQPCCSDATCTQERNENGHTVYYCRA" (SEQ ID NO: 192); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0362] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“LSQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 195); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0363] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89%96641601282Attorney Docket No. 277702-582798identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“NSQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 197); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0364] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“ALQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 200); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0365] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97%97641601282Attorney Docket No. 277702-582798identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“EAQYCVPVDQPCSLNTQPCCSDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 202); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0. 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0366] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:“GSQYCVPVDQPCSLNTQPCCKDAVCTQERNENGHTVYYCRA" (SEQ ID NO: 211); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0367] In some embodiments, an HVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:98641601282Attorney Docket No. 277702-582798“GSQYCVPVDQPCSLNTQPCCEPNFCTQERNENGHTVYYCRA" (SEQ ID NO: 244); wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0368] In some embodiments, the polynucleotide is operable to encode an HVP that can comprise, consist essentially of, or consist of, a homopolymer or heteropolymer of two or more HVPs, wherein the amino acid sequence of each HVP is the same or different.

[0369] In some embodiments, the polynucleotide is operable to encode an HVP that can comprise, consist essentially of, or consist of, an HVP that is a fused protein comprising two or more HVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each HVP may be the same or different.

[0370] In some embodiments, the linker is a cleavable linker.

[0371] In some embodiments, the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 102-111.

[0372] In some embodiments, the linker is cleavable inside at least one of (i) the gut or hemolymph of an insect, and (ii) cleavable inside the gut of a mammal.

[0373] Stringent hybridization

[0374] In some embodiments, the present disclosure contemplates a polynucleotide that can stringently hybridize to a complementary polynucleotide of the present disclosure, the polynucleotide is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of 99641601282Attorney Docket No. 277702-582798SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2is L. Y or S; X3is Y, A, or F; X4is I, T or V; Xs is M or V; X6is G, H, N. Q or D; X7is Q or R,; X8is D, G. N or S; X9is P, W, Y or L; Xio is H, K or N; Xu is C, P or T, or absent X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E. or K; X23 is G, or D; X24 is H, N, or L; X25 is T. V, P, L, W, or G; X26 is Y, or K; or a complementary polynucleotide sequence thereof

[0375] In some embodiments, a polynucleotide of the present disclosure is operable to encode an HVP that comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52. 55-56, 58-61, 65 82, 93, 154, 190, 192, 195. 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof; or (b) said HVP comprises an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244; or (c) said polynucleotide segment hybridizes to a polynucleotide having a polynucleotide segment operable to encode an HVP having an amino acid sequence as set forth in SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244.

[0376] In one embodiment, the present disclosure provides a method comprising contacting a sample of nucleic acids with a nucleic acid probe that hybridizes under stringent hybridization conditions with a polynucleotide comprising a polynucleotide segment encoding an HVP or fragment thereof as provided herein, and does not hybridize under such hybridization conditions with a polynucleotide that does not comprise the segment, wherein the probe is homologous or complementary to a polynucleotide encoding any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14. 16. 20-22, 24-25, 27, 32-35. 38-39, 41-42, 45-48. 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or a polynucleotide encoding 100641601282Attorney Docket No. 277702-582798an HVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical amino acid sequence to SEQ ID NOs: 3, 4, 6, 8-9, 13, 14. 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154. 190, 192. 195, 197, 200, 202, 211. and 244. The method may further comprise (a) subjecting the sample and probe to stringent hybridization conditions; and (b) detecting hybridization of the probe with polynucleotide of the sample.

[0377] In one embodiment, the present disclosure provides a method comprising contacting a sample of nucleic acids with a nucleic acid probe that hybridizes under stringent hybridization conditions with a polynucleotide comprising a polynucleotide segment encoding an HVP or fragment thereof as provided herein, and does not hybridize under such hybridization conditions with a polynucleotide that does not comprise the segment, wherein the probe is homologous or complementary to a polynucleotide encoding any one of SEQ ID NOs: 3, 4, 6. 8-9, 13, 14. 16. 20-22, 24-25, 27, 32-35. 38-39, 41-42, 45-48. 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or a polynucleotide encoding an HVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical amino acid sequence to SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202, 211, and 244. The method may further comprise (a) subjecting the sample and probe to stringent hybridization conditions: and (b) detecting hybridization of the probe with polynucleotide of the sample.101641601282Attorney Docket No. 277702-582798

[0378] In addition, the present disclosure describes a vector comprising a polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising an ammo acid sequence that is at least 95% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2is L, Y or S; X3is Y, A, or F; X4is I, T or V; Xs is M or V; X6is G, H, N, Q or D; X7 is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G. H, I, K, L, N, P, Q, R, S. T, V or D; Xis is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or a complementary polynucleotide sequence thereof.

[0379] HVP-insecticidal proteins

[0380] In some embodiments, an HVP-insecticidal protein can be any protein, peptide, polypeptide, amino acid sequence, configuration, construct, or arrangement, comprising: (1) at least one HVP, or two or more HVPs; and (2) one or more additional non-HVP peptides, polypeptides, or proteins. For example, in some embodiments, these additional non-HVP peptides, polypeptides, or proteins may have the ability to increase the mortality and / or inhibit the grow th of insects exposed to the HVP-insecticidal protein, relative to the HVP alone; increase the expression of the HVP-insecticidal protein, e.g., in a host cell; and / or affect the post-translational processing of the HVP-insecticidal protein.

[0381] In some embodiments, an HVP-insecticidal protein can be a polymer comprising two or more HVPs. In yet other embodiments, an HVP-insecticidal protein can be a polymer comprising two or more HVPs, wherein the HVPs are operably linked via a linker peptide, e.g., a cleavable and / or a non-cleavable linker. Here, the linker peptide falls under the category of the additional non-HVP peptide described above.

[0382] In some embodiments, an HVP-insecticidal protein can refer to a one or more HVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and / or any other combination thereof.102641601282Attorney Docket No. 277702-582798

[0383] In some embodiments, an HVP-insecticidal protein can be a polymer of amino acids that, when properly folded or in its most natural thermodynamic state, exerts an insecticidal activity against one or more insects.

[0384] In some embodiments, an HVP-insecticidal protein can be a polymer comprising two or more HVPs that are different. In other embodiments, an insecticidal protein can be a polymer of two or more HVPs that are the same.

[0385] In yet other embodiments, an HVP-insecticidal protein can comprise one or more HVPs, and one or more peptides, polypeptides, or proteins, that may assist in the HVP-insecticidal protein’s folding.

[0386] In some embodiments, an HVP-insecticidal protein can comprise one or more HVPs, and one or more peptides, polypeptides, or proteins, wherein the one or more peptides, polypeptides, or proteins are protein tags that help stability or solubility. In other embodiments, the peptides, polypeptides, or proteins can be protein tags that aid in affinity purification.

[0387] In some embodiments, an HVP-insecticidal protein can refer to a one or more HVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker; one or more heterologous peptides; one or more additional polypeptides; and / or any other combination thereof. In some embodiments, an insecticidal protein can comprise a one or more HVPs as disclosed herein.

[0388] In some embodiments, an HVP-insecticidal protein can comprise an HVP homopolymer, e.g., two or more HVP monomers that are the same HVP. In some embodiments, the insecticidal protein can comprise an HVP heteropolymer, e.g., two or more HVP monomers, wherein the HVP monomers are different.

[0389] In some embodiments, an HVP-insecticidal protein can comprise, consist essentially of, or consist of one or more HVPs wherein each HVP comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at 103641601282Attorney Docket No. 277702-582798least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L. Y or S; X3 is Y, A. or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R.; X8is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N. or L; X25 is T, V, P, L, W, or G; X26 is Y, or K: or a complementary polynucleotide sequence thereof

[0390] In some embodiments, an HVP-insecticidal protein can comprise, consist essentially of, or consist of one or more HVPs having an amino acid sequence set forth in SEQ ID NOs: 3, 4. 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39. 41-42, 45-48, 50-52.55-56, 58-61, 65 82. 93, 154, 190. 192, 195. 197, 200, 202. 211, and 244, wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof.

[0391] In some embodiments, the HVP-insecticidal protein may comprise an HVP having an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202, 211, and 244 w herein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; or an agriculturally acceptable salt thereof.

[0392] Examples of linkers include, but not limited to, the follo ing sequences: IGER (SEQ ID NO: 119), EEKKN, (SEQ ID NO: 120), and ETMFKHGL (SEQ ID NO: 121). or combinations thereof.104641601282Attorney Docket No. 277702-582798

[0393] In some embodiments, the linker can be one or more of the following:ALKFLV (SEQ ID NO: 102), ALKLFV (SEQ ID NO: 103), IFVRLR (SEQ ID NO: 104). LFAAPF (SEQ ID NO: 105), ALKFLV GS (SEQ ID NO: 106), ALKLFV GS (SEQ ID NO: 107), IFVRLRGS (SEQ ID NO: 108), LFAAPFGS (SEQ ID NO: 109), LFVRLRGS (SEQ ID NO: 110), and / or LGERGS (SEQ ID NO: 111).

[0394] Exemplary methods for the generation of cleavable and non-cleavable linkers can be found in U. S. Patent Application No. 15 / 727,277; and PCT Application No.PCT / US2013 / 030042, the disclosures of which are incorporated herein by reference in their entireties.

[0395] Exemplary ERSPs and STAs and their methods of use are provided in U. S. Patent No. 9.567,381, the disclosure of which is incorporated herein by reference in its entiret.

[0396] Detailed methods concerning ERSPs, STAs, and linkers, are described below.

[0397] MEANS FOR BINDING THE nAChR SUBUNIT Bl

[0398] The present disclosure provides means for binding an insect nicotinic acetylcholine receptor (nAChR) subunit [31, wherein binding the nAChR subunit [31 results in insect mortality.

[0399] Nicotinic acety lcholine receptors are a class of ligand-gated ion channels that mediate fast synaptic transmission in the nervous system (e g., of an insect). These receptors are integral to the functioning of cholinergic synapses, where they facilitate the rapid actions of acetylcholine (ACh) by allowing the flow of cations through their central pore. The cholinergic pathway is a critical excitatory neurotransmission system in the insect nervous system. Nicotinic acetylcholine receptors (nAChRs) are prototypical members of the Cys-loop ligand-gated ion channel superfamily, which includes ionotropic GABA, glycine and serotonin receptors. nAChRs are ligand-gated ion channels that serve as the foundation for rapid synaptic communication in insects, and mediate the fast actions of acetylcholine (ACh) in the nervous system and at neuromuscular junctions. Briefly, the primary function of insect nAChRs is to mediate cholinergic synaptic transmission by responding to the binding of ACh. Upon ACh binding, the receptor undergoes a conformational change that opens the central pore, permitting an influx of cations. The resulting depolarization of the neuron propagates the synaptic signal, thereby enabling rapid communication within the nervous system.

[0400] Typically, nAChRs assemble as pentamers of homologous subunits arranged around a central ion channel. Each nAChR subunit has four transmembrane domains (TM1- 105641601282Attorney Docket No. 277702-5827984) and possesses an N-terminal extracellular domain containing the characteristic Cys-loop motif consisting of two disulfide bond-forming cysteines separated by 13 amino acid residue...

Claims

1. Attorney Docket No. 277702-582798CLAIMS1. A hexatoxin variant peptide (HVP) having insecticidal activity against one or more insect species, said HVP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4 is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; Xi4is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or an agriculturally acceptable salt thereof.

2. The HVP of claim 1, wherein the HVP comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244, or an agriculturally acceptable salt thereof.

3. The HVP of any one of claims 1 to 2, wherein the HVP comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202, 211, and 244.

4. The HVP of any one of claims 1 to 3, wherein the HVP has 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions.419641601282Attorney Docket No. 277702-5827985. The HVP of claim 1, wherein the HVP is a homopolymer or heteropolymer of two or more HVPs, wherein the amino acid sequence of each HVP is the same or different, or a mixture of identical and different HVPs.

6. The HVP of claim 1, wherein the HVP is a fused protein comprising two or more HVPs separated by a cleavable or non-cleavable linker.

7. The HVP of claim 6, wherein the linker is a cleavable linker.

8. The HVP of claim 7, wherein the linker has an amino acid sequence as set forth in any one of SEQ IDNOs: 102-111.

9. A combination comprising, consisting essentially of, or consisting of, two or more HVPs of any one of claims 1-8.

10. A composition comprising, consisting essentially of, or consisting of, one or more HVPs of any one of claims 1-8 and an agriculturally acceptable excipient.

11. A polynucleotide operable to encode a hexatoxin variant peptide (HVP), said HVP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2is L, Y or S; X3is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; X8is D, G, N or S; X9 is P, W, Y or L; X10 is H, K orN; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; Xi4is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or420641601282Attorney Docket No. 277702-582798L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or a complementary polynucleotide sequence thereof.

12. The polynucleotide of claim 11, wherein the polynucleotide encodes an HVP comprising, consisting essentially of, or consisting of, an amino sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244.

13. The polynucleotide of any one of claims 11 to 12, wherein the polynucleotide encodes a HVP further comprising 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions.

14. A vector comprising the polynucleotide of any one of claims 11-13.

15. A polynucleotide operable to hybridize under stringent hybridization conditions with a polynucleotide segment encoding the HVP of any one of claims 1-8.

16. A yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an HVP, said HVP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24is H, N, or L; X25 is T, V, P, L, W, or G; X26is Y, or K; or a complementary nucleotide sequence thereof.421641601282Attorney Docket No. 277702-58279817. The yeast strain of claim 16, wherein the HVP comprises an amino sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244.

18. The yeast strain of claim 16, wherein the HVP comprises an amino sequence as set forth in any one of SEQ ID NOs: 13, 38, 39, 48, 50, 190, 192, 195, 197, 200, 202, 211, and 244.

19. The polynucleotide of any one of claims 16 to 18, wherein the HVP further comprises 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions.

19. The yeast strain of claim 16, wherein the yeast strain is selected from any species belonging to the genera Saccharomyces Pi chia. Kluyveromyces, Hansenula, Yarrow ia or Schizosaccharomyces.

20. The yeast strain of claim 19, wherein the yeast strain is selected from the group consisting of Kluyver omyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

21. The yeast strain of claim 20, wherein the yeast strain is Kluyveromyces lactis or Kluyveromyces marxianus.

22. A method of producing an HVP, the method comprising:(a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide operable to encode an HVP, or a complementary nucleotide sequence thereof, said HVP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4- P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21- X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO:422641601282Attorney Docket No. 277702-5827981; and wherein Xi is D, E, L, N, Q, S, T or G; X2is L, Y or S; X3is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7is Q or R,; Xs is D, G, N or S; X9is P, W, Y or L; Xio is H, K or N; Xu is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6 is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, orK;(b) introducing the vector into a host cell; and(c) growing the host cell in a growth medium under conditions operable to enable expression of the HVP and secretion into the growth medium.

23. The method of claim 22, wherein the FIVP comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244.

24. The method of claim 23, wherein the HVP is a homopolymer or heteropolymer of two or more HVPs, wherein the amino acid sequence of each HVP is the same or different, or a mixture of identical and different HVPs.

25. The method of claim 24, wherein the HVP is a fused protein comprising two or more HVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each HVP may be the same or different, or a mixture of identical and different HVPs.

26. The method of claim 25, wherein the linker is a cleavable linker.

27. The method of claim 26, wherein the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 102-111.

28. The method of claim 22, wherein the vector is a plasmid comprising an alpha-MF signal.423641601282Attorney Docket No. 277702-58279829. The method of claim 22, wherein the host cell is a yeast strain.

30. The method of claim 29, wherein the yeast strain is selected from any species belonging to the genera Saccharomyces Pichia, Kluyveromyces, Hansenula, Yarrow ia, or Schizosaccharomyces.

31. The method of claim 30, wherein the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

32. The method of claim 31, wherein the yeast strain is Kluyveromyces lactis.

33. The method of claim 22, wherein expression of the HVP provides a yield of: at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, at least 190 mg / L, at least 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of HVP per liter of yeast culture medium.

34. The method of claim 22, wherein expression of the HVP in the medium results in the expression of an HVP polymer comprising two or more HVP polypeptides in the medium.424641601282Attorney Docket No. 277702-58279835. The method of claim 22, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the HVP of the first expression cassette.

36. The method of claim 22, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the HVP of the first expression cassette, or an HVP of a different expression cassette.

37. The method of claims 35 or 36, wherein the expression cassette is operable to encode an HVP as set forth in any one of SEQ ID NOs: 3, 4, 6, 8-9, 13, 14, 16, 20-22, 24-25, 27, 32-35, 38-39, 41-42, 45-48, 50-52, 55-56, 58-61, 65 82, 93, 154, 190, 192, 195, 197, 200, 202, 211, and 244.

38. A method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the hexatoxin variant peptide (HVP) of any one of claims 1-8, the combination of claim 8, or the composition of claim 9, to: the pest, a locus of the pest, a food supply of the pest, a habitat of the pest, or a breeding ground of the pest; a plant, a seed, a plant part, a locus of a plant, or an environment of a plant that is susceptible to an attack by the pest; an animal, a locus of an animal, or an environment of an animal susceptible to an attack by the pest; or a combination thereof.

39. The method of claim 38, wherein the pest is selected from the group consisting of: group consisting of: Eumorpha ache mon, Colias eurytheme; Caudra Cantella, Amorbia humerosana, Psendaletia nnipuncta; Platyptilia cardnidactyla; Datana major, Thyridopteryx ephemeraeformis; Hypercompe scribonia, Erionota thrax; Acleris gloverana; Phryganidia californica; Paleacrita merriccata, Grapholita packardi; Nymphula stagnata; Xylomyges curialis; Cydia pomonella, Acrobasis vaccinii; Evergestis rimosalis; Noctuid species; Agrotis ipsilon, Orgyia psendotsngata; Erinnyis ello, Ennomos subsignaria; Lobesia botrana;Thyme liens lineola, Melissopus latif err eanus; Archips rosanus; Archips argyrospiha;Paralobesia viteana; Platynota stultana; Elarrisina americana; Plathypena scabra, Dryocampa rubicunda; Batrachedra comosae, Lymantria dispar, Lambdina fiscellaria, Mandnca quinquemaculata; Mandnca sexta; Pieris rapae; Antomeris io; Choristonenra pinus; Epiphyas425641601282Attorney Docket No. 277702-582798postvittana; Diaphania hyalinata; Homadaula anisocentra; Chori stoneura rosaceana.Syntomeida epilais Playnota stultana Sabulodes aegrotata; Papilio cresphontes; Argyrotaenia citrana Grapholita molester, Anarsia lineatellcr, Neophasia menapicr, Argyrotaenia velutinana Schizura concinna; Sibine stimulea; Heterocampa giittivitta; Estigmene acrea; Crambus sp.; Ennomos subsignaria Alsophila pometaria; Choristoneura fumiferana; Lasiocampidae sp.; Thecla basilides; Ephestia elutella; Platynota idaeusalis; Anarsia lineatelia,' Peridroma saucia; Platynota flavedana; Anticar sia gemmatalis; Datana integerrima,' Hyphantria cunea; Orgyia vetusta; Southern Diatraea crambidoides; Cylas formicarius, Anthonomus eugenii; Diaprepes abbrevia s,' Otiorhynchus ovates; Curculio caryae; Curculio occidentis; Lissorhoptrus oryzophilus; Hypera postica; Hypera zoilus; Euwallacea fornicates,' Euetheola humilis;Hypothenemus hamper, Listronotus maculicollis; Maladera castanea; Rhizotroqus majalis; Cotinis nitida; Popillia japonica; Phyllophaga sp.; Cyclocephala borealis,' Anomala orientalis; Cyclocephala lurida; Sphenophorus parvulus; Sphenophorus apicalis; Sphenophorus cariosus, Sphenophorus inaequalis; Sphenophorus minimus,' Aedes aegypti; Busseola fusca; Chilo suppressalis; Culex pipiens; Culex quinquefasciatus; Diabrotica virgifera; Diatraea saccharalis; Helicoverpa armigera; Helicoverpa zea; Heliothis virescens; Leptinotarsa decemlineata;Ostrinia furnacalis; Ostrinia nubilalis; Pectinophora gossypiella; Plodia interpunctella; Plutella xylostella; Pseudoplusia includens; Spodoptera exigua; Spodoptera frugiperda Spodoptera littoralis; Trichoplusia ni; and Xanthogaleruca luteola.

40. The method of claim 39, wherein the pest is selected from the group consisting of: Aedes aegypti,' Busseola fusca, Chilo suppressalis, Culex pipiens, Culex quinquefasciatus,' Diabrotica virgifera, Diatraea saccharalis,' Helicoverpa armigera, Helicoverpa zea,' Heliothis virescens,' Leptinotarsa decemlineata,' Ostrinia furnacalis,' Ostrinia nubilalis,' Pectinophora gossypiella, Plodia interpunctella, Plutella xylostella, Pseudoplusia includens, Spodoptera exigua, Spodoptera frugiperda,' Spodoptera littoralis,' Trichoplusia ni; and Xanthogaleruca luteola.

41. A method of combating an insect, the method comprising administering to the insect, a locus of the insect, or to a plant or animal susceptible to an attack by the insect:means for binding an insect nicotinic acetylcholine receptor (nAChR) subunit 01; wherein binding the nAChR subunit 01 results in insect mortality.426641601282Attorney Docket No. 277702-58279842. The method of claim 41, wherein the means for binding the insect nAChR subunit 01 binds at least one nAChR subunit 01 amino acid residue selected from T153, 1154, D155, V156, T157, Y158, Q163, T165, 1167, K169, 1205, E206, V207, P208, Y210, N212, Y214, E223, Y229, 1231, T292, L294, V295, P503, H504, E507, Y508 and V509 of SEQ ID NO: 1, or their homologous, or functional or analogous equivalent amino acid residues in other nAChR 01 proteins from other insect pest nAChR proteins.

43. The method of claim 41, wherein the means for binding the insect nAChR subunit 01 binds at least one nAChR subunit 01 amino acid residue selected from E206 and 1231 of SEQ ID NO: 1, or their homologous, or functional or analogous equivalent amino acid residues in other nAChR 01 proteins from other insect pest nAChR proteins.

44. The method of claim 42 or 43, wherein the means for binding the insect nAChR subunit 01 comprises an insecticidal peptide.

45. The method of claim 42 or 43, wherein the insecticidal peptide is a hexatoxin variant peptide (HVP).

46. The method of claim 45, wherein the HVP comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3 is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; X8is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or427641601282Attorney Docket No. 277702-582798R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or an agriculturally acceptable salt thereof.

47. The method of claim 41, wherein the insect is an insect belonging to an order selected from: Blattodea, Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Lxodida, Megaloptera, Orthoptera, Siphonaptera, Psocodea, Thysanoptera, and Trombidiformes.

48. The method of claim 47, wherein the insect is of the order Diptera.

49. The method of claim any one of claims 41-48, wherein the insect is selected from the group consisting of: Amyelois transitella, Eumorpha achemon, Colias eurytheme,' Caudra cautella, Amorbia humerosana, Pseudaletia unipuncta, Platyptilia carduidactyla,' Datana major, Thyridopteryx ephemeraeformis, Hypercompe scribonia, Erionota thrax, Acleris gloverana, Phryganidia californica, Paleacrita merriccata, Grapholita packardi, Nymphula stagnata, Xylomyges curialis, Cydia pomonella, Acrobasis vaccinii,' Evergestis rimosalis,' Nocteid species; Agrotis ipsilon, Orgyia pseudotsugata, Erinnyis ello, Ennomos subsignaria, Lobesia botrana, Thymelicus lineola, Melissopus latif err eanus,' Archips rosanus,' Archips argyrospilia, Paralobesia viteana, Platynota stultana,' Elarrisina americana, Plathypena scabra, Dryocampa rubicunda, Batrachedra comosae, Lymantria dispar, Lambdina fiscellaria, Manduca quinquemaculata, Manduca sexta, Pieris rapae, Automeris io,' Choristoneura pinus,' Epiphyas postvittana, Diaphania hyalinata, Homadaula anisocentra, Choristoneura rosaceana, Syntomeida epilais, Playnota stultana, Sabulodes aegrotata, Papilio cresphontes,' Argyrotaenia citrana, Grapholita molesta, Anar si a lineatelkr, Neophasia menapia, Argyrotaenia vehilinana, Schizura concinna, Sibine stimulea, Heterocampa guttivitta, Estigmene acrea, Crambus sp.; Ennomos subsignaria, Alsophila pometaria, Choristoneura fumiferana, Lasiocampidae sp.; Thecla basil ides, Ephestia elutelkr, Platynota idaeusalis, Anarsia lineatella,' Peridroma saucia, Platynota flavedana, Anticar sia gemmatalis, Datana integerrima, Hyphantria cunea, Orgyia vetusta,' Southern Diatraea crambidoides,' Cylas formicarius,' Anthonomus eugenir, Diaprepes abbreviates,' Otiorhynchus ovatus; Curculio caryae,' Curculio occidentis,' Lissorhoptrus oryzophilus, Hypera postica, Hypera zoilus,' Euwallacea fornicates,' Euetheola humilis, Hypothenemus hamper, Listronotus maculicollis,' Maladera castanea, Rhizotroqus majalis,'428641601282Attorney Docket No. 277702-582798Cotinis nitida; Popillia japonica; Phyllophaga sp.; Cyclocephala borealis., Anomala orientalise Cyclocephala lurida; Sphenophorus parvulus; Sphenophorus apicalis, Sphenophorus cariosus; Sphenophorus inaequalis Sphenophorus minimus,' Periplaneta americana; Aquatica leii;Ceutorhynchus assimilis; Diabrotica virgifera virgifera Photinus pyralis; Tribolium castaneum; Anopheles gambiae; Bradysia odoriphaga; Drosophila melanogaster, Musca domestica; Aphis gossypii; Bemisia tabaci; Macrosteles quadrilineatus; Myzus persicae; Nilaparvata lugens; Aphidius gifuensis; Apis mellifera; Bombus impatiens; Bombus terrestris; Camponotus floridanus; Cephus cinctus; Ceratina calcarata; Lasius niger; Neodiprion lecontei; Nylanderia fulva; Atta cephalotes; Bombyx mori; Ixodes scapularis; Protohermes xanthodes; Anabrus simplex,' Locusta migratoria; Ctenocephalides felis; Pediculus humanus corporis,' Thrips palmi, Tetranychus urticae; Aedes aegypti; Busseola fusca; Chilo suppressalis; Culex pipiens; Culex quinquefasciatus; Diabrotica virgifera, Diatraea saccharalis; Helicoverpa armigera;Helicoverpa zea; Heliothis virescens; Leptinotarsa decemlineata, Ostrinia fur nacalis; Ostrinia nubilalis; Pectinophora gossypiella, Plodia interpunctella; Plutella xylostella, Pseudoplusia includens; Spodoptera exigua; Spodoptera frugiperda; Spodoptera littoralis; Trichoplusia ni; and Xanthogaleruca luteola.

50. The method of 49, wherein the insect is selected from the group consisting of: Aedes aegypti; Busseola fusca; Chilo suppressalis; Culex pipiens; Culex quinquefasciatus; Diabrotica virgifera; Diatraea saccharalis; Helicoverpa armigera; Helicoverpa zea; Heliothis virescens; Leptinotarsa decemlineata; Ostrinia furnacalis; Ostrinia nubilalis; Pectinophora gossypiella; Plodia interpunctella; Plutella xylostella; Pseudoplusia includens; Spodoptera exigua;Spodoptera frugiperda; Spodoptera littoralis; Trichoplusia ni; and Xanthogaleruca luteola.

51. The method of claim 41, wherein the insect nAChR subunit 1 comprises an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the mature amino acid sequence according to SEQ ID NO: 249.

52. The method of claim 41, wherein the insect nAChR subunit 01 comprises an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the mature amino acid sequence according to Figure 19.429641601282Attorney Docket No. 277702-58279853. The method any one of claims 41-52, wherein administering the means for binding the insect nAChR subunit 1 comprises administering the means for binding the insect nAChR subunit pi in a composition comprising at least one excipient.

54. The method of claim 53, wherein the at least one excipient is selected from the group consisting of: trehalose; maltodextrin; potassium phosphate dibasic anhydrous (K2HPO4); potassium phosphate monobasic (KH2PO4); BIT; and fermentation solids.

55. The method of claim 54, wherein the composition comprises the means for binding the insect nAChR subunit pi in a concentration ranging from about 0.001% to about 16% w / w of the total weight of the composition.

56. The method of claim 54, wherein the composition comprises trehalose in a concentration ranging from about 5% to about 40% w / w; BIT in a concentration ranging from about 0.01% to about 0.1% w / w; maltodextrin in a concentration ranging from about 10% to about 50% w / w; potassium phosphate dibasic anhydrous (K2HPO4) in a concentration ranging from about 1% to about 5% w / w; potassium phosphate monobasic (KH2PO4) in a concentration ranging from about 0.10% to about 1% w / w; and fermentation solids in a concentration ranging from about 15% to about 40% w / w; of the total weight of the composition.

57. A composition comprising:means for binding an insect nicotinic acetylcholine receptor (nAChR) subunit 1, andat least one excipient;wherein binding the nAChR subunit 01 results in insect mortality.

58. The composition of claim 57, wherein the means for binding the insect nAChR subunit 01 binds at least one nAChR subunit 01 amino acid residue selected from T153, 1154, D155, V156, T157, Y158, Q163, T165, 1167, K169, 1205, E206, V207, P208, Y210, N212, Y214, E223, Y229, 1231, T292, L294, V295, P503, H504, E507, Y508 and V509 of SEQ ID NO: 249,430641601282Attorney Docket No. 277702-582798or their homologous, or functional or analogous equivalent amino acid residues in other nAChR pi proteins from other insect pest nAChR proteins.

59. The composition of claim 58, wherein the means for binding the insect nAChR subunit i comprises an insecticidal peptide.

60. The composition of claim 59, wherein the insecticidal peptide is an HVP.

61. The composition of claim 60, wherein the HVP comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-Q-X3-C-X4-P-X5-X6-X7-P-C-X8-X9-X10-X11-X12-X13-C-C-X14-X15-X16-X17-C-T-X18-X19-X20-X21-X22-N-X23-X24-X25-V-X26-Y-C-R-A; wherein the HVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the HVP does not comprise the entire amino acid sequence of SEQ ID NO: 1; and wherein Xi is D, E, L, N, Q, S, T or G; X2 is L, Y or S; X3is Y, A, or F; X4is I, T or V; X5is M or V; X6is G, H, N, Q or D; X7 is Q or R,; Xs is D, G, N or S; X9 is P, W, Y or L; X10 is H, K or N; X11 is C, P or T, or absent; X12 is Q or C; X13 is P or W; X14 is E, G, H, I, K, L, N, P, Q, R, S, T, V or D; X15 is G, N, P, Q or D; Xi6is L, N, R, T, W or A; X17 is F, H, I, Q, S, V or T; Xis is Q or E; X19 is D, K, Q or E; X20 is F, H, L, M, Q or R; X21 is N or D; X22 is E, or K; X23 is G, or D; X24 is H, N, or L; X25 is T, V, P, L, W, or G; X26 is Y, or K; or an agriculturally acceptable salt thereof.

62. The composition of claim 57, wherein the at least one excipient is selected from the group consisting of: trehalose; maltodextrin; potassium phosphate dibasic anhydrous (K2HPO4); potassium phosphate monobasic (KH2PO4); BIT; and fermentation solids.

63. The composition of claim any one of claims 57-62, wherein the means for binding the insect nAChR subunit 01 is in a concentration ranging from about 2% to about 16% w / w of the total weight of the composition.431641601282Attorney Docket No. 277702-58279864. The composition of claim 63, wherein the composition comprises trehalose in a concentration ranging from about 5% to about 40% vt / r, BIT in a concentration ranging from about 0.01% to about 0.1% w / w; maltodextrin in a concentration ranging from about 10% to about 50% w / w; potassium phosphate dibasic anhydrous (K2HPO4) in a concentration ranging from about 1% to about 5% w / w; potassium phosphate monobasic (KH2PO4) in a concentration ranging from about 0.10% to about 1% w / w; and fermentation solids in a concentration ranging from about 15% to about 40% w / w; of the total weight of the composition.

65. The composition of claim 64, wherein the concentration of the means for binding the insect nAChR subunit pi ranges from about 7% to about 9% w / w; and wherein the concentration of trehalose ranges from about 20% to about 30% w / w; BIT ranges from about 0.025% to about 0.075% w / w; maltodextrin ranges from about 30% to about 40% w / w; potassium phosphate dibasic anhydrous (K2HPO4) ranges from about 2% to about 3% w / w; potassium phosphate monobasic (KH2PO4) ranges from about 0.2% to about 0.6%; and fermentation solids range from about 20% to about 30%, of the total weight of the composition.

66. The composition of claim 65, wherein the concentration of the means for binding the insect nAChR subunit pi is about 8.5% w / w; and wherein the concentration of trehalose is about 25% w / w; BIT is about 0.05% w / w; maltodextrin is about 36.3% w / w; potassium phosphate dibasic anhydrous (K2HPO4) is about 2.6% w / w; potassium phosphate monobasic (KH2PO4) is about 0.4% il r, and fermentation solids are about 26.85% w / w, of the total weight of the composition.

67. The composition of claim 65, wherein the composition consists essentially of the following: an amount of the means for binding the insect nAChR subunit p i that is 8.5% w / w; an amount of trehalose that is 25% w / w; an amount of BIT that is 0.05% w / w; an amount of maltodextrin that is 36.3% w / w; an amount of potassium phosphate dibasic anhydrous (K2HPO4) that is 2.6% w / w; an amount of potassium phosphate monobasic (KH2PO4) that is 0.4% w / w; and an amount of fermentation solids that is 26.85% w / w, of the total weight of the composition.432641601282Attorney Docket No. 277702-58279868. The composition of claim 65, wherein the composition consists of the following: an amount of the means for binding the insect nAChR subunit 1 that is 8.5% w / w; an amount of trehalose that is 25% w / w; an amount of BIT that is 0.05% w / w; an amount of maltodextrin that is 36.3% w / w; an amount of potassium phosphate dibasic anhydrous (K2HPO4) that is 2.6% w / w; an amount of potassium phosphate monobasic (KH2PO4) that is 0.4% w / w; and an amount of fermentation solids that is 26.85% w / w, of the total weight of the composition.433641601282

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