Omega hexatoxin variant peptides and methods of use thereof

Abstract

New pesticidal peptides, pesticidal 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 Omega Variant Peptides (OVPs), which are non-natural peptide variations based upon the wild-type Omega-ACTX-Hvla protein and the non-natural recombinant protein, GS+Omega-ACTX-Hvla. Here, we describe: genes encoding OVPs; combinations, mixtures, and compositions comprising OVP peptides and / or pesticidal proteins; and methods using the same that are useful for the control of pests such as Varroa mites.

Description

Omega Hexatoxin Variant Peptides and Methods of Use ThereofCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit under 35 U.S.C. §1. 19(e) of U.S. Provisional Application No. 63 / 516,698 filed on July 31, 2023, and U.S. Provisional Application No. 63 / 637,325 filed on April 22, 2024, the contents of the aforementioned applications are incorporated herein by reference in their entireties.SEQUENCE LISTING

[0002] This application incorporates by reference in its entirety the Sequence Listing XML entitled “277702-544030. xml” (84,514 bytes), which was created on July 30, 2024, and filed electronically herewith.TECHNICAL FIELD

[0003] New and useful pesticidal proteins, nucleotides, peptides, their expression in plants, methods of producing the pesticidal proteins, new processes, production techniques, new formulations, and combinations of new and known organisms for the control of pests, deleterious insects, and Varroa mites, are described and claimed.BACKGROUND

[0004] Pests, such as deleterious insect pests, represent a worldwide threat to human health and food security. Insect pests 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 aey pii. 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. Blowflies (Chrysomya megacephala) and houseflies (Musca domestica) 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 wake. Eye gnats in the genus Hippelates can carry the spirochaete pathogen that causes yaws (Treponema perlenue). and may also spread conjunctivitis (pinkeye). Tsetse flies in the genus Glossina 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 (Leishmania spp.) that cause Leishmaniasis.

[0006] Likewise, insect pests directly and / or indirectly threaten global human food security. 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 some instances, the damage inflicted by insect pests can even lead to malnutrition or starvation. 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. One way insect pests compromise human food security is through their detrimental effects on bees.

[0007] Bees are beneficial insects that play a crucial role in world agriculture and economics. The industrial production of commercially relevant crops, vegetables, and fruits, are dependent on bees. Indeed, bees are responsible for the pollination of the majority' of both agricultural crops and wild plants. Moreover, bees are an economically important producer of commodities having diverse uses in food and medicine.

[0008] Without bees there would be a significant decrease in the availability and variety of fresh produce, thereby jeopardizing global human nutrition and health. Crops once effortlessly pollinated by bees, would instead require costly robotic or hand pollination; consequently, without these costly measures, many crops would be lost, or persist only with the dedication of passionate hobbyists.

[0009] In other words, our fates are intertwined with the health and survival of bees.

[0010] While bee health is threatened by many factors, there is no threat more significant or concerning than Varroa mites (e.g., Varroa destructor). Varroa mites are ectoparasitic mites that parasitize bees such as Apis cerana and Apis mellifera.

[0011] Varroa mites affect both the pupae and adult bees. The mites use specialized mouthparts to puncture the bees' cuticle and feed on the hemolymph found within; this injury and feeding subsequently weakens the bees — and injured bees are prone to more infections. Accordingly, Varroa mites believed to be vectors for a number of bee pathogens such as:acute bee paralysis virus (ABPV), black queen cell virus (BQCV), deformed wing virus (DWV), and Kashmir bee virus (KBV).

[0012] An infestation of Varroa mites (which is considered a disease and called “varroosis”) leads to a wide range of detrimental effects on individual bees and colony health as a whole. Indeed, a significant Varroa mite infestation will lead to the death of a bee colony. Because Varroa mites are one of the most significant stressors causing a decline in world-wide bee populations, they have a profound impact on agriculture in general, and the beekeeping industry in particular. Moreover, Varroa mites may be one of the key factors responsible for bee colony losses, including colony collapse disorder (CCD).

[0013] Currently, there are no reliable, simple, and / or long-lasting, treatments for combatting the Varroa mite. And, a reliable and effective treatment is needed that would not only control or inhibit Varroa mites, but more importantly, would not affect the health of the bees being parasitized by the mites.SUMMARY

[0014] The present disclosure describes novel, recombinant Omega Variant Peptides (OVPs) having pesticidal activity, and agriculturally acceptable salts thereof.

[0015] In addition, the present disclosure describes an Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%. or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S- C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2, wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is A, D, E, F, G. H, I, K, L, M, N, P, Q. R, V, W, or Y; or an agriculturally acceptable salt thereof. Each of the single letters of Formulas (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (II), and (III) denotes an amino acid residue in its single letter format.

[0016] In some embodiments, the present disclosure describes an Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2, wherein Xi and X2 are each independently any amino acid, or each areindependently optionally absent; and wherein X3 is D. E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof. In some embodiments, the OVP of Formulas (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (II), and (III) are deglycosylated.

[0017] In addition, the present disclosure describes an Omega Variant Peptide (OVP) having pesticidal activity , said OVP comprising or consisting of an amino acid sequence that is at least 90%. 95%. 96%. 97%. 97%. 98%. 99%. or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety'.

[0018] In a further aspect, the present disclosure describes an Omega V ariant Peptide (OVP) having pesticidal activity, said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence as set forth in any one of SEQ ID NOs: 15, 16, 19, 34, 35 and 38, or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety’. In related embodiments, an OVP of the present disclosure for use in preparing a composition, for example, an agricultural formulation, for creating a vector, a host cell, for preparing a yeast strain, or for treating a pest, for example, an insect or mite, can include an OVP, consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 15, 16, 19, 34, 35 and 38, or an agriculturally acceptable salt thereof.

[0019] In addition, the present disclosure describes a combination comprising two or more Omega Variant Peptide (OVPs) having pesticidal activity, wherein each of the two or more OVPs independently comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P-X?-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V- K-R-C-D (SEQ ID NO: 70); wherein each of the OVPs independently comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2, wherein Xi and X2are each independently any amino acid, or each are independently optionally absent; and wherein X3 is A, D. E, F. G, H. I. K, L. M. N, P. Q, R. V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0020] In addition, the present disclosure describes a composition comprising an Omega Variant Peptide (OVP) having pesticidal activity, and an agriculturally acceptable excipient; wherein the OVP comprises or consists of an amino acid sequence that is at least90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K- E-N-E-N-G-N-T-V-K-R-C-D; wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0021] In addition, the present disclosure describes a composition comprising an Omega Variant Peptide (OVP) having pesticidal activity', and an agriculturally acceptable excipient; wherein the OVP comprises or consists of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F- K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0022] In addition, the present disclosure describes a composition comprising a combination of two or more Omega Variant Peptide (OVPs), each having pesticidal activity, and an agriculturally acceptable excipient; wherein each of the two or more OVPs independently comprises, consists essentially of, or consists of: an amino acid sequence that is at least 90%. 95%. 96%. 97%. 97%. 98%. 99%. or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S- C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein each of the OVPs independently comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2, wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is A, D. E, F, G, H. I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0023] In addition, the present disclosure describes a polynucleotide operable to encode a Omega Variant Peptide (OVP), said OVP comprising or consisting of an amino acidsequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D. E, F, G, H. I. K, L. M, N, P, Q, R, V, W, or Y; or a complementary polynucleotide sequence thereof.

[0024] In addition, the present disclosure describes a polynucleotide operable to encode a Omega Variant Peptide (OVP), said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is D, E, F, G, H, I, K. L, M, N, P, Q, R, V, W, or Y; or a complementary polynucleotide sequence thereof.

[0025] In addition, the present disclosure describes a vector comprising a polynucleotide operable to encode a Omega Variant Peptide (OVP), said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P- X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A. D, E. F, G. H, I, K, L, M, N, P, Q, R, V, W, or Y; or a complementary polynucleotide sequence thereof.

[0026] In addition, the present disclosure describes a vector comprising a polynucleotide operable to encode a Omega Variant Peptide (OVP), said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (ID): X1-X2-S-P-T-C-I- P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is D, E. F, G. H, I, K, L, M, N, P, Q, R, V, W, or Y; or a complementary polynucleotide sequence thereof.

[0027] In addition, the present disclosure describes a polynucleotide operable to hybridize under stringent hybridization conditions with a polynucleotide segment encoding an Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P- X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2: wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y.

[0028] In addition, the present disclosure describes a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an OVP, said OVP comprising or consisting of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): Xi- X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C- D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D, E, F, G, H, I, K. L, M, N, P, Q, R, V, W, or Y; or a complementary' nucleotide sequence thereof.

[0029] In addition, the present disclosure describes a method of producing an OVP, 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 OVP. or a complementary nucleotide sequence thereof, said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally independently absent; and wherein X3 is A, D, E, F, G. H, I, K, L, M, N, P, Q. R, V, W, or Y; (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 OVP and secretion into the growth medium.

[0030] In addition, the present disclosure describes a method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of anOmega Variant Peptide (OVP) as described herein; a combination comprising two or more OVPs as described herein; a composition comprising an OVP and an agriculturally acceptable excipient, as described herein; and / or a composition comprising a combination of two or more OVPs and an agriculturally acceptable 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 thereofBRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 depicts an HPLC chromatogram showing GS+Omega-ACTX-hvla (SEQ ID NO: 1) (“Omega+2”). Here, tw o heterogeneous species of the peptide (identified as Omega 1 and Omega 2) indicate the presence of a glycosylated form.

[0032] FIG. 2 depicts an HPLC chromatogram showing the Omega peptide and nonglycosylated mutants. Each line corresponds to an Omega Variant Peptide (“OVP”) having an amino acid substitution at a residue containing a potential glycosylation site. Omega peptide and the OVP having an S9A mutation are indicated with arrows. The OVP having an S9A mutation is the only variant peptide that possessed a sharp peak in the chromatogram. Here, pink = S3A; orange = S9A; black = S21 A; blue = S23A. The short orange arrow7indicates a second peak that appeared after the primary glycosylation site has been removed.

[0033] FIG. 3 depicts the percent mortality of houseflies injected with GS+Omega (“Omega+2”) and S9A OVP after 24-hours. As shown here, the LD50 for Omega+2 was 4.4 ng / pL, whereas the S9A OVP had an LD50 of 4.25 ng / pL.

[0034] FIG. 4 depicts an MS chromatogram for the Omega Variant Peptide (OVP) of SEQ ID NO: 3 (S9A OVP). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0035] FIG. 5 depicts an MS chromatogram for S9D OVP (SEQ ID NO: 4). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0036] FIG. 6 depicts an MS chromatogram for S9E OVP (SEQ ID NO: 5). The expected m / z value (shown in circles) is based on the average mass provided in the tablebelow the chromatogram. The columns named “+2 species’'; “+3 species"’; and “+4 species”; refer to different charge states of the ionic species.

[0037] FIG. 7 depicts an MS chromatogram for S9F OVP (SEQ ID NO: 6). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species"’; and “+4 species”; refer to different charge states of the ionic species.

[0038] FIG. 8 depicts an MS chromatogram for S9G OVP (SEQ ID NO: 7). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0039] FIG. 9 depicts an MS chromatogram for S9H OVP (SEQ ID NO: 8). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0040] FIG. 10 depicts an MS chromatogram for S9I OVP (SEQ ID NO: 9). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ‘'+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0041] FIG. 11 depicts an MS chromatogram for S9K OVP (SEQ ID NO: 10). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ‘"+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0042] FIG. 12 depicts an MS chromatogram for S9L OVP (SEQ ID NO: 11). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ”+3 species”; and ”+4 species”; refer to different charge states of the ionic species.

[0043] FIG. 13 depicts an MS chromatogram for S9M OVP (SEQ ID NO: 12). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named "+2 species”; "‘+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0044] FIG. 14 depicts an MS chromatogram for S9N OVP (SEQ ID NO: 13). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named ”+2 species”; ”+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0045] FIG. 15 depicts an MS chromatogram for S9P OVP (SEQ ID NO: 14). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ‘'+3 species”; and '‘+4 species”; refer to different charge states of the ionic species.

[0046] FIG. 16 depicts an MS chromatogram for S9Q OVP (SEQ ID NO: 15). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ”+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0047] FIG. 17 depicts an MS chromatogram for S9R OVP (SEQ ID NO: 16). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named "+2 species”; "‘+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0048] FIG. 18 depicts an MS chromatogram for Omega+2 (SEQ ID NO: 17). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The arrows indicate the m / z values corresponding to the glycosylated form. The columns named ”+2 species”; "+3 species”; and ‘‘+4 species”; refer to different charge states of the ionic species.

[0049] FIG. 19 depicts an MS chromatogram for S9T OVP (SEQ ID NO: 18). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The arrows indicate the m / z values corresponding to the glycosylated form. The columns named '‘+2 species”; “+3 species”; andc’+4 species”; refer to different charge states of the ionic species.

[0050] FIG. 20 depicts an MS chromatogram for S9V OVP (SEQ ID NO: 19). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ”+3 species”; and ”+4 species”; refer to different charge states of the ionic species.

[0051] FIG. 21 depicts an MS chromatogram for S9W OVP (SEQ ID NO: 20). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named "+2 species”; "‘+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0052] FIG. 22 depicts an MS chromatogram for S9Y OVP (SEQ ID NO: 21). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named "+2 species”; ”+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0053] FIG. 23 depicts an MS chromatogram for S7A OVP (SEQ ID NO: 22). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ‘'+3 species”; and '‘+4 species”; refer to different charge states of the ionic species.

[0054] FIG. 24 depicts an MS chromatogram for S7D OVP (SEQ ID NO: 23). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ”+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0055] FIG. 25 depicts an MS chromatogram for S7E OVP (SEQ ID NO: 24). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named "+2 species”; ”+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0056] FIG. 26 depicts an MS chromatogram for S7F OVP (SEQ ID NO: 25). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named "+2 species”; ”+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0057] FIG. 27 depicts an MS chromatogram for S7G OVP (SEQ ID NO: 26). The expected m / z value (shown in circles) is based on the average mass provided in the table below7the chromatogram. The columns named "+2 species”; "+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0058] FIG. 28 depicts an MS chromatogram for S7H OVP (SEQ ID NO: 27). The expected m / z value (shown in circles) is based on the average mass provided in the table below7the chromatogram. The columns named " i 2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0059] FIG. 29 depicts an MS chromatogram for S7I OVP (SEQ ID NO: 28). The expected m / z value (shown in circles) is based on the average mass provided in the table below7the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0060] FIG. 30 depicts an MS chromatogram for S7K OVP (SEQ ID NO: 29). The expected m / z value (shown in circles) is based on the average mass provided in the table below' the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0061] FIG. 31 depicts an MS chromatogram for S7L OVP (SEQ ID NO: 30). The expected m / z value (shown in circles) is based on the average mass provided in the tablebelow the chromatogram. The columns named “+2 species’'; “+3 species"’; and “+4 species”; refer to different charge states of the ionic species.

[0062] FIG. 32 depicts an MS chromatogram for S7M OVP (SEQ ID NO: 31). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species"’; and “+4 species”; refer to different charge states of the ionic species.

[0063] FIG. 33 depicts an MS chromatogram for S7N OVP (SEQ ID NO: 32). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0064] FIG. 34 depicts an MS chromatogram for S7P OVP (SEQ ID NO: 33). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0065] FIG. 35 depicts an MS chromatogram for S7Q OVP (SEQ ID NO: 34). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ‘'+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0066] FIG. 36 depicts an MS chromatogram for S7R OVP (SEQ ID NO: 35). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; ‘"+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0067] FIG. 37 depicts an MS chromatogram for WT Omega (SEQ ID NO: 36). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The arrows indicate the m / z values corresponding to the glycosylated form. The columns named '‘+2 species”; “+3 species”; and ‘’+4 species”; refer to different charge states of the ionic species.

[0068] FIG. 38 depicts an MS chromatogram for S7T OVP (SEQ ID NO: 37). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The arrows indicate the m / z values corresponding to the glycosylated form. The columns named “+2 species”; “+3 species”; and ‘'+4 species”; refer to different charge states of the ionic species.

[0069] FIG. 39 depicts an MS chromatogram for S7V OVP (SEQ ID NO: 38). The expected m / z value (shown in circles) is based on the average mass provided in the tablebelow the chromatogram. The columns named “+2 species’'; “+3 species"’; and “+4 species”; refer to different charge states of the ionic species.

[0070] FIG. 40 depicts an MS chromatogram for S7W OVP (SEQ ID NO: 39). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species"’; and “+4 species”; refer to different charge states of the ionic species.

[0071] FIG. 41 depicts an MS chromatogram for S7Y OVP (SEQ ID NO: 40). The expected m / z value (shown in circles) is based on the average mass provided in the table below the chromatogram. The columns named “+2 species”; “+3 species”; and “+4 species”; refer to different charge states of the ionic species.

[0072] FIG. 42 depicts a graph showing Varroa mite mortality after treatment with wild-type Omega-ACTX-hvla (SEQ ID NO: 2). Here, Varroa mites were either untreated (control), or treated with 0.5 pL of a 20 mg / mL solution of wild-type Omega-ACTX-hvla (SEQ ID NO: 2). Percent mortality was then determined after incubation for 18 hours 32.3°C, 58% relative humidity, and no lights.

[0073] FIG. 43 depicts a graph showing honeybees effected after injection with WT Omega (SEQ ID NO: 2) and Omega+2 (SEQ ID NO: 1). Here, a dose response was conducted to calculate and ED50 for the different treatments with results corrected for control mortality. Percent effected was then determined after incubation for 20 hours at 32.3°C, 58% relative humidity, and no lights.

[0074] FIG. 44 depicts a graph showing houseflies effected after injection with WT Omega (SEQ ID NO: 2) and Omega+2 (SEQ ID NO: 1). Here, a dose response was conducted to calculate and ED50 for the different treatments with results corrected for control mortality. Percent effected was then determined after incubation for 20 hours at 32.3°C, 58% relative humidity, and no lights.DETAILED DESCRIPTION

[0075] DEFINITIONS

[0076] 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.

[0077] “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 hostorganism in order to achieve successful genetic modification of the host organism’s chromosomal locus.

[0078] “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. The Hexathelidae family of spiders formerly contained he 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 peptides isolated from Atracidae family species are the Omega- ACTX, Kappa- ACTX, and U-ACTX peptides. See HXTX below.

[0079] “ADN1 promoter” refers to the DNA segment comprised of the promoter sequence derived from the Schizosaccharomyces pombe adhesion defective protein 1 gene.

[0080] “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.

[0081] “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.

[0082] “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.

[0083] “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.

[0084] “Agroinfection” means a plant transformation method where DNA is introduced into a plant cell by using Agrobacteria A. tumefaciens or A. rhizogenes.

[0085] ’‘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., CLUSTALW2, 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 Accelrys (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 similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.

[0086] “Alpha-MF signal” or “aMF secretion signal” refers to a protein that directs nascent recombinant polypeptides to the secretory' pathway.

[0087] “Ameliorate” or “amelioration” includes the arrest, prevention, decrease, or improvement in one or more the symptoms, signs, and features of the disease being treated, both temporary and long-term.

[0088] “Apiary ” refers to a location where two or more beehives are kept and / or managed by beekeepers. As used herein, the term “apiary” refers to both the physical space used to keep bees and the collection of hives within that space.

[0089] “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, applying can refer to, e.g., application of an OVP or an agriculturally acceptable salt thereof; or application of an OVP-pesticidal protein or agriculturally acceptable salt thereof, as a combination, a mixture, or a composition further comprising one or more excipients, e.g., a spray able 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 OVP and a second OVP, wherein the first and second OVP can be the same or different, can be applied or administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

[0090] As used herein, the phrase "an amino acid substitution" refers to a substitution of one amino acid for another different amino acid in an amino acid chain. An amino acid substitution, does not include an amino acid addition and does not include an amino acid deletion. For purposes of illustration, when the phrase "an OVPs independently comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2;", this means that at least one amino acid in the sequence of SEQ ID NO: 1: GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD is substituted with a different amino acid. With reference to Formula (I), X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C- C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70), the amino acid substitution can occur at position (or residue number) 9 i.e. at X3. Additional amino acid substitutions (for example, conservative amino acid substitutions) may also occur, and such OVPs with for example, one to five substitutions (for example, conservative amino acid substitutions) of amino acids along the sequence of SEQ ID NO: 1 other than at positions Xi, X2 and X3 relative to SEQ ID NO: 1, are included within the definition of an OVP.

[0091] “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: 41 (NCBI Accession No. AAA32925.1).

[0092] “Beneficial insect” refers to an insect that confers a benefit to humans (e.g., increased food production, an economic benefit, etc.). For example, in some embodiments, a beneficial insect may be an insect that pollinates economically relevant crops. In someembodiments, a beneficial insect can be a bee, e.g., Apis mellifera, Apis car ana. Apis dorsata. Apis flor ea, Apis labortosa. Apis andreniformis . or Apis koschevnikovi .

[0093] “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 OVPs of the present disclosure provide increased bioavailability of an OVP. In some embodiments, bioavailability7is affected by the extent and rate at which the active moiety7(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 wall, 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.

[0094] “Biomass” refers to any measured plant product.

[0095] “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.

[0096] “bp’’ or “base pair” refers to a molecule comprising two 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), cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.

[0097] “Bt-resistanf ’ or “Bt-resistance” or “Bt-resistant insect” or “Bacillus thuringiensis-toxm-res siant 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.

[0098] “cDNA” 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.

[0099] “CEW” refers to Com earworm.

[0100] “Cleavable Linker” see Linker.

[0101] “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.

[0102] “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 codingsequence. 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 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, 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.

[0103] “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.

[0104] “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 OVP, and one or more additional OVPs; wherein the first OVP and one or more additional OVPs are the same or different.

[0105] 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 OVP, and one or more additional OVPs; wherein the first OVP and one or more additional OVPs are the same or different; and wherein the first OVP and one or more additional OVPs are present in a single entity (e.g., an admixture for simultaneous administration).

[0106] 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 additionalcomponents, 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 be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

[0107] Accordingly, in some embodiments, a combination can refer to the association of a first OVP, and one or more additional OVPs; wherein the first OVP and one or more additional OVPs are the same or different; and wherein the first OVP and one or more additional OVPs are present separately (e.g., different units for separate, sequential, simultaneous, concurrent or chronologically-staggered administration).

[0108] 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 OVP, and one or more additional OVPs; wherein the first OVP and one or more additional OVPs are the same or different).

[0109] For example, in some embodiments, a “combination” refers to the result of a simultaneous application of both a first OVP, and one or more additional OVPs; wherein the first OVP and one or more additional OVPs are the same or different.

[0110] In another embodiment, a “combination” refers to the result of a separate application of a first OVP, and one or more additional OVPs; wherein the first OVP and one or more additional OVPs are the same or different.

[0111] 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 OVP, followed by a second application of one or more additional OVPs (wherein the first OVP and one or more additional OVPs 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.

[0112] “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 polynucleotidewhose sequence 5’-TATAC-3' is complementary to a polynucleotide whose sequence is 5‘- GTATA-3’.

[0113] ’‘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.

[0114] “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.

[0115] “Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.

[0116] “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).

[0117] “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 some embodiments, an LN subunit and an Lc subunit can be fused via a peptide bond, thus forming a cyclic protein.

[0118] “Cysteine residue” refers to a cysteine amino acid.

[0119] “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.

[0120] “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.

[0121] “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 agiven polypeptide with a particular activity; such functionally equivalent variants are contemplated herein.

[0122] “Deglycosylation” or “de-glycosylation” or “non-glycosylation” (all used interchangeably) refers to the absence, removal, or deletion of one or more sugar (e.g., carbohydrate) moieties from a protein. Likewise, the term “glycosylation” refers to the presence, addition, or attachment of one or more sugar (e.g., carbohydrate) moieties to a protein. In addition, the terms “deglycosylation” or “glycosylation” includes qualitative changes in the glycosylation or deglycosylation of proteins involving a change in the type and proportions (amount) of the various sugar (e.g., carbohydrate) moieties present. For example, in some embodiments, the term “glycosylation” refers to refers to the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules, including but not limited to: N-linked glycosylation, O-linked glycosylation (O — N- acetylgalactosamine (O-GalNAc), O-fucose, O-glucose, O — N-acetylglucosamine (O- GlcNAc), O — N-acetylglucosamine, O-mannose, Collagen Glycosylation, Hydroxy proline Glycosylation, Glycosylation of Glycogenin, Glycosylation of Ceramide. Proteoglycans), phospho-Serine Glycosylation and C-mannosylation. In some embodiments, the sugars attached to a glycosylation site can be glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N- acetylglucosamine (GlcNAc), and sialic acid (e.g., N-acetyl-neuraminic acid (NeuAc or NANA)).

[0123] In some embodiments, deglycosylation and / or glycosylation can be achieved by modification of an amino acid residue. For example, in some embodiments, deglycosylation can be achieved by removing one or more glycosylation sites (i.e. , an amino acid residue with, e.g., a functional hydroxyl group, wherein said glycosylation site is operable to receive a carbohydrate attached via a covalent bond), that may or may not be present in the native sequence. In some embodiments, atypically glycosylated amino acid residue can be substituted for a residue that may not be glycosylated. Accordingly the removal of amino acid residues containing glycosylation sites, or the addition of amino acid residues that do not possess glycosylation sites, or vice versa, can be accomplished by altering the amino acid sequence. For example, in some embodiments, deglycosylation can be achieved by altering an amino acid sequence such as by the removal of, or substitution of, one or more serine (“S”) or threonine (“T”) residues (e.g., O-linked glycosylation sites) or asparagine (“N”) residues (e.g., N-linked glycosylation sites).

[0124] As used herein, the terms “non-glycosylated” or “deglycosylated” or “nonglycosylated peptide” or “deglycosylated peptide” or “deglycosylated protein” or “resistant toglycosylation” or “non-glycosylated OVP” or “deglycosylated OVP” (e.g., “. . . wherein the OVP is deglycosylated' all refer to the condition of a peptide, polypeptide, or protein comprising the removal of one or more native amino acid residues operable to be glycosylated (i.e., an amino acid residue containing a glycosylation site); and / or the substitution of one or more native amino acid residues operable to be glycosylated, with one or more non-native amino acid residues that are not operable to be glycosylated (e.g., an amino acid residue that does not contain a glycosylation site).

[0125] Thus, in some embodiments, a deglycosylated peptide of the present disclosure refers to a peptide comprising the removal of one or more native amino acid residues operable to be glycosylated, resulting in a level of glycosylation in the deglycosylated peptide that is reduced relative to the native state. In yet other embodiments, a deglycosylated peptide of the present disclosure refers to a peptide comprising the substitution of one or more native amino acid residues operable to be glycosylated, with one or more non-native amino acid residues that are not operable to be glycosylated, resulting in a level of glycosylation in the deglycosylated peptide that is reduced relative to the native state. Accordingly, when a peptide of the present disclosure has a native ammo acid residue operable to be glycosylated removed, or substituted with a non-native amino acid residue that is not operable to be glycosylated, the peptide is referred to as being “resistant to glycosylation” or a peptide that is “deglycosylated” (“. . . wherein the OVP is deglycosylated:' .

[0126] In some embodiments, an OVP of the present disclosure is resistant to N- and / or O-linked glycosylation at one or more residues, e.g., because the native residues are removed or substituted. Accordingly, as used herein, the phrase “resistant,” when used in the context of glycosylation (e.g., “resistant to glycosylation”), refers to the removal or substitution of a native amino acid residue that is operable to be glycosylated, with an ammo acid residue that does not possess a glycosylation site). N-linked glycosylation occurs in the endoplasmic reticulum (ER). N-linked glycosylation occurs at an N-X-S or N-X-T motif wherein X cannot be P or D amino acid residues. N-linked glycosylation results in the addition of a very large sugar complex, which is subsequently pruned down, but is nevertheless still often very large. O-linked glycosylation occurs in the Golgi. Here, a glycosylation occurs at the hydroxyl oxygen of S or T amino acid residues. O-linked glycosylation results in the addition of sugars sequentially, and not usually more than a few' such sugars. For example, mannosylation occurs in yeast, and is O-linked (e.g.. inmannosylation there is often the addition of a mannose sugar moiety, resulting in the increase of an additional +162 Daltons added per mannose sugar moiety).

[0127] In some embodiments, glycosylation of a heterologous peptide (e.g., an OVP as described herein) can occur when said heterologous peptide is expressed in a recombinant cell culture system using standard fermentation techniques (e.g., such as those described herein). The glycosylation of the heterologous peptide results in the production of variants, alternate forms, or “species” of the heterologous peptide. Thus, rather than the production of a single, homogenous species of the heterologous peptide, glycosylation results in the production of one or more heterogeneous species of the heterologous peptide. In some embodiments, these one or more heterogeneous species of the heterologous peptide can be a “glycosylated species” or a “deglycosylated species,” which can be observed / measured to the methods described herein (e.g., as separate peaks using HPLC; based on molecular weight; mass spectrometry, etc.).

[0128] Accordingly, the present disclosure contemplates heterologous peptides (i.e., OVPs or OVP-pesticidal proteins) that are resistant to post-translation modifications (e.g., glycosylation), and whose expression results in the production of a single, homogenous species of the heterologous peptide, when expressed in a recombinant cell culture system using standard fermentation techniques as described herein.

[0129] “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 synthetic 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 an known / 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).

[0130] 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 havinga 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 a known protein or polynucleotide are known to one of skill in the art.

[0131] 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.

[0132] “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. In some embodiments, a disulfide bond occurs via the oxidative folding of tw o 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.

[0133] “Double expression cassette” refers to two OVP expression cassettes contained on the same vector.

[0134] “Double transgene peptide expression vector” or “double transgene expression vector” means a yeast expression vector that contains two copies of the OVP expression cassette.

[0135] “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 LCJ), which can be arranged in single-stranded or double-stranded form. For example, one or more nucleotides creates a polynucleotide.

[0136] “dNTPs” refers to the nucleoside triphosphates that compose DNA and RNA.

[0137] “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.

[0138] 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.

[0139] “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.

[0140] “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.

[0141] “ER” or “Endoplasmic reticulum” is a subcellular organelle common to all eukaryotes where some post translation modification processes occur.

[0142] “ERSP” or “Endoplasmic reticulum signal peptide” is an N-terminus sequence of amino acids that — during protein translation of the mRNA molecule encoding an OVP — 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 inj ected into the ER.

[0143] "ersp" refers to a polynucleotide encoding the peptide, ERSP.

[0144] “ER trafficking” means transportation of a cell expressed protein into ER for post-translational modification, sorting and transportation.

[0145] “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.

[0146] "Expression cassette’7refers to (1 ) a DNA sequence of interest, e.g.. a polynucleotide operable to encode an OVP; 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 OVP. In alternative embodiments, there are two expression cassettes, each comprising a polynucleotide operable to encode an OVP (i.e., a double expression cassette). In other embodiments, there are three expression cassettes operable to encode an OVP (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 a second expression cassette. Methods concerning expression cassettes and cloning techniques are well-known in the art and described herein. See also OVP expression cassette.

[0147] “FECT” means a transient plant expression system using Foxtail mosaic virus with elimination of coating protein gene and triple gene block.

[0148] “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 OVP 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 solelylimited to strict anaerobic conditions, as the term “fermentation” used herein may also occur processes that occur in the presence of oxygen.

[0149] “GFP” means a green fluorescent protein from the jellyfish, Aequorea victoria.

[0150] "Grow th medium” refers to a nutrient medium used for growing cells in vitro.

[0151] “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.

[0152] "Hexathehdae" refers to a family of mygalomorph spiders that previously contained the genera: Atracidae, Macrothelidae and Porrhothelidac, however. Atracidae. Macrothelidae and Porrhothelidae have since been classified as their O TI 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.

[0153] “Homolog” refers to a polynucleotide (nucleotide sequence) or peptide (amino acid sequence) possessing a high degree of sequence relatedness to a subject sequence. Such relatedness may be quantified by determining the degree of identity and / or similarity' between the sequences being compared. Falling within the term “homolog” are the terms “ortholog”, meaning a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide in another species; and “paralog” meaning a functionally similar sequence when considered within the same species. Paralogs present in the same species or orthologs of omega-ACTX genes in other species can readily be identified without undue experimentation, by molecular biological techniques well known in the art.

[0154] “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 tw o DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of 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 x l00. 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 oftwo 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 .

[0155] There may be partial homology , or complete homology and thus identical. “Sequence identity7’ 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” above.

[0156] “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 ty pe 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 ty pes, 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.Homologous 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 replacementevent in which nucleic acid sequences contained within the targeting construct have replaced specific sequences present within the endogenous genomic sequences.

[0157] “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 he 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 peptides isolated from Atracidae family species are the Omega- ACTX, Kappa- ACTX, and U-ACTX peptides.

[0158] “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.

[0159] “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, the 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-strandedpolynucleotides 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.

[0160] “IC50” or “ICAO” 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.

[0161] “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.

[0162] “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 Press, 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 areincorporated 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.

[0163] “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur wi thin a natural environment.

[0164] ’‘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.

[0165] “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 thereol) less in size, amount, intensity, or degree. For example, in some embodiments, the application of a pesticidally effective amount of an OVP or agriculturally acceptable salt thereof, or an agricultural composition comprising an OVP 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 thenumber 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 growth; 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 OVP or agriculturally acceptable salt thereof, or an agricultural composition comprising an OVP or agriculturally acceptable salt thereof.

[0166] In another embodiment, the application of a pesticidally effective amount of a wild-type omega-ACTX-Hvla peptide, OVP, OVP-pesticidal protein, or an 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 contacted or pollinated by a beneficial insect; (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 growth; becomes confused (e g., with regard to navigation, locating food, sleeping behaviors, and / or mating); fails to pupate if applicable; interferes with reproduction and / or fecundity of the pest (e.g., interferes with or precludes the pest from producing offspring and / or interferes with or precludes the pest from producing fertile offspring); and / or weakens, destroys, or otherwise disrupts the pest’s eggs — relative to the number of pests or the level of the pest’s activities that have not been exposed to a pesticidally effective amount of a wild-type omega-ACTX-Hvla peptide OVP, OVP- pesticidal protein, or an agriculturally acceptable salt thereof, or an agricultural composition comprising the same and one or more excipients.

[0167] 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.

[0168] 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 OVP, OVP-pesticidal protein, or WT Omega peptide of the present disclosure, or an agricultural composition comprising the same, 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 6%, 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; ability to produce offspringand / or ability to produce fertile offspring) that have not received a pesticidally effective amount of an OVP, OVP-pesticidal protein, or WT Omega peptide of the present disclosure, or an agricultural composition thereof.

[0169] “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 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.

[0170] “Insect” includes all organisms in the class “Insecta.” The term “pre-adult” 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.

[0171] “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.

[0172] “Insect hemolymph environment” means the specific pH and proteinase conditions of found within an insect or insect larva.

[0173] “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.

[0174] “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 readingframe in the ORF to separate the upstream and downstream DNA sequences. For example, in some 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 hemolymph and lepidopteran hemolymph environment.

[0175] "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).

[0176] “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.

[0177] “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 ofAecfes aegypti (mosquito).

[0178] “kDa” refers to kilodalton, a unit equaling 1,000 daltons; a “Dalton” or “dalton” is a unit of molecular weight (MW).

[0179] “Knock in” or “knock-in” or “knocks-in” or “knocking-in” refers to the replacement of an endogenous gene with 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”).

[0180] “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 geneencoding a peptide, polypeptide, or protein. As a result, the deletion may render a gene inactive, 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 knock ed-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”).

[0181] “ / ” or “ / infer” refers to a nucleotide encoding intervening linker peptide.

[0182] “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 OVP, and / or multiple OVPs. When referring to amino acids, “L” can also mean leucine.

[0183] ‘LAC4 promoter” or “Lac4 promoter” or “pLac4” refers to a DNA segment comprised of the promoter sequence derived from the K. lactis P-galactosidase gene. The LAC4 promoters is strong and inducible reporter that is used to drive expression of exogenous genes transformed into yeast.

[0184] “LAC4 terminator” or “Lac4 terminator” refers to a DNA segment comprised of the transcriptional terminator sequence derived from the K. lactis P-galactosidase gene.

[0185] “LD20” refers to a dose required to kill 20% of a population.

[0186] “LD50” refers to lethal dose 50 which means the dose required to kill 50% of a population.

[0187] “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.

[0188] “Lepidopteran hemolymph environment” means the specific pH and proteinase conditions of found within lepidopteran insect or larva.

[0189] “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 plantprotease (e.g., papain, bromelain, ficin, actinidin, zingibain, and / or cardosins), an insect protease, 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 tertian- 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.

[0190] “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.

[0191] “Locus of a plant” refers to any place in which a plant is grow ing; 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.

[0192] “Locus of an animal” refers to any place where animals live, eat, breed, sleep, or are otherwise present.

[0193] “Medium” (plural “media”) refers to a nutritive solution for culturing cells in cell culture.

[0194] “MO A” refers to mechanism of action.

[0195] “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:Migration distance of the protein Migration distance of the dye frontFormula (a)

[0196] 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.

[0197] “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.

[0198] “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.

[0199] “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 ammo acid substitutions when compared to a peptide with a disclosed and / or claimed sequence, as indicated by a SEQ ID NO.

[0200] “N-terminus” or “N-terminal” refers to the free amine group (i.e., -NH2) that is positioned on beginning or start of a polypeptide.

[0201] “NCBI” refers to the National Center for Biotechnology Information.

[0202] “nm” refers to nanometers.

[0203] “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.

[0204] ■‘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).

[0205] “OD” refers to optical density. Typically, OD is measured using a spectrophotometer. When measuring growth 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.

[0206] “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).

[0207] “Omega” or “Omega peptide” or “omega toxin” or “omega hexatoxin” or “WT Omega” or “WT Omega peptide” (all used interchangeably) all refer to a wild-type (WT) Omega- ACTX peptide. In some embodiments, a WT Omega peptide can be a wildtype Omega-ACTX-Hv la peptide, or a wild-type Omega- ACTX-Hv2a peptide. The wildtype Omega-ACTX-Hv la peptide was first isolated from the Sydney Funnel-web Spider, Atrax robustus. An exemplary wild-type Omega-ACTX-Hv la peptide has an amino acid sequence: “SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO: 2). The wild-type Omega-ACTX-Hv2a peptide is isolated from Hadronyche versuta. An exemplary wild-type Omega-ACTX-Hv2a peptide has an amino acid sequence: “LLACLFGNGRCSSNRDCCELTPVCKRGSCVSSGPGLVGGILGGIL” (SEQ ID NO: 69).

[0208] An exemplary description a WT Omega peptide is provided in Chong et al., The omega-atracotoxins: selective blockers of insect M-LVA and HVA calcium channels. Bi ochem Pharmacol. 2007 Aug 15;74(4):623-38; and U.S. Patent Nos. 8.703,910; and 9,567,381; the disclosures of which are incorporated herein by reference in their entireties.

[0209] “Omega+2” or “O+2” or “co+2-ACTX-Hvl a+2” or Omega+2-ACTX-Hvl a (all used interchangeably) all refer to a non-natural, recombinant peptide having the amino acid sequence “GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO:1). An exemplary' description of Omega+2 is provided in U.S. Patent Nos. 8,703,910; and 9,567,381; the disclosures of which are incorporated herein by reference in their entireties.

[0210] ’‘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, arginine=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.

[0211] “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).

[0212] 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 codingsequence does not contain introns); however, as used herein, the terms ‘‘coding sequence”; “CDS”; “open reading frame”; and “ORF / are used interchangeably.

[0213] “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 embodiments, 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).

[0214] “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.

[0215] “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.”

[0216] “OVP” or “Omega Variant Peptide” or “Omega hexatoxin variant peptide” or “Omega mutant” or “Omega mutant peptide” (all used interchangeably) refer to peptides having one or more mutations relative to the Omega+2 peptide having an amino acid sequence: “GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO: 1).

[0217] In some embodiments, an OVP can have an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I):X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-DFormula (I) (SEQ ID NO: 70) wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2;; and wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0218] "OVP 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 OVP, e.g., an OVP ORF. For example, one example of an OVP expression cassette is one or more segments of DNA that contains a polynucleotide segment operable to express an OVP, a ADH1 promoter, a LAC4 terminator, and an alpha- MF secretory signal. An OVP expression cassette contains all of the nucleic acids necessary to encode an OVP or an OVP-pesticidal protein.

[0219] “OVP ORF” refers to a polynucleotide operable to encode an OVP, or an OVP-pesticidal protein.

[0220] “OVP ORF diagram” refers to the composition of one or more OVP ORFs, as written out in diagram or equation form. For example, a “OVP 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 “OVP ORF diagram” may describe the polynucleotide segments encoding the ERSP, LINKER, STA, and OVP, by diagramming in equation form the DNA segments as “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “linker” or “L” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and “ovp” (i.e., the polynucleotide sequence encoding an OVP), respectively. An example of an OVP ORF diagram is “er p-sta-(linkeri-ovpj)N,” or “ersp-(ovpj-linkeri)N- sta” and / or any combination of the DNA segments thereof.

[0221] “OVP-pesticidal protein’" or “OVP-pesticidal polypeptide"’ or “pesticidal protein” or “pesticidal polypeptide” refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, comprising: (1) at least one OVP, or two or more OVPs; and (2) additional peptides, polypeptides, or proteins that are not an OVP. For example, in some embodiments, an OVP-pesticidal protein can be a fusion protein comprising at least one OVP, and at least one additional peptide or protein that is not a OVP, wherein at least one OVP is linked directly or indirectly to a non-OVP protein.

[0222] In some embodiments, an OVP-pesticidal protein can be a fusion protein comprising at least one OVP, and at least one additional peptide, polypeptide, or protein that is not a OVP, wherein at least one OVP is linked directly or indirectly to a non-OVP 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 OVP-pesticidal protein, relative to an OVP alone; increase the expression of said OVP- pesticidal protein, e.g., in a host cell or an expression system; and / or affect the post- translational processing of the OVP-pesticidal protein.

[0223] In some embodiments, an OVP-pesticidal protein can be a polymer comprising two or more OVPs. In some embodiments, an OVP-pesticidal protein can be a polymer comprising two or more OVPs, wherein the OVPs are operably linked via a linker peptide, e.g., a cleavable and / or non-cleavable linker. In some embodiments, an OVP- pesticidal protein can refer to a one or more OVPs 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 OVP-pesticidal protein can be a non-naturally occurring protein comprising (1) an OVP; and (2) additional peptides, polypeptides, or proteins, e.g.. an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.

[0224] “OVP construct” refers to the three-dimensional arrangement / orientation of peptides, polypeptides, and / or motifs of operably linked polypeptide segments (e.g., an OVP- pesticidal protein). For example, an OVP ORF can include one or more of the following components or motifs: an OVP; 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 “OVP construct” is used to describe the designation and / or orientation of the structural motif. In other words, the OVP construct describes the arrangement and orientation of the components or motifs contained within a given OVP ORF. For example, in some embodiments, an OVP construct describes, without limitation, the orientation of one ofthe following OVP-pesticidal proteins: ERSP-OVP; ERSP-(OVP)N; ERSP-OVP-L; ERSP- (OVP)N-L; ERSP-(OVP-L)N; ERSP-L-OVP; ERSP-L-(OVP)N; ERSP-(L-OVP)N; ERSP- STA-OVP; ERSP-STA-(OVP)N; ERSP-OVP-STA; ERSP-(OVP)N-STA; ERSP-(STA- OVP)N; ERSP-(OVP-STA)N; ERSP-L-OVP-STA; ERSP-L-STA-OVP; ERSP-L-(OVP- STA)N; ERSP-L-(STA-OVP)N; ERSP-L-(OVP)N-STA; ERSP-(L-OVP)N-STA; ERSP-(L- STA-OVP)N: ERSP-(L-OVP-STA)N; ERSP-(L-STA)N-OVP; ERSP-(L-OVP)N-STA; ERSP- STA-L-OVP; ERSP-STA-OVP-L; ERSP-STA-L-(OVP)N; ERSP-(STA-L)N-OVP; ERSP- STA-(L-OVP)N; ERSP-(STA-L-OVP)N; ERSP-STA-(OVP)N-L; ERSP-STA-(OVP-L)N; ERSP-(STA-OVP)N-L; ERSP-(STA-OVP-L)N; ERSP-OVP-L-STA; ERSP-OVP-STA-L; ERSP-(OVP)N-STA-L ERSP-(OVP-L)N-STA; ERSP-(OVP-STA)N-L; ERSP-(OVP-L- STA)N: or ERSP-(OVP-STA-L)N; wherein N is an integer ranging from 1 to 200. See also “Structural motif.”

[0225] “Peptide yield” means the pesticidal 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 HPEC chromatograph, for example, mAu.sec.

[0226] “Pest” refers to any organism that is annoying, troublesome, detrimental, and / or bothersome, and which is directly or indirectly deleterious, harmful and / or causes stress, damage, or destruction to people, plants, crops, pets, livestock, and / or a beneficial insect. In some embodiments, a pest can attack, spread disease, and / or parasitize a beneficial insect, and thereby indirectly harm or damage plants, crops, pets, and / or livestock, and / or people.

[0227] In some embodiments, a pest can be a deleterious insect, nematode, or mite.

[0228] 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 effect on 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.

[0229] 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.

[0230] 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., planttissues, plant cells, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, propagules, embryos and progeny of the same.

[0231] In some embodiments, a pest can have a harmful direct effect on a beneficial insect. For example, in some embodiments, a pest can be an animal, such as an arthropod (e.g., an arthropod parasite), that causes a harmful direct effect on the beneficial insect by feeding on the hemolymph of adult beneficial insects and / or their developing brood, and / or transmitting viruses to the beneficial insect.

[0232] In yet other embodiments, a pest can be an animal, such as an arthropod (e g., an arthropod parasite), that causes a harmful indirect effect on a beneficial insect, e.g., via transmission of a disease agent (e.g.. a virus, bacteria, etc.) from the pest to the beneficial insect. Accordingly, in some embodiments, the pest serves as a vector for pathogen transmission to beneficial insects.

[0233] In some embodiments, a pest can be an animal, such as an arthropod (e.g., an arthropod parasite), that causes a harmful indirect effect on people, pets, and / or livestock, e.g., via the weakening and / or death of the beneficial insects, and the subsequent inability of the beneficial insects to confer their beneficial effects (e.g., the pollination of commercially relevant crops) to people. Accordingly, the weakening and / or death of the beneficial insects may result in food scarcity and / or economic hardship, which in turn wi 11 have a negative effect on people, pets, and / or livestock.

[0234] In some embodiments, a pest can be a mite, e.g.. a Varroa mite. In some embodiments, the pest can be any species belonging to genus: Varroa, such as Varroa destructor, Varroa jacobsoni, Varroa rindereri, or Varroa underwoodi.

[0235] As used herein, “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 pests.

[0236] “Pesticidal activity” means that upon or after exposing the pest to an OVP, OVP-pesticidal protein, or WT Omega peptide of the present disclosure, or a composition of the present disclosure, the pest 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.

[0237] “Pesticidal activity assay” refers to an assay wherein one or more pests are treated with either an agent (e.g., an OVP, OVP-pesticidal protein, or WT Omega peptide ofthe present disclosure, or a composition of the present disclosure), or a comparator or control, and wherein the pesticidal 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, which is required to achieve 100% pest mortality, i.e., 100% of pests 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 up-ended.

[0238] 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 is treated with the agent (e.g., an OVP, OVP-pesticidal protein, or WT Omega peptide of the present disclosure, or a composition 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% pest mortality (i.e., 100% of the pests are dead, unmoving, or unable to hold on to the side of a container or host, when said container or host is up-ended).

[0239] Accordingly, where an agent such as an OVP, OVP-pesticidal protein, or WT Omega peptide of the present disclosure has a pesticidal activity that is greater than a comparator or control, e.g., a comparator such as a vehicle, water, etc. — as measured via a 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.

[0240] For example, in some embodiments, a pesticidal activity assay can be performed as follows: one or more pests (e.g., adult Varroa mites, or another deleterious insect pest as described herein) can be immobilized via CO2 for 10 minutes, and then transferred to a CO2 pad to keep them immobilized. Varroa mites 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., an OVP, OVP-pesticidal protein, or WT Omega 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 peptide solution containing the agent, or a control solution containing water, can be topically applied to each Varroa mite, or the environment (e.g., host environment). Here,the peptide solution containing the agent is topically applied to pests in a first group, and the control solution containing water is topically applied to pests in a second group. The treated pests and untreated pests 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). “Pest mortality,” i.e., pest knock-down and pest death, can then be assessed at 24-hours post-treatment of the single dose; here, “pest mortality” is the dose (ng / pL) required for 100% of the pests 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.

[0241] “Pesticidal effect” refers to the removal or the reduction of harm of pests. The concept of “pesticidal effect” includes reducing of the target pest, killing of pests (extermination), pest proliferation inhibition, pest development inhibition, pest growth inhibition, repelling of pests (repellence), reducing of the survival rate of the target pest and the removal or the reduction of harm of pests (for example, inhibition of ingestion capacity of agricultural pests). “Pesticidal effect” includes killing of any individual or group of pests.

[0242] “Pesticidally-effective amount” refers to an amount of (1) an OVP, OVP- pesticidal protein, WT Omega peptide, or an agriculturally acceptable salt thereof; and / or (2) a pesticidal composition comprising: an OVP, OVP-pesticidal protein, WT Omega peptide, or an agriculturally acceptable salt thereof, and an excipient; that is sufficient to: partially or completely inhibit a pest (e.g., an insect pest such as a Varroa mite or another deleterious insect as described herein); bring about the death of at least one pest; partially or completely reduce or decrease pest growth, feeding, or normal physiological development; partially or completely inhibit or decrease the normal pest cellular processes, including maintenance and growth; and / or attenuate or decrease the severity of a pest infestation. This amount will vary depending on such factors including but not limited to: the specific target pest 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 pesticidally- effective amount may also vary with respect to climatic conditions, environmental considerations, and / or frequency of application and / or severity of pest infestation. In some embodiments, pesticidally-effective amounts can be measured by use of assays that measure the reduction in growth or decline in pest populations. One measure of reduction can be to express the decrease in population in logarithmic scale typical of a specific pest species, i.e., a 1 log reduction is equivalent to a 90% reduction versus a control, a 2 log reduction is a 99% reduction, etc.

[0243] “Pharmaceutically acceptable salt’' is synonymous with agriculturally acceptable salt, and as used herein refers to a compound that is modified by making acid or base salts thereof.

[0244] “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).

[0245] “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.

[0246] “Plant-incorporated protectant” or “PIP” means a pesticidal protein produced by transgenic plants, and the genetic material necessary for the plant to produce the protein.

[0247] “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.

[0248] “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.

[0249] “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).

[0250] “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.

[0251] “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 RNA; 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.

[0252] In yet other embodiments, a polynucleotide can refer to a polymeric-form of nucleotides operable to encode the open reading frame of a gene.

[0253] In some embodiments, a polynucleotide can refer to cDNA.

[0254] 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 j oined by a phosphodiester bond between their 3’ and 5’ carbons. How ever, 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.

[0255] 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.

[0256] In some embodiments, a poly nucleotide 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-nucleotidecomponents. 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.

[0257] 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 polynucleotide is RNA. Uracil can also be used in DNA. Thus, the term “sequence7’ refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and non-natural bases.

[0258] The term “RNA molecule’’ or ribonucleic acid molecule refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and typically 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.

[0259] In some embodiments, a polynucleotide can further comprise one or more heterologous regulator ' 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.

[0260] “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.

[0261] “Promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

[0262] “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 which 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 modificationsincluding, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0263] ’‘Ratio” refers to the quantitative relation between two amounts showing the number of times one value contains or is contained within the other.

[0264] “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 DNA molecule. 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.

[0265] “Recombinant DNA” or “rDNA” refers to DNA that is comprised of two or more different DNA segments.

[0266] “Recombinant vector” means a DNA plasmid vector into which foreign DNA has been inserted.

[0267] “Regulator}’ 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 regulator}' element can be found at the transcriptional and post- transcriptional level. Regulator}’ 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; poly adenylation 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.

[0268] “Restriction enzyme” 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.

[0269] “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 enzyme.

[0270] “Secondary pesticidal agent” refers to any one or more agents that is not an OVP. OVP-pesticidal protein, or WT Omega peptide of the present disclosure, e.g., any one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, poisons, insecticides, pesticides, organic compounds, inorganiccompounds, prokaryote organisms and / or the products produced therefrom, or eukaryote organisms and / or the products produced therefrom, wherein the secondary pesticidal agent is not an OVP, OVP-pesticidal protein, or WT Omega peptide of the present disclosure. Nonlimiting examples of secondary pesticidal agents include: essential oils (e.g., thymol, eucalyptol, menthol, or any combination thereof), formic acid, and / or chemical pesticides (e.g., a pyrethroid, amitraz, or an organophosphate such as Coumaphos).

[0271] "Selection gene” means a gene which confers an advantage for a genetically modified organism to grow under the selective pressure.

[0272] “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.

[0273] “sp ” refers to species.

[0274] “ssp.” or “subsp.” refers to subspecies.

[0275] “Subcloning” or “subcloned” refers to the process of transferring DNA from one vector to another, usually advantageous vector. For example, polynucleotide encoding an OVP can be subcloned into a pLB102 plasmid subsequent to selection of yeast colonies transformed with pKLACl plasmids.

[0276] “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.

[0277] “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 a pesticidal protein or an OVP in the ORF. The operably- linked STA can either be upstream or downstream of the OVP and can have any intervening sequence between the two sequences (STA and OVP) as long as the intervening sequencedoes not result in a frame shift of either DNA sequence. The translational stabilizing protein can also have an activity which increases delivery of the OVP across the gut wall and into the hemolymph of the insect.

[0278] “sta” means a nucleotide encoding a translational stabilizing protein.

[0279] “Stable composition’" refers to a composition or formulation that maintains the chemical and / or physical stability of the active ingredient (e.g., an OVP or OVP -pesticidal 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.

[0280] “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).

[0281] Stringent hybridization conditions are sequence- and length-dependent, and will be different in different circumstances. Similarly, stringent hybridization conditions depend on % (percent)-identi ty (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 sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 or 500 nucleotides in length

[0282] 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, ty pically 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.

[0283] 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 1 x to 2*SSC (20xSSC=3.0 M NaCl / 0.3 M trisodiumcitrate) 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 I xSSC 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.

[0284] 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)A . 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 typically 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.

[0285] 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.

[0286] 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).

[0287] 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 York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter2 (Greene Publishing and Wiley-Interscience, New York); the disclosures of which are incorporated herein by reference in their entireties.

[0288] In some embodiments, a polynucleotide of the present disclosure can stringently hybridize to a polynucleotide encoding an OVP, or a complementary nucleotide sequence thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0289] "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.

[0290] “Susceptible to attack by a pest(s),” refers to plants or animal subjects susceptible to attack by a pest, and / or that targets for pest infections or parasitization. For example, in one embodiment, an example of a pest infection is parasitization of the animal susceptible to attack by a pest, i.e., the animal is a host organisms that is targeted for exploitation by parasites. In some embodiments, an animal susceptible to attack by a pest can be a beneficial insect, e.g., any species belonging to the genus: Apis, such as a bee. In some embodiments, an animal susceptible to attack by a pest can be a species selected from: Apis mellifera, Apis cerana. Apis dorsata. Apis florea, Apis laboriosa. Apis andreniformis, or Apis koschevnikovi.

[0291] “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

[0292] “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 OVP) into a host organism (e.g., a prokaryote 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 eukaryotic 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 prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).

[0293] “Transgene” means a heterologous and / or exogenous DNA sequence encoding a protein which is transformed into a plant.

[0294] “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.

[0295] “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.

[0296] “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.

[0297] “Triple expression cassette refers to three OVP expression cassettes contained on the same vector.

[0298] “TRBO” means a transient plant expression system using Tobacco mosaic virus with removal of the viral coating protein gene.

[0299] ’Trypsin cleavage” means an in vitro assay that uses the protease enzy me 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 try psin enzyme cleaving that site.

[0300] “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.

[0301] “UBI” refers to ubiquitin. For example, in some embodiments, UBI can refer to a ubiquitin monomer isolated from Zea mays.

[0302] '‘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.

[0303] “Vector” refers to the DNA segment that accepts a foreign gene of interest. The gene of interest is known as an “insert” or “transgene.”

[0304] “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.

[0305] “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 ormedian 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.

[0306] 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.

[0307] 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.

[0308] The present disclosure is performed yvithout 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 Enzy mology (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,E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis. Spring er-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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] Omega Variant Peptides (OVPs)

[0313] In some embodiments, an OVP 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-type or originating DNA sequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein from which the OVP was derived.

[0314] In some embodiments, an OVP comprises 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-type or originating DNAsequence, polynucleotide, amino acid sequence, peptide, polypeptide, or protein from which the OVP was derived.

[0315] An exemplar}' originating protein from which OVPs 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 “WT-Omega,” which has an amino acid sequence: “SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO: 2).

[0316] Another exemplary originating protein from which OVPs 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 “Omega+2,’' which has an amino acid sequence of “GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD’’ (SEQ ID NO: 1).

[0317] Exemplary OVPs

[0318] In some embodiments, an Omega Variant Peptide (OVP) can be a mutant or variant that differs from wild type Omega 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 OVP. The result of this variation is a non- naturally occurring polypeptide and / or polynucleotide sequence encoding the same, relative to WT Omega, that possesses pesticidal activity.

[0319] In some embodiments, an Omega Variant Peptide (OVP) can be a mutant or variant that differs from a Omega+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 OVP. The result of this variation is a non-naturally occurring polypeptide and / or polynucleotide sequence encoding the same, relative to Omega+2, that possesses pesticidal activity.

[0320] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth inSEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D. E, F, G, H. I. K, L. M, N, P, Q, R. V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0321] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; and wherein the OVP is deglycosylated.

[0322] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO; 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4. or 5 amino acid substitutions that are conservative amino acid substitutions.

[0323] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity' comprises, consists essentially of, or consists of, an amino acid sequence that is 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, atleast 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is A, D, E, F, G. H, I, K, L, M, N, P, Q. R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1 , 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 1 , 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety and wherein the OVP is deglycosylated.

[0324] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IA): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 71); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is any amino acid and X2 is serine (S), or each independently are optionally absent; and wherein X is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0325] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity’ comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IB): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 72); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is any amino acid, or wherein Xiand X2 are independently optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M,N, P, Q, R. V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of theO, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety and wherein the OVP is deglycosylated.

[0326] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IC): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 73); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S). or wherein Xi and X2 are independently optionally absent; and wherein X is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3. 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety and wherein the OVP is deglycosylated.

[0327] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H. I, K, L, M. N, P, Q, R. V,W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety’.

[0328] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence according to Formula (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N- G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2. 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety .

[0329] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IE): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 75); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is any amino acid and X2 is serine (S); and wherein X3 is D, E, F, G, H, I, K. L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0330] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IF): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 76); wherein the OVPcomprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is any amino acid; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0331] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IG): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 77); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S); and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2. 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety'.

[0332] In some embodiments, the OVPs of Formulas (ID), (IE), (IF), and (IG), are deglycosylated peptides.

[0333] As used herein, the term “conservative amino acid substitutions” refers to amino acid substitutions to a molecule that do not affect the functional and / or chemical characteristics of the molecule (i.e., the OVP). Accordingly, conservative amino acid substitutions are generally therefore based on the relative similarity of the amino acid sidechain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary conservative amino acid substitutions are well known to those having ordinary skill in the art. For example, in some embodiments, conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g.,lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic, acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic, nonpolar or slightly polar residues (e.g., alanine, serine, threonine, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cystine); beta-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); and large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0334] In some embodiments, where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

[0335] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; and wherein if Xi and X2 are each absent, then X3 is not alanine (A); or an agriculturally acceptable salt thereof; and with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0336] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of. or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; and wherein if Xi and X2 are each absent, then X3 is not alanine (A); or an agriculturally acceptable salt thereof; and wherein the OVP is deglycosylated.

[0337] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; and wherein if Xi and X2 are each absent, then X3 is not alanine (A); or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions.

[0338] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; and wherein if Xi and X2 are each absent, then X3 is not alanine (A); or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 1, 2, 3, 4, or 5 amino acidsubstitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety’ and wherein the OVP is deglycosylated.

[0339] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 Formula (IC): X1-X2-S-P-T-C-I-P-X3-G-Q-P- C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 73); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S), or wherein Xi and X2 are optionally independently absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2. 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; and wherein the OVP is deglycosylated, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety', and wherein if Xi and X2 are each absent, then X3 is not alanine (A).

[0340] A summary of illustrative OVPs are provided in the table below.

[0341] Table 1. Summary of illustrative OVPs. Table 1 provides a summary of OVPs that produce a single, homogenous species (i.e., a non-glycosylated form or nonglycosylated species) of the OVP when expressed in a recombinant cell culture system (e.g., a recombinant yeast cell culture system) — relative to Omega+2 (SEQ ID NO: 1).

[0342] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprises an amino acid sequence that is 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 OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0343] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, or consists of an amino acid sequence of Table 1, or an agriculturally acceptable salt thereof.

[0344] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprises an amino acid sequence that is 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 OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; and wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety’.

[0345] In some embodiments, the OVP 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: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof; wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety'.

[0346] In some embodiments, the OVP 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 least99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety .

[0347] In some embodiments, the OVP consists essentially of an amino sequence as set forth in any one of SEQ ID NOs: 3-16. 19-35, or 38-40, or an agriculturally acceptable salt thereof; wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety .

[0348] In some embodiments, the OVP consists of an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof; and wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety .

[0349] In some embodiments, the OVP consists of an amino sequence as set forth in any one of SEQ ID NOs: 15, 16, 19, 34, 35 and 38, or an agriculturally acceptable salt thereof; and wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions that are conservative amino acid substitutions, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety .

[0350] In some embodiments, an OVP of the present disclosure, consists of an amino acid sequence according to any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof, and wherein the OVP is deglycosylated.

[0351] In some embodiments, an OVP of the present disclosure, consists of an amino acid sequence according to any one of SEQ ID NOs: 15, 16, 19, 34, 35 and 38, or an agriculturally acceptable salt thereof, and wherein the OVP is deglycosylated.

[0352] In some embodiments, the OVP consists of an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof.

[0353] In some embodiments, an OVP of the present disclosure can comprise, consist essentially of, or consist of, a homopolymer or heteropolymer of two or more OVPs, wherein the amino acid sequence of each OVP is the same or different.

[0354] In some embodiments, an OVP of the present disclosure can comprise, consist essentially of, or consist of, an OVP that is a fused protein comprising two or more OVPsseparated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each OVP may be the same or different.

[0355] In some embodiments, the linker is a cleavable linker.

[0356] In some embodiments, the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 42-51.

[0357] 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.

[0358] Detailed methods concerning linkers are described below.

[0359] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-Zi-G-N-T-V-K-R-C-D (SEQ ID NO: 78); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; wherein X3 is A, D, E, F, G, H, I, K. L, M, N, P, Q, R, V, W, or Y; and wherein Zi is A, R. N, D. C, E. Q, G, H, I, L, K, M, F. P, S, T, W. Y, or V; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0360] In some embodiments, an Omega Variant Peptide (OVP) having pesticidal activity comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (III): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-Zi-G-N-T-V-K-R-C-D (SEQ ID NO 79); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; wherein X3 is D, E, F, G, H, I, K, L, M, N, P. Q, R, V, W, or Y; andwherein Zi is A, L. P. or T; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0361] Polynucleotides encoding OVPs

[0362] In some embodiments, a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an Omega Variant Peptide (OVP).

[0363] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P- C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D, E, F, G, H, I, K. L, M, N, P, Q, R, V, W, or Y; or a complementary nucleotide sequence thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0364] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP having pesticidal activity, wherein the OVP comprises, consists essentially of, or consists of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G- N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; and wherein if Xi and X2 are each absent, then X3 is not alanine (A); or an agriculturally acceptable salt thereof.

[0365] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino acid sequencehaving pesticidal activity , the OVP comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IC): X1-X2-S- P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 73); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S), or wherein Xi and X2 are optionally absent; and wherein X3 is A, D, E, F, G, H, I. K, L. M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; and wherein the OVP is deglycosylated, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety’.

[0366] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (ID): X1-X2-S-P-T-C-I-P-X3-G-Q- P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0367] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (ID): X1-X2-S-P-T-C-I-P-X3-G-Q- P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is D, E, F. G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1 , 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0368] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IE): X1-X2-S-P-T-C-I-P-X3-G-Q- P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 75); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is any amino acid and X2 is serine (S); and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0369] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino acid sequence that is 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 (IF): X1-X2-S-P-T-C-I-P-X3-G-Q- P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 76); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is any aminoacid; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W. or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0. 1, 2, 3. 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0370] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of. an amino acid sequence that is 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 (IG): X1-X2-S-P-T-C-I-P-X3-G-Q- P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 77); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S). and wherein X3 is D. E, F. G, H. 1. K, L. M. N, P, Q, R. V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety’.

[0371] In some embodiments, a polynucleotide is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or a complementary nucleotide sequence thereof.

[0372] In some embodiments, polynucleotides of the present disclosure encode an OVP, wherein the polynucleotide hybridizes under stringent conditions to a polynucleotide which encodes an OVP having an amino acid sequence of SEQ ID NOs: 33-16, 19-35, or 38- 40, or a complementary nucleotide sequence thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0373] Nucleotide sequence homologs, e.g.. OVPs 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 OVP or fragment thereof, and hybridizes to the second polynucleotide. In suchcase, the second polynucleotide can be any of the polynucleotides operable to encode an OVP of the present disclosure, under stringent hybridization conditions.

[0374] In some embodiments, a polynucleotide of the present disclosure is operable to encode an Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprising an amino acid sequence that is 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 OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2 and 2, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0375] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP that comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3-16. 19-35, or 38-40, or a complementary’ nucleotide sequence thereof, wherein the OVP further comprises 0, 1, 2. 3. 4, or 5 amino acid substitutions that are conservative amino acid substitutions, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0376] In some embodiments, a poly nucleotide of the present disclosure is operable to encode an OVP that consists of an amino sequence as set forth in any one of SEQ ID NOs: 3- 16, 19-35, or 38-40, or a complementary nucleotide sequence thereof.

[0377] In some embodiments, a polynucleotide of the present disclosure is operable to encode an OVP, the OVP consists of an amino sequence as set forth in any one of SEQ ID NOs: 15, 16, 19, 34, 35, and 38, or a complementary’ polynucleotide thereof.

[0378] In some embodiments, the polynucleotide is operable to encode an OVP that can comprise, consist essentially of, or consist of, a homopolymer or heteropolymer of two or more OVPs, wherein the amino acid sequence of each OVP is the same or different.

[0379] In some embodiments, the poly nucleotide is operable to encode an OVP that can comprise, consist essentially of, or consist of, an OVP that is a fused protein comprising two or more OVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each OVP may be the same or different.

[0380] In some embodiments, the linker is a cleavable linker.

[0381] In some embodiments, the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 42-51.

[0382] 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.

[0383] Stringent hybridization

[0384] In some embodiments, a polynucleotide of the present disclosure can stringently hybridize to a polynucleotide encoding an OVP, or a complementary sequence thereof, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 according to Formulas (I), (IA), (IB), (IC), (ID). (IE), (IF), (IG), (II), (III), an OVP of Table 1, or an OVP having an amino acid sequence of any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof.

[0385] In some embodiments, a poly nucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide segment encoding an OVP or fragment thereof, wherein: (a) said OVP comprises an amino acid sequence set forth in SEQ ID NOs: 3-16, 19-35, or 38-40; or (b) said OVP comprises an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NOs: 3-16, 19-35, or 38-40; or (c) said polynucleotide segment hybridizes to a polynucleotide having a polynucleotide segment operable to encode an OVP having an amino acid sequence as set forth in SEQ ID NOs: 3-16, 19-35, or 38-40, or a complementary nucleotide sequence thereof.

[0386] 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 OVP 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: 3-16. 19-35, or 38-40, or a polynucleotide encoding an OVP comprising an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NOs: 33-16, 19-35, or 38-40. 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.

[0387] OVP-pesticidal proteins

[0388] In some embodiments, an OVP-pesticidal protein can be any protein, peptide, polypeptide, amino acid sequence, configuration, construct, or arrangement, comprising: (1) at least one OVP, or two or more OVPs (wherein the two or more OVPs are the same or different); and (2) one or more additional non-OVP peptides, polypeptides, or proteins. For example, in some embodiments, these additional non-OVP peptides, polypeptides, or proteins may have the ability to increase the mortality and / or inhibit the growth of insects exposed to the OVP-pesticidal protein, relative to the OVP alone; increase the expression of the OVP- pesticidal protein, e g., in a host cell; and / or affect the post-translational processing of the OVP-pesticidal protein.

[0389] In some embodiments, an OVP-pesticidal protein can be a polymer comprising two or more OVPs. In yet other embodiments, an OVP-pesticidal protein can be a polymer comprising two or more OVPs, wherein the OVPs 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-OVP peptide described above.

[0390] In some embodiments, an OVP-pesticidal protein can refer to a one or more OVPs 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

[0391] In some embodiments, an OVP-pesticidal protein can be a polymer of amino acids that, when properly folded or in its most natural thermodynamic state, exerts a pesticidal activity against one or more insects.

[0392] In some embodiments, an OVP-pesticidal protein can be a polymer comprising two or more OVPs that are different. In other embodiments, a pesticidal protein can be a polymer of two or more OVPs that are the same.

[0393] In some embodiments, an OVP-pesticidal protein can comprise, consist essentially of, or consist of one or more OVPs having an amino acid sequence set forth in SEQ ID NOs: 3-16, 19-35, or 38-40, wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0394] In some embodiments, the OVP polymers (homor polymer or heteropolymer) can include polymers separated or linked with a linker as described herein. Examples of linkers include, but not limited to, the following linker amino acid sequences: IGER (SEQ IDNO:59), EEKKN, (SEQ ID NO:60), and ETMFKHGL (SEQ ID NO:61), or combinations thereof.

[0395] In some embodiments, the linker can be one or more of the following: ALKFLV (SEQ ID NO: 42), ALKLFV (SEQ ID NO: 43), IFVRLR (SEQ ID NO: 44), LFAAPF (SEQ ID NO: 45), ALKFLV GS (SEQ ID NO: 46), ALKLFVGS (SEQ ID NO: 47), IFVRLRGS (SEQ ID NO: 48), LFAAPFGS (SEQ ID NO: 49), LFVRLRGS (SEQ ID NO: 50), and / or LGERGS (SEQ ID NO: 51).

[0396] Exemplary methods for the generation of cleavable and non-cleavable linkers can be found in U.S. Patent No. 11,447,531, the disclosure of which is incorporated herein by reference in its entirety.

[0397] 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 entirety.

[0398] Detailed methods concerning ERSPs, STAs, and linkers, are likewise described below.

[0399] METHODS FOR PRODUCING AN OVP

[0400] Methods of producing proteins are well known in the art, and there are a variety of techniques available. For example, in some embodiments, proteins can be produced using recombinant methods, cell-free expression systems, cell extracts, or chemically synthesized.

[0401] In some embodiments, an OVP of the present disclosure can be created using any known method for producing a protein. For example, in some embodiments, and without limitation, an OVP can be created using a recombinant expression system, such as yeast expression system or an bacterial expression system. However, those having ordinary skill in the art will recognize that other methods of protein production are available.

[0402] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a method of producing an OVP using a recombinant expression system.

[0403] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a method of producing an OVP. said method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide operable to encode an OVP, or a complementary nucleotide sequence thereof, (b) introducing the vector into a host cell, for example a bacteria or a yeast, or an insect, or a plant cell, or an animal cell; and (c) growing the yeast strain in a growthmedium under conditions operable to enable expression of the OVP and secretion into the growth medium. In some related embodiments, the host cell, is a yeast cell.

[0404] The invention is practicable in a wide variety of host cells (see host cell section below). Indeed, an end-user of the invention can practice the teachings thereof in any host cell of his or her choosing. Thus, in some embodiments, the host cell can be any host cell that satisfies the requirements of the end-user; i.e., in some embodiments, the expression of an OVP may be accomplished using a variety of host cells, and pursuant to the teachings herein. For example, in some embodiments, a user may desire to use one specific type of host cell (e.g., a yeast cell or a bacteria cell) as opposed to another; the preference of a given host cell can range from availability to cost.

[0405] For example, in some embodiments, in some embodiments, the present disclosure comprises, consists essentially of, or consists of, a method of producing an OVP, said method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide operable to encode an OVP, or a complementary nucleotide sequence thereof; (b) introducing the vector into a host cell, for example a bacteria or a yeast, or an insect, or a plant cell, or an animal cell; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the OVP and secretion into the grow th medium. In some related embodiments, the host cell, is a yeast cell.

[0406] In some embodiments, a method of producing an OVP comprises: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide encoding an OVP, or a complementary sequence thereof, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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), or Formula (IA), Formula (IB), Formula (IC), Formula (ID), Formula (IE), Formula (IF). Formula (IG), Formula (II), or Formula (III), with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety; (b) introducing the vector into a host cell, for example a bacteria or a yeast, or an insect, or a plant cell, or an animal cell; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the OVP and secretion into the growth medium. In some related embodiments, the host cell, is a yeast cell.

[0407] In some embodiments, the present disclosure provides a method of producing an OVP, said method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide operable to encode an OVP, or a complementary nucleotide sequence thereof, said OVP 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 set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40; wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety; (b) introducing the vector into a host cell, for example a bacteria or a yeast, or an insect, or a plant cell, or an animal cell; and (c) growing the yeast strain in a grow th medium under conditions operable to enable expression of the OVP and secretion into the growth medium. In some related embodiments, the host cell, is a yeast cell.

[0408] In some embodiments, the method of producing an OVP produces a homopolymer, wherein each OVP has the same amino acid sequence.

[0409] In some embodiments, the method of producing an OVP produces a homopolymer, wherein each OVP has a different amino acid sequence.

[0410] In some embodiments, the method of producing an OVP, wherein the OVP is a fused protein comprising two or more OVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each OVP may be the same or different.

[0411] In some embodiments, the method of producing an OVP, wherein the linker is a cleavable linker.

[0412] In some embodiments, the method of producing an OVP, wherein the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 42-51.

[0413] In some embodiments, the method of producing an OVP, wherein 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.

[0414] In some embodiments, the method of producing an OVP provides for a vector, wherein the vector is a plasmid. In some embodiments, the plasmid my comprise an alpha- MF signal.

[0415] In some embodiments, the present disclosure provides a method of producing an OVP, said method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide operable to encode an OVP, or a complementary nucleotide sequence thereof, (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 OVP and secretion into the growth medium, wherein the vector is transformed into a microorganism, e.g., a yeast or a bacteria.

[0416] In some embodiments, the present disclosure provides a method of producing an OVP, wherein the host cell can be a yeast strain.

[0417] In some embodiments, the yeast strain is selected from any species belonging to the genera Saccharomyces, Pichia, Kluyveromyces , Hansenula, Yarrowia, or Schizosaccharomyces .

[0418] In some embodiments, the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pas tor is.

[0419] In some embodiments, the yeast strain is Kluyveromyces lactis.

[0420] In some embodiments, the yeast strain is Kluyveromyces marxianus.

[0421] In some embodiments, the OVP is secreted into the growth medium.

[0422] In some embodiments, the OVP is secreted into the growth medium in a cell culture or fermentation of a suitably transformed host cell incorporating a polynucleotide operable to encode the OVP. wherein expression of the OVP provides a yield of: at least 30 mg / L, at least 40 mg / L, at least 50 mg / L, at least 60 mg / L, 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 least15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least18,000 mg / L, at least 19.000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L of OVP per liter of yeast culture medium.

[0423] In some embodiments, the expression of the OVP in the medium results in the expression of a single OVP in the medium.

[0424] In some embodiments, the expression of the OVP in the medium results in the expression of an OVP polymer comprising two or more OVP polypeptides in the medium.

[0425] In some embodiments, the vector comprises two or three expression cassettes, each expression cassette operable to encode the OVP of the first expression cassette.

[0426] In some embodiments, the vector comprises two or three expression cassettes, each expression cassette operable to encode the OVP of the first expression cassette, or an OVP of a different expression cassette.

[0427] In some embodiments, an expression cassette of the present disclosure is operable to encode an OVP as set forth in any one of SEQ ID NOs: 3-16. 19-35, or 38-40.

[0428] Isolating and mutating proteins

[0429] In various illustrative embodiments, an OVP can be obtained by creating an OVP polynucleotide sequence, which in turn can be created by generating a mutation in a wild-type polynucleotide sequence operable to encode WT Omega, e.g., “SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO: 2); or generating a mutation in a Omega+2 polynucleotide sequence operable to encode Omega+2, e.g., “GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO: 1) (i.e.. creating an OVP polynucleotide sequence); inserting that OVP polynucleotide (oyp) sequence into the appropriate vector; transforming a host organism in such a way that the polynucleotide encoding an OVP is expressed; culturing the host organism to generate the desired amount of OVP; and then purifying the OVP from in and / or around host organism.

[0430] Wild-type spider toxins can be isolated from spider venom, which in turn can be isolated from the venom glands of spiders, e.g., Atrax robustus or Haydr onyche versuta. using any of the techniques known to those having ordinary skill in the art. For example, in some embodiments, venom can be isolated according to the methods described in U.S. Patent No 5,688,764, the disclosure of which is incorporated herein by reference in its entirety.

[0431] In some embodiments, a wild-type Omega polynucleotide sequence can be obtained by screening a genomic library using primer probes directed to the Omegapolynucleotide sequence. Alternatively, wild-type Omega polynucleotide sequence, an Omega+2 polynucleotide sequence, and / or OVP polynucleotide sequences can be chemically synthesized. For example, a wild-type Omega polynucleotide sequence, an Omega+2 polynucleotide sequence, and / or OVP polynucleotide sequence can be generated using the oligonucleotide synthesis methods such as the phosphoramidite; triester, phosphite, or H- Phosphonate methods (see Engels. J. W. and Uhlmann, E. (1989), Gene Synthesis [New Synthetic Methods (77)]. Angew. Chem. Int. Ed. Engl., 28: 716-734. the disclosure of which is incorporated herein by reference in its entirety).

[0432] Chemically synthesizing OVP polynucleotides

[0433] In some embodiments, the polynucleotide sequence encoding the OVP can be chemically synthesized using commercially available polynucleotide synthesis sendees such as those offered by Genewiz® (e.g., TurboGENE™; PriorityGENE; and FragmentGENE), or Sigma- Aldrich® (e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos). Exemplan method for generating DNA and or custom chemically synthesized polynucleotides are well known in the art, and are illustratively provided in U.S. Patent No. 5,736,135, Serial No. 08 / 389,615, filed on Feb. 13, 1995. the disclosure of which is incorporated herein by reference in its entirety. See also Agarwal, et al.. Chemical synthesis of polynucleotides. Angew Chem Int Ed Engl. 1972 Jun; 11(6):451-9; Ohtsuka et al., Recent developments in the chemical synthesis of polynucleotides. Nucleic Acids Res. 1982 Nov 11; 10(21): 6553-6570; Sondek & Shortle. A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. Proc Natl Acad Sci U S A. 1992 Apr 15; 89(8): 3581-3585; Beaucage S. L., et al., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. Tetrahedron, Elsevier Science Publishers, Amsterdam, NL, vol. 48, No. 12, 1992, pp. 2223-2311; Agrawal (1993) Protocols for Oligonucleotides and Analogs: Synthesis and Properties; Methods in Molecular Biology Vol. 20, the disclosures of which are incorporated herein by reference in their entireties.

[0434] Producing a mutation in a wild-type Omega polynucleotide sequence and / or a Omega+2 polynucleotide sequence can be achieved by various means that are well known to those having ordinary skill in the art. Methods of mutagenesis include Kunkel’s method; cassette mutagenesis; PCR site-directed mutagenesis; the “perfect murder” technique (delitto perfetto),- direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker; direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker using long homologous regions; transplacement “pop-in pop-out” method; andCRISPR-Cas 9. Exemplar}' methods of site-directed mutagenesis can be found in Ruvkun & Ausubel, A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1; 289(5793): 85-8; Wallace et al., Oligonucleotide directed mutagenesis of the human betaglobin gene: a general method for producing specific point mutations in cloned DNA.Nucleic Acids Res. 1981 Aug 11; 9(15)3647-56; Dalbadie-McFarland et al., Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A. 1982 Nov; 79(21):6409-13; Bachman. Site-directed mutagenesis. Methods Enzymol. 2013; 529:241 -8; Carey et al., PCR-mediated site-directed mutagenesis. Cold Spring Harb Protoc. 2013 Aug 1; 2013(8):738-42; and Cong et al., Multiplex genome engineering using CRISPR / Cas systems. Science. 2013 Feb 15; 339(6121):819-23. the disclosures of all of the aforementioned references are incorporated herein by reference in their entireties.

[0435] Chemically synthesizing polynucleotides allows for a DNA sequence to be generated that is tailored to produce a desired polypeptide based on the arrangement of nucleotides within said sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A] or thymine [T] molecules); the mRNA sequence that is transcribed from the chemically synthesized DNA polynucleotide can be translated to a sequence of amino acids, each amino acid corresponding to a codon in the mRNA sequence. Accordingly, the amino acid composition of a polypeptide chain that is translated from an mRNA sequence can be altered by changing the underlying codon that determines which of the 20 amino acids will be added to the growing polypeptide; thus, mutations in the DNA such as insertions, substitutions, deletions, and frameshifts may cause amino acid insertions, substitutions, or deletions, depending on the underlying codon.

[0436] In some embodiments, a polynucleotide can be chemically synthesized, wherein said polynucleotide harbors one or more mutations. In some embodiments, an mRNA can be created from the template DNA sequence. In yet other embodiments, the mRNA can be cloned and transformed into a competent cell.

[0437] Recombinant expression, vectors, and transformation

[0438] Obtaining an OVP from a chemically synthesized DNA polynucleotide sequence and / or a wild-type DNA polynucleotide sequence that has been altered via mutagenesis can be achieved by cloning the DNA sequence into an appropriate vector. There are a variety of expression vectors available, host organisms, and cloning strategies known to those having ordinary skill in the art. For example, the vector can be a plasmid, which can introduce a heterologous gene and / or expression cassette into yeast cells to be transcribed andtranslated. The term ‘‘vector’ is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A vector may contain “vector elements” such as an origin of replication (ORI); a gene that confers antibiotic resistance to allow for selection; multiple cloning sites; a promoter region; a selection marker for non-bacterial transfection; and a primer binding site. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated herein by reference in their entireties. In addition to encoding an OVP polynucleotide, a vector may encode a targeting molecule. A targeting molecule is one that directs the desired nucleic acid to a particular tissue, cell, or other location.

[0439] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a vector comprising a polynucleotide operable to encode an OVP of the present disclosure.

[0440] In some embodiments, a vector of the present disclosure comprises a polynucleotide operable to encode an OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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-S-P- T-C-I-P-Xs-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D, E, F. G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or a complementary nucleotide sequence thereof, withthe proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0441] In some embodiments, a vector of the present disclosure comprises a polynucleotide or complementary sequence thereof, that can stringently hybridize to a polynucleotide or segment thereof operable to encode an OVP, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N- C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D. E, F, G, H. I. K, L. M, N, P, Q, R, V, W, or Y, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0442] In some embodiments, a vector of the present disclosure comprises a polynucleotide or complementary sequence thereof, that can stringently hybridize to a polynucleotide or segment thereof operable to encode an OVP, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0443] In some embodiments, a vector of the present disclosure comprises a polynucleotide or complementary sequence thereof, that can stringently hybridize to apolynucleotide or segment thereof operable to encode an OVP, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is D. E, F, G, H. I. K, L. M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2, 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0444] In some embodiments, a vector of the present disclosure comprises a polynucleotide or complementary sequence thereof, that can stringently hybridize to a polynucleotide or segment thereof operable to encode an OVP, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 (IE): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 75); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is any amino acid and X2 is serine (S); and wherein X3 is D, E, F, G, H, I, K. L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0445] In some embodiments, a vector of the present disclosure comprises a polynucleotide or complementary sequence thereof, that can stringently hybridize to a polynucleotide or segment thereof operable to encode an OVP, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 (IF): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 76); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is any amino acid; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1. 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2. 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0446] In some embodiments, a vector of the present disclosure comprises a polynucleotide or complementary sequence thereof, that can stringently hybridize to a polynucleotide or segment thereof operable to encode an OVP, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 (IG): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 77); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is senne (S); and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions, for example, wherein one or more of the 0, 1, 2. 3, 4, or 5 amino acid substitutions are conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety'.

[0447] In some embodiments, a plant, plant tissue, plant cell, plant seed, or part thereof, can be transformed with a polynucleotide encoding an OVP, or a complementary nucleotide sequence thereof, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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

[0448] In some embodiments, a vector of the present disclosure comprises a polynucleotide operable to encode an OVP, said OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 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: 3-16, 19-35. or 38-40; wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; or a complementary' nucleotide sequence thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0449] In some embodiments, a polynucleotide operable to encode an OVP or an OVP-pesticidal protein, or a complementary nucleotide sequence thereof, can be transformed into a host cell.

[0450] In some embodiments, a polynucleotide operable to encode an OVP or an OVP-pesticidal protein, or a complementary nucleotide sequence thereof, can be cloned into a vector, and transformed into a host cell.

[0451] In some embodiments, an OVP ORF can be transformed into a host cell. In some embodiments, an OVP ORF can be cloned into a vector (e.g., a plasmid) and subsequently transformed into a host cell.

[0452] In addition to a polynucleotide sequence operable to encode an OVP (e.g., an OVP ORF) or an OVP-pesticidal protein, additional DNA segments known as regulatory elements can be cloned into a vector that allow for enhanced expression of the foreign DNA or transgene; examples of such additional DNA segments include (1) promoters, terminators, and / or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulator}'elements. The combination of a DNA segment of interest (e.g., ovp) with any one of the foregoing cis-acting elements is called an "‘expression cassette.”

[0453] In some embodiments, an expression cassette or OVP expression cassette can contain one or more polynucleotides operable to encode one or more OVPs, and / or one or more OVP-pesticidal proteins.

[0454] In some embodiments, an expression cassette or OVP expression cassette can contain one or more polynucleotides operable to encode one or more OVPs, and / or one or more OVP-pesticidal proteins; and, optionally, one or more additional regulatory' elements such as: (1) promoters, terminators, and / or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements.

[0455] In some embodiments, a single expression cassette can contain one or more of the aforementioned regulatory elements, and a polynucleotide operable to express an OVP. For example, in some embodiments, an OVP expression cassette can comprise polynucleotide operable to encode an OVP, and an a-MF signal; Kex2 site; LAC4 terminator; ADN1 promoter; and an acetamidase (amdS) selection marker — flanked by LAC4 promoters on the 5 ’-end and 3 ’-end.

[0456] 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 express an OVP. In alternative embodiments, there are two expression cassettes operable to encode an OVP (i.e. , a double expression cassette).In other embodiments, there are three expression cassettes operable to encode an OVP (i.e., a triple expression cassette).

[0457] In some embodiments, a double expression cassette can be generated by subcloning a second OVP expression cassette into a vector containing a first OVP expression cassette.

[0458] In some embodiments, a triple expression cassette can be generated by subcloning a third OVP expression cassette into a vector containing a first and a second OVP expression cassette.

[0459] In some embodiments, one, two, three, or more expression cassettes can be cloned into a vector, wherein each expression cassette comprises: (1) a DNA sequence of interest, e.g., a polynucleotide operable to encode an OVP; and one or more of the following: (2) promoters, terminators, and / or enhancer elements; (3) an appropriate mRNA stabilizingpolyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and / or (6) post-transcriptional regulatory elements.

[0460] In some embodiments, one, two, three, or more expression cassettes can be cloned into a vector, wherein each expression cassette comprises a polynucleotide encoding an OVP, wherein each of the OVPs are the same or different.

[0461] In some embodiments, one, two, three, or more expression cassettes can be cloned into a vector, wherein each expression cassette comprises a polynucleotide encoding an OVP ORF, wherein each of the OVP ORFs are the same or different.

[0462] Methods of cloning, recombinant expression, the preparation of vectors, and transformation techniques are known in the art. For example, in some embodiments, an OVP polynucleotide can be cloned into a vector (for example, a cloning vector or an expression vector known in the art) using a variety of cloning strategies, and commercial cloning kits and materials readily available to those having ordinary skill in the art, such as the SnapFast; Gateway; TOPO; Gibson; LIC; InFusionHD; or Electra strategies. Likewise, there are numerous commercially available vectors that can be used to produce OVP, e.g., an OVP polynucleotide can be generated using polymerase chain reaction (PCR). and combined with a pCR1MII-TOPO vector, or a PCR1M2.1 -TOPO® vector (commercially available as the TOPO® TA Cloning ® Kit from Invitrogen) for 5 minutes at room temperature; the TOPO® reaction can then be transformed into competent cells, which can subsequently be selected based on color change. See Janke et al., A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004 Aug; 21(ll):947-62; and Adams et al. Methods in Yeast Genetics. Cold Spring Harbor, NY, 1997, the disclosures of which are incorporated herein by reference in their entireties.

[0463] In some embodiments, a polynucleotide encoding an OVP or multiple copies of OVPs (either the same or different) can be cloned into a vector such as a plasmid, cosmid, virus (bacteriophage, animal viruses, and plant viruses), and / or artificial chromosome (e.g., YACs).

[0464] In some embodiments, a polynucleotide encoding an OVP can be inserted into a vector, for example, a plasmid vector using E. coll as a host, using methods known in the art.

[0465] In some embodiments a polynucleotide encoding an OVP (e.g., an OVP ORF), along with other DNA segments together composing an OVP expression cassette can be designed for secretion from host yeast cells. An illustrative method of designing an OVP expression cassette is as follows: the cassette can begin with a signal peptide sequence.followed by a DNA sequence encoding a Kex2 cleavage site (Lysine- Arginine), and subsequently followed by the OVP polynucleotide transgene (OVP ORF), with the addition of glycine-serine codons at the 5’-end, and finally a stop codon at the 3’-end. All these elements will then be expressed to a fusion peptide in yeast cells as a single open reading frame (ORF). An a-mating factor (aMF) signal sequence is most frequently used to facilitate metabolic processing of the recombinant pesticidal peptides through the endogenous secretion pathway of the recombinant yeast, i.e. the expressed fusion peptide will typically enter the Endoplasmic Reticulum, wherein the a -mating factor signal sequence is removed by signal peptidase activity, and then the resulting pro-pesticidal peptide will be trafficked to the Golgi Apparatus, in which the Lysine- Arginine dipeptide mentioned above is completely removed by Kex2 endoprotease, after which the mature, polypeptide (i.e., OVP), is secreted out of the cells.

[0466] In some embodiments, polypeptide expression levels in recombinant yeast cells can be enhanced by optimizing the codons based on the specific host yeast species. Naturally occurring frequencies of codons observed in endogenous open reading frames of a given host organism need not necessarily be optimized for high efficiency expression. Furthermore, different yeast species (for example, Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, etc.) have different optimal codons for high efficiency expression. Hence, codon optimization should be considered for the OVP expression cassette, including the sequence elements encoding the signal sequence, the Kex2 cleavage site and the OVP, because they are initially translated as one fusion peptide in the recombinant yeast cells.

[0467] In some embodiments, a codon-optimized OVP expression cassette can be ligated into a yeast-specific expression vectors for yeast expression. There are many expression vectors available for yeast expression, including episomal vectors and integrative vectors, and they are usually designed for specific yeast strains. One should carefully choose the appropriate expression vector in view of the specific yeast expression system which will be used for the peptide production. In some embodiments, integrative vectors can be used, which integrate into chromosomes of the transformed yeast cells and remain stable through cycles of cell division and proliferation. The integrative DNA sequences are homologous to targeted genomic DNA loci in the transformed yeast species, and such integrative sequences include pLAC4, 25S rDNA, pAOXl, and TRP2, etc. The locations of pesticidal peptide transgenes can be adjacent to the integrative DNA sequence (Insertion vectors) or within the integrative DNA sequence (replacement vectors).

[0468] In some embodiments, the expression vectors or cloning vectors can contain E. coll elements for DNA preparation in E. coli. for example, E. coll replication origin, antibiotic selection marker, etc. In some embodiments, vectors can contain an array of the sequence elements needed for expression of the transgene of interest, for example, transcriptional promoters, terminators, yeast selection markers, integrative DNA sequences homologous to host yeast DNA, etc. There are many suitable yeast promoters available, including natural and engineered promoters, for example, yeast promoters such as pLAC4. pAOXl, pUPP, pADHl , pTEF, pGall , etc., and others, can be used in some embodiments.

[0469] In some embodiments, selection methods such as acetamide prototrophy selection; zeocin-resistance selection; geneticin-resistance selection; nourseothricin- resistance selection; uracil deficiency selection; and / or other selection methods may be used. For example, in some embodiments, the Aspergillus nidulans amdS gene can be used as selectable marker. Exemplary methods for the use of selectable markers can be found in U.S. Patent Nos. 6,548,285 (filed Apr. 3, 1997); 6,165,715 (filed June 22, 1998); and 6,110,707 (filed Jan. 17, 1997), the disclosures of which are incorporated herein by reference in their entireties.

[0470] In some embodiments, a polynucleotide encoding an OVP can be inserted into a pKLACl vector. The pKLACl is commercially available from New England Biolabs® Inc., (item no. NEB &E1000). The pKLACl vector is designed to accomplish high-level expression of recombinant protein (e.g., OVP) in the yeast Kluyveromyces lactis. The pKLACl plasmid can be ordered alone, or as part of a '. lactis Protein Expression Kit. The pKLACl plasmid can be linearized using the SacII or BstXI restriction enzymes, and possesses a MCS downstream of an aMF secretion signal. The aMF secretion signal directs recombinant proteins to the secretory pathway, which is then subsequently cleaved via Kex2 resulting in peptide of interest, for example, an OVP. Kex2 is a calcium-dependent serine protease, which is involved in activating proproteins of the secretory pathway, and is commercially available (PeproTech®; item no. 450-45).

[0471] In some embodiments, a poly nucleotide encoding an OVP can be inserted into a pLB102 plasmid, or subcloned into a pLB102 plasmid subsequent to selection of yeast colonies transformed with pKLACl plasmids ligated with polynucleotide encoding an OVP. Yeast, for example K. lactis, transformed with a pKLACl plasmids ligated with polynucleotide encoding an OVP can be selected based on acetamidase (amdS), which allows transformed yeast cells to grow in YCB medium containing acetamide as its only nitrogen source.

[0472] In some embodiments, a polynucleotide encoding an OVP can be inserted into other commercially available plasmids and / or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (anon-profit plasmid repository); GenScript®; Takara®; Qiagen®; and Promega™

[0473] In some embodiments, a yeast cell transformed with one or more OVP expression cassettes can produce an OVP in a yeast culture with a yield of; at least 30 mg / L, at least 40 mg / L. at least 50 mg / L. at least 60 mg / L. at least 70 mg / L. at least 80 mg / L. at least 90 mg / L, at least 100 mg / L, at least 1 10 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 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 OVP per liter of medium.

[0474] In some embodiments, two expression cassettes comprising a polynucleotide operable to express an OVP can be inserted into a vector, for example a pKS022 plasmid, resulting in a yield of about 2 g / L of OVP (supernatant of yeast fermentation broth).Alternatively, in some embodiments, three expression cassettes comprising a polynucleotide operable to express an OVP can be inserted into a vector, for example a pLB103bT plasmid.

[0475] In some embodiments, multiple OVP expression cassettes can be transfected into yeast in order to enable integration of one or more copies of the optimized OVP transgene into the K. lactis genome. An exemplary' method of introducing multiple OVP expression cassettes into aK lactis genome is as follows: an OVP expression cassette DNA sequence is synthesized, comprising an intact LAC4 promoter element, a codon-optimized OVP ORF element and a pLAC4 terminator element; the intact expression cassette is ligated into the pLB103b vector between Sal I and Kpn I restriction sites, downstream of the pLAC4 terminator of pLB10V5, resulting in the double transgene OVP expression vector, pKS022; the double transgene vectors, pKS022, are then linearized using Sac II restrictionendonuclease and transformed into YCT306 strain of K. lactis by electroporation. The resulting yeast colonies are then grown on YCB agar plate supplemented with 5 rnM acetamide, which only the acetamidase-expressing cells could use efficiently as a metabolic source of nitrogen. To evaluate the yeast colonies, about 100 to 400 colonies can be picked from the pKS022 yeast plates. Inoculates from the colonies are each cultured in 2.2 mL of the defined K. Iciclis media with 2% sugar alcohol added as a carbon source. Cultures are incubated at 23.5°C, with shaking at 280 rpm. for six days, at which point cell densities in the cultures will reach their maximum levels as indicated by light absorbance at 600 nm (OD600). Cells are then removed from the cultures by centrifugation at 4,000 rpm for 10 minutes, and the resulting supernatants (conditioned media) are filtered through 0.2 pM membranes for HPLC yield analysis.

[0476] Chemically synthesizing OVPs

[0477] Peptide synthesis or the chemical synthesis or peptides and / or polypeptides can be used to generate OVPs: these methods can be performed by those having ordinary skill in the art, and / or through the use of commercial vendors (e.g., GenScript®; Piscataway. New Jersey). For example, in some embodiments, chemical peptide synthesis can be achieved using Liquid phase peptide synthesis (LPPS), or solid phase peptide synthesis (SPPS).

[0478] In some embodiments, peptide synthesis can generally be achieved by using a strategy’ wherein the coupling the carboxyl group of a subsequent amino acid to the N- terminus of a preceding amino acid generates the nascent polypeptide chain — a process that is opposite to the type of polypeptide synthesis that occurs in nature.

[0479] Peptide deprotection is an important first step in the chemical sy nthesis of polypeptides. Peptide deprotection is the process in which the reactive groups of amino acids are blocked through the use of chemicals in order to prevent said amino acid’s functional group from taking part in an unwanted or non-specific reaction or side reaction; in other words, the amino acids are “protected” from taking part in these undesirable reactions.

[0480] Prior to synthesizing the peptide chain, the amino acids must be “deprotected” to allow the chain to form (i.e., amino acids to bind). Chemicals used to protect the N-termini include 9-fluorenylmethoxy carbonyl (Fmoc), and tert-butoxy carbonyl (Boc), each of which can be removed via the use of a mild base (e.g., piperidine) and a moderately strong acid (e.g., trifluoracetic acid (TFA)), respectively.

[0481] The C-terminus protectant required is dependent on the type of chemical peptide synthesis strategy used: e.g., LPPS requires protection of the C-terminal amino acid.whereas SPPS does not owing to the solid support which acts as the protecting group. Side chain amino acids require the use of several different protecting groups that vary based on the individual peptide sequence and N-terminal protection strategy; typically, however, the protecting group used for side chain amino acids are based on the tert-butyl (tBu) or benzyl (Bzl) protecting groups.

[0482] Amino acid coupling is the next step in a peptide synthesis procedure. To effectuate amino acid coupling, the incoming amino acid’s C-terminal carboxylic acid must be activated: this can be accomplished using carbodiimides such as diisopropylcarbodiimide (DIC), or di cyclohexylcarbodiimide (DCC), which react with the incoming amino acid’s carboxyl group to form an O-acylisourea intermediate. The O-acy lisourea intermediate is subsequently displaced via nucleophilic attack via the primary amino group on the N- terminus of the growing peptide chain. The reactive intermediate generated by carbodiimides can result in the racemization of amino acids. To avoid racemization of the amino acids, reagents such as 1 -hydroxybenzotriazole (HOBt) are added in order to react with the O- acylisourea intermediate. Other couple agents that may be used include 2-(lH-benzotriazol-l- yl)-1.1.3.3-tetramethyluronium hexafluorophosphate (HBTU), and benzotriazol- 1-yl-oxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP), with the additional activating bases. Finally, following amino acid deprotection and coupling,

[0483] At the end of the synthesis process, removal of the protecting groups from the polypeptide must occur — a process that usually occurs through acidolysis. Determining which reagent is required for peptide cleavage is a function of the protection scheme used and overall synthesis method. For example, in some embodiments, hydrogen bromide (HBr); hydrogen fluoride (HF); or trifluoromethane sulfonic acid (TFMSA) can be used to cleave Bzl and Boc groups. Alternatively, in other embodiments, a less strong acid such as TFA can effectuate acidolysis of tBut and Fmoc groups. Finally, peptides can be purified based on the peptide’s physiochemical characteristics (e.g., charge, size, hydrophobicity, etc.). Techniques that can be used to purify peptides include Purification techniques include Reverse-phase chromatography (RPC); Size-exclusion chromatography; Partition chromatography; High- performance liquid chromatography (HPLC); and Ion exchange chromatography (IEC).

[0484] Exemplary methods of peptide synthesis can be found in Anderson G. W. and McGregor A. C. (1957) T-butyloxy carbonylamino acids and their use in peptide synthesis. Journal of the American Chemical Society. 79, 6180-3; Carpino L. A. (1957) Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1, l-disubstituted-2- arenesulfonhydrazidesl-4. Journal of the American Chemical Society. 79, 4427-31; McKayF. C. and Albertson N. F. (1957) New amine-masking groups for peptide synthesis. Journal of the American Chemical Society. 79, 4686-90; Merrifield R. B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society. 85, 2149-54; Carpino L. A. and Han G. Y. (1972) 9-fluorenylmethoxy carbonyl amino-protecting group. The Journal of Organic Chemistry. 37, 3404-9; and A Lloyd-Williams P. et al. (1997) Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press. 278; U.S. Patent Nos: 3.714,140 (filed Mar. 16, 1971); 4.411,994 (filed June 8, 1978); 7,785,832 (filed Jan. 20, 2006); 8,314,208 (filed Feb. 10, 2006); and 10,442,834 (filed Oct, 2, 2015); and United States Patent Application 2005 / 0165215 (filed Dec. 23, 2004), the disclosures of which are incorporated herein by reference in their entireties.

[0485] CELL CULTURE AND TRANSFORMATION TECHNIQUES

[0486] The terms “transformation’’ and “transfection” both describe the process of introducing exogenous and / or heterologous polynucleotide (e.g., DNA or RNA) to a host organism. Generally, those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and / or heterologous polynucleotide (e.g.. 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 polynucleotide (e.g., 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 prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).

[0487] In some embodiments, a host organism can be transformed with a polynucleotide operable to encode an OVP. In some embodiments, the host organism can be an microorganism, e.g., a cell.

[0488] In some embodiments, a vector comprising an OVP expression cassette can be cloned into an expression plasmid and transformed into a host cell. In some embodiments, the host cell can be selected from any host cell described herein.

[0489] In some embodiments, a host cell can be transformed using the following methods: electroporation; cell squeezing; microinjection; impalefection; the use of hydrostatic pressure; sonoporation; optical transfection; continuous infusion; lipofection; through the use of viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus; the chemical phosphate method; endocytosis via DEAE- dextran or polyethylenimine (PEI); protoplast fusion; hydrodynamic deliver; magnetofection; nucleoinfection; and / or others. Exemplary methods regarding transfection and / ortransformation techniques can be found in Makrides (2003), Gene Transfer and Expression in Mammalian Cells, Elvesier; Wong, TK & Neumann, E. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107, 584-587 (1982); Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER: Unit-9.3; Kim & Eberwine, Mammalian cell transfection: the present and the future. Anal Bioanal Chem. 2010 Aug; 397(8): 3173-3178, each of these references are incorporated herein by reference in their entireties.

[0490] Electroporation is an exemplary method for transforming host cells. Electroporation is a technique in which electricity is applied to cells causing the cell membrane to become permeable; this in turn allows exogenous DNA to be introduced into the cells. Electroporation is readily known to those having ordinary skill in the art, and the tools and devices required to achieve electroporation are commercially available (e.g., Gene Pulser Xcell™ Electroporation Systems, Bio-Rad®; Neon® Transfection System for Electroporation, Thermo-Fisher Scientific; and other tools and / or devices). Exemplary methods of electroporation are illustrated in Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER: Unit-9.3; Saito (2015) Electroporation Methods in Neuroscience. Springer press; Pakhomov et al., (2017) Advanced Electroporation Techniques in Biology and Medicine. Taylor & Francis; the disclosure of which is incorporated herein by reference in its entirety.

[0491] In some embodiments, electroporation can be used transform a cell with one or more vectors containing a polynucleotide operable to encode one or more OVPs or OVP- pesticidal proteins. For example, in some embodiments, electroporation can be used transform a cell with one or more vectors containing one or more OVP expression cassettes.

[0492] Exemplary description of yeast transformation and culture methods

[0493] In some embodiments, electroporation can be used transform a yeast cell with one or more vectors containing one or more OVP expression cassettes, which can produce OVP in a yeast culture with a yield of: at least 10 mg / L, at least 20 mg / L, at least 30 mg / L, at least 40 mg / L, at least 50 mg / L, at least 60 mg / L, 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 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 7,000mg / 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 least16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least40,000 mg / L, at least 50.000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least80.000 mg / L, at least 90.000 mg / L, or at least 100,000 mg / L of OVP per liter of medium.

[0494] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding an OVP into yeast, for example, in some embodiments, an OVP expression cassette cloned into a plasmid, and transformed into yeast cells via electroporation.

[0495] In some embodiments, an OVP expression cassette cloned into a plasmid, and transformed a host cell (e.g., a yeast cell) via electroporation can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) with a suitable yeast species, for example. Kluyveromyces lactis, Kluyveromyces marxlanus, Saccharomyces cerevisiae. Pichia pastoris. and others yeast species as described herein.

[0496] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding an OVP into yeast, for example, an OVP cloned into a plasmid, and transformed into K. lactis cells via electroporation. Methods of K. lactis transformation with plasmids is known in the art, and described herein.

[0497] In some embodiments, using the illustrated methods described herein, i.e., vectors of the present disclosure utilizing yeast, and methods transformation and fermentation, may result in production of OVP in amounts of: at least 10 mg / L, or at least 100,000 mg / L of OVP per liter of medium, and all values inbetween.

[0498] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding an OVP into plant protoplasts by incubating sterile plant material in a protoplast solution (e.g., around 8 mL of 10 mM 2-| / V- morpholino] ethanesulfonic acid (MES), pH 5.5; 0.01% (w / v) pectylase; 1% (w / v) macerozyme; 40 mM CaCh: and 0.4 M mannitol). Methods of introducing a vector containing a polynucleotide into plant protoplasts are known to those having ordinary skill in the art.

[0499] Host Cells and Host Organisms

[0500] The methods, compositions, OVPs, and OVP-pesticidal proteins of the present disclosure may be implemented in any host organism. For example, in some embodiments.the host organism can be a cell. In some embodiments, the cell can be, e.g., a eukaryotic or prokaryotic cell.

[0501] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein is a prokaryote. For example, in some embodiments, the host cell may be an Archaebacteria or Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli). Bacilli (e.g., B. sublilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium. Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, ox Paracoccus.

[0502] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be a unicellular cell. For example, in some embodiments, the host cell may be bacterial cells such as gram positive bacteria.

[0503] In some embodiments, the host cell may be a bacteria selected from the following genera consisting of: Candidatus Chloracidobacterium, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Mycobacterium, Propionibacterium, Streptomyces, Aquifex Bacteroides, Porphyromonas, Bacteroides, Porphyromonas, Flavobacterium, Chlamydia. Prosthecobacter, Verrucomicrobium, Chlor oflexus. Chroococcus, Merismopedia, Synechococcus, Anabaena, Nostoc, Spirulina, Trichodesmium, Pleurocapsa, Prochlorococcus, Prochloron, Bacillus, Listeria, Staphylococcus, Clostridium, Dehalobacter, Epulopiscium, Ruminococcus, Enterococcus, Lactobacillus, Streptococcus, Erysipelothrix, Mycoplasma. Leptospirillum, Nitrospira, Thermodesulfobactenum, Gemmata, Pirellula, Planctomyces, Caulobacter, Agrobacterium, Bradyrhizobium, Brucella. Methylobacterium. Prosthecomicrobium, Rhizobium, Rhodopseudomonas, Sinorhizobium, Rhodobacter, Roseobacter, Acetobacter, Rhodospirillum, Rickettsia. Rickettsia conorii, Mitochondria, Wolbachia, Erythrobacter, Erythromicrobium, Sphingomonas, Alcaligenes. Burkholderia, Leptothrix. Sphaerotilus, Thiobacillus. Neisseria, Nitrosomonas, Gallionella. Spirillum, Azoarcus, Aeromonas, Succinomonas, Succinivibrio. Ruminobacter. Nitrosococcus.Thiocapsa, Enterobacter, Escherichia, Klebsiella, Salmonella, Shigella, Wigglesworthia, Yersinia, Coxiella, Legionella, Halomonas, Pasteurella, Acinetobacter, Azotobacter, Pseudomonas, Psychrobacter, Beggiatoa, Thiomargarita. Vibrio. Xanthomonas, Bdellovibrio, Campylobacter. Helicobacter, Myxococcus, Desulfosarcina, Geobacter, Desulfur omonas, Borrelia, Leptospira, Treponema, Petrotoga, Thermotoga, Deinococcus, or Thermus.

[0504] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be selected from one of the following bacteria species: Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis , Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptomyces lividans, Streptomyces murinus, Streptomyces coelicolor, Streptomyces albicans, Streptomyces griseus, Streptomyces plicatosporus , Escherichia albertii, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia senegalensis, Escherichia vulneris. Pseudomonas abietaniphila. Pseudomonas agarici. Pseudomonas agarolyticus, Pseudomonas alcaliphila, Pseudomonas alginovora. Pseudomonas andersonii, Pseudomonas antarctica. Pseudomonas asplenii, Pseudomonas azelaica, Pseudomonas batumici, Pseudomonas borealis, Pseudomonas brassicacearum, Pseudomonas chloritidismutans, Pseudomonas cremoricolorata. Pseudomonas diterpeniphila, Pseudomonas filiscindens , Pseudomonas frederiksber gensis. Pseudomonas gingeri. Pseudomonas graminis, Pseudomonas grimontii, Pseudomonas halodenitrificans, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas hydrogenovora, Pseudomonas indica. Pseudomonas japonica, Pseudomonas jessenii, Pseudomonas kilonensis, Pseudomonas koreensis, Pseudomonas Uni, Pseudomonas lurida, Pseudomonas lutea, Pseudomonas marginata, Pseudomonas meridiana, Pseudomonas mesoacidophila. Pseudomonas pachastr ellae, Pseudomonas palleroniana, Pseudomonas parafulva, Pseudomonas pavonanceae, Pseudomonas proteolyica, Pseudomonas psychrophila, Pseudomonas psychrotolerans, Pseudomonas pudica, Pseudomonas rathonis, Pseudomonas reactans, Pseudomonas rhizosphaerae. Pseudomonas salmononii. Pseudomonas thermaerum, Pseudomonas thermocarb oxy dovorans, Pseudomonas thermotolerans, Pseudomonas thivervalensis, Pseudomonas umsongensis, Pseudomonas vancouverensis, Pseudomonas wisconsinensis, Pseudomonas xanthomarina Pseudomonas xiamenensis. Pseudomonas aeruginosa. Pseudomonas alcaligenes. Pseudomonas anguilliseptica, Pseudomonas citronellolis, Pseudomonas flavescens, Pseudomonas jinjuensis, Pseudomonas mendocina, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas straminae. Pseudomonas aurantiaca. Pseudomonas chlor or aphis. Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas taetrolens Pseudomonas azotoformans, Pseudomonas brenneri. Pseudomonas cedrina, Pseudomonas congelans, Pseudomonas corrugata, Pseudomonas costantinii, Pseudomonas extremorientalis, Pseudomonas fluorescens, Pseudomonas fulgida, Pseudomonas gessardii, Pseudomonas libanensis. Pseudomonas mandelii, Pseudomonas marginalis. Pseudomonas mediterranea. Pseudomonas migulae, Pseudomonas mucidolens,Pseudomonas orientalis, Pseudomonas poae, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas tolaasii, Pseudomonas trivialis, Pseudomonas veronii Pseudomonas denitriflcans, Pseudomonas pertucinogena, Pseudomonas fulva, Pseudomonas monteilii, Pseudomonas mosselii. Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas balearica, Pseudomonas luteola, or Pseudomonas stutzeri. Pseudomonas avellanae, Pseudomonas cannabina, Pseudomonas caricapapyae, Pseudomonas cichorii, Pseudomonas coronafaciens. Pseudomonas fuscovaginae, Pseudomonas tremae, or Pseudomonas viridiflava.

[0505] In some embodiments, the host cell used to produce a OVP or OVP -pesticidal protein can be eukaryote.

[0506] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be a cell belonging to the clades: Opisthokonta; Viridiplantae (e.g., algae and plant); Amebozoa; Cercozoa; Alveolata; Marine flagellates; Heterokonta; Discicristata; or Excavata.

[0507] In some embodiments, the procedures and methods described herein can be accomplished using a host cell that is, e.g., a Metazoan, a Choanoflagellata, or a fungi.

[0508] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a fungi. For example, in some embodiments, the host cell may be a cell belonging to the eukaryote phyla: Ascomycota, Basidiomycota, Chytridiomycota, Microsporidia, or Zygomycota.

[0509] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a fungi belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces , Cryptococcus, or Ustilago.

[0510] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a fungi belonging to one of the following species: Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces uvarum; Aspergillus flavus, A. terreus, A. awamori; Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum. and Cladosporium Cladosporioides; Magnaporthe grise. Magnaporthe oryzae. Magnaporthe rhizophila; Morchella deliciosa, Morchella esculenta, Morchella conica; Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma; Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[0511] In some embodiments, the host cell used to produce a OVP or OVP -pesticidal protein may be a fungi belonging to one of the following genera: Aspergillus. Cladosporium. Magnaporthe, Morchellct, Neurospora, Penicillium, Saccharomyces , Cryptococcus, or Ustilago.

[0512] In some embodiments, the host cell used to produce a OVP or OVP -pesticidal protein may be a member of the Saccharomycelaceae family. For example, in some embodiments, the host cell may be one of the following genera within the Saccharomycelaceae family: Brettanomyces. Candida. Citeromyces, Cyniclomyces. Debaryomyces, Issatchenkia, Kazachstania, Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora. Tetrapisispora, Vanderwaltozyma. Torulaspora, Williopsis.Zygosaccharomyces, or Zygotorulaspora.

[0513] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium herbarum, Cladosporium sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae. Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa. Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[0514] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be a species within the Candida genus. For example, the host cell may be one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila. Candida carvajalis, Candida cerambycidarum. Candida chauliodes. Candida corydalis. Candida dosseyi, Candida dubliniensis , Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candida kefyr.

[0515] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be any species within the genera Kluyveromyces .

[0516] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be a species in the genera Kluyveromyces, e.g., the host cell may be one of thefollowing: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus. Kluyveromyces nonfermentans , or Kluyveromyces wickerhamii.

[0517] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be a species within the Pichia genus. For example, the host cell may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica. or Pichia subpelliculosa.

[0518] In some embodiments, the host cell used to produce a OVP or OVP-pesticidal protein may be a species within the Saccharomyces genus. For example, the host cell may be one of the following: Saccharomyces arboricolus. Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii. Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus , Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis. Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[0519] In some embodiments, the procedures and methods described here can be accomplished using a host cell that is a Kluyveromyces lactis. Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica. Schizosaccharomyces pombe, or Hansenula anomala.

[0520] The use of yeast cells as a host organism to generate recombinant OVP is an exceptional method, well known to those having ordinary skill in the art. In some embodiments, the methods and compositions described herein can be performed with any species of yeast, including but not limited to any species of the genus Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces and the species Saccharomyces includes any species of Saccharomyces, for example Saccharomyces cerevisiae species selected from following strains: INVScl. YNN27, S150-2B. W303-1B, CG25, W3124, JRY188, BJ5464, AH22, GRF18, W303-1A and BJ3505. In some embodiments, members of the Pichia species including any species of Pichia, for example the Pichia species, Pichia pastoris, for example, the Pichia pastoris is selected from following strains: Bg08, Y-11430, X-33, GS115, GS190, JC220, JC254, GS200, JC227, JC300, JC301, JC302, JC303, JC304, JC305, JC306, JC307, JC308, YJN165, KM71,MC100-3, SMD1163, SMD1165, SMD1168, GS241, MS105, any pep4 knock-out strain and any prbl knock-out strain, as well as Pichia pastoris selected from following strains: Bg08, X-33, SMD1168 and KM71. In some embodiments, any Kluyveromyces species can be used to accomplish the methods described here, including any species of Kluyveromyces, for example, Kluyveromyces lactis, and we teach that the stain of Kluyveromyces lactis can be but is not required to be selected from following strains: GG799, YCT306, YCT284, YCT389. YCT390, YCT569, YCT598, NRRL Y-l 140. MW98-8C. MS I, CBS293.91. Y721, MD2 / 1 , PM6-7A, WM37, K6, K7, 22AR1, 22A295-1, SD1 1, MG1 / 2, MSK110, JA6, CMK5, HP101, HP 108 and PM6-3C, in addition to Kluyveromyces lactis species is selected from GG799, YCT306 and NRRL Y-l 140.

[0521] In some embodiments, the host cell used to produce a OVP or a OVP- pesticidal protein can be an Aspergillus oryzae.

[0522] In some embodiments, the host cell used to produce a OVP or a OVP- pesticidal protein can be an Aspergillus japonicas.

[0523] In some embodiments, the host cell used to produce a OVP or a OVP- pesticidal protein can be an Aspergillus niger.

[0524] In some embodiments, the host cell used to produce a OVP or a OVP- pesticidal protein can be a Bacillus licheniformis.

[0525] In some embodiments, the host cell used to produce a OVP or a OVP- pesticidal protein can be a Bacillus subtilis.

[0526] In some embodiments, the host cell used to produce a OVP or a OVP- pesticidal protein can be a Trichoderma reesei.

[0527] In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Hansenula species including any species of Hansenula and preferably Hansenula polymorpha. In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Yarrowia species for example, Yarrowia lipolytica. In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Schizosaccharomyces species including any species of Schizosaccharomyces and preferably Schizosaccharomyces pombe.

[0528] In some embodiments, the host cell used to produce a OVP or a OVP- pesticidal protein can be a species selected from: Saccharomyces cerevisiae, Pichia pastoris.Hansenula polymorpha, Yarrowia lipolytica. Arxula adeninivorans , Kluyveromyces lactis, or Schizosaccharomyces pombe.

[0529] In some illustrative embodiments, yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pas tor is. and others, can be used as a host organism. Yeast cell culture techniques are well know n to those having ordinary' skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols. Springer (1996); Bill, Recombinant Protein Production in Yeast. Springer (2012); Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press (201 ); Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture. Biosci Biotechnol Biochem. 2014; 78(6): 1090-3; Dymond. Saccharomyces cerevisiae growth media. Methods Enzymol. 2013; 533: 191-204; Looke et al., Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 2011 May; 50(5):325-8; and Romanos et al., Culture of yeast for the production of heterologous proteins. Curr Protoc Cell Biol. 2014 Sep 2; 64:20.9. 1-16, the disclosures of which are incorporated herein by reference in their entireties.

[0530] Recipes for yeast cell fermentation media and stocks are described herein.

[0531] Yeast strains

[0532] The present disclosure contemplates the creation of yeast strains operable to express an OVP or an OVP -pesticidal protein. For example, in some embodiments, a host cell can be transformed with a polynucleotide operable to encode an OVP (e.g.. by using any of the vectors described herein). In some embodiments, that host cell can be yeast strain.

[0533] In some embodiments, a yeast strain can be produced by preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a OVP or complementary nucleotide sequence thereof.

[0534] In some embodiments, a yeast strain of the present disclosure comprises, consists essentially of, or consists of: a first expression cassette comprising a polynucleotide operable to encode an OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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-S-P- T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 70); wherein the OVP comprises at least one ammo acid substitution relative to theamino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are optionally absent; and wherein X3 is A, D, E, F. G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or a complementary nucleotide sequence thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety .

[0535] In some embodiments, a yeast strain of the present disclosure comprises, consists essentially of, or consists of: a first expression cassette comprising a polynucleotide operable to encode an OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 (ID): X1-X2-S- P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 isD, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

[0536] In some embodiments, a yeast strain of the present disclosure comprises, consists essentially of, or consists of: a first expression cassette comprising a polynucleotide operable to encode an OVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is 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 ...

Claims

CLAIMS1. An Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E- N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2, wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

2. The Omega Variant Peptide (OVP) of claim 1, wherein the OVP comprises or consists of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IE): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 75); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2, wherein Xi is any amino acid and X2 is serine (S); and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

3. The Omega Variant Peptide (OVP) of claim 1, wherein the OVP comprises or consists of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IF): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 76); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi is glycine (G) and X2 is any amino acid; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

4. The Omega Variant Peptide (OVP) of claim 1, wherein the OVP comprises or consists of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IG): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N- E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 77); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S); and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety; and wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions.

5. An Omega Variant Peptide (OVP), wherein the OVP comprises or consists of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IC): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N- C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 73); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S), or wherein Xi and X2 are independently optionally absent; and wherein X is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

6. An Omega Variant Peptide (OVP), wherein the OVP comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof.

7. A composition comprising, consisting essentially of, or consisting of, one or more OVPs of any one of claims 1-6, and an agriculturally acceptable excipient.

8. A polynucleotide operable to encode an Omega Variant Peptide (OVP), said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2 and 2, wherein Xi and X2are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

9. The polynucleotide of claim 8, wherein the polynucleotide encodes an OVP comprising, consisting essentially of, or consisting of, an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or a complementary sequence thereof.

10. A vector comprising the polynucleotide of claim 9.

11. A polynucleotide operable to hybridize under stringent hybridization conditions with a polynucleotide segment encoding the OVP of any one of claims 1-6.

12. A yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an OVP, said OVP comprising or consisting of an amino acid sequence of any one of claims 1-6.

13. A yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an OVP, said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (ID): XI-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G- N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2, wherein Xi and X2are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

14. A yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an OVP, said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IA): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G- N-T-V-K-R-C-D (SEQ ID NO: 71); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is any amino acid and X2 is serine (S), or each independently are optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

15. A yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an OVP, said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IC): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G- N-T-V-K-R-C-D (SEQ ID NO: 73); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi is glycine (G) and X2 is serine (S), or each are optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or a complementary nucleotide sequence thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

16. The yeast strain of any one of claims 13-15, wherein the OVP comprises, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40.

17. The yeast strain of any one of claims 13-16, wherein the yeast strain is selected from any species belonging to the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrow ia or Schizosaccharomyces.

18. The yeast strain of claim 17, wherein the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia pastoris.

19. A method of producing an OVP, 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 OVP, or a complementary nucleotide sequence thereof, said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to (ID): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C- C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D; wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2, wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety;(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 OVP and secretion into the growth medium.

20. The method of claim 19, wherein the OVP comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof.

21. A method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the Omega Variant Peptide (OVP) as provided in any one of: Formulas (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (II), (III), an OVP as provided in any one of SEQ ID NO: 3-16, 19-35, or 38-40, or an OVP as provided in Table 1, or an agriculturally acceptable salt thereof of any of the aforementioned OVPs, or the composition of claim 7 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 issusceptible 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.

22. The method of claim 21, wherein the pest is selected from the group consisting of: Eumorpha achemon, Colias eurytheme,' Caudra cautella, Amorbia humerosana, Pseudaletia unipuncta, Platyptilia carduidactyla, Datana major., Thyridopteryx ephemeraeformis,' Hypercompe scribonia, Erionota thrax,' Acleris gloverana,' Phryganidia califomica,' Paleacrita merriccata, Grapholita packardi, Nymphula stagnata, Xylomyges curtails, Cydia pomonella, Acrobasis vaccinii', Evergestis rimosalis, Noctuid species; Agrotis ipsilorr, 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,' Elomadaula anisocentra,' Choristoneura rosaceana, Syntomeida epilais,' Playnota stultana,' Sabulodes aegrotata, Papilio cresphontes,' Argyrotaenia citrana, Grapholita molesta, Anarsia lineatella, Neophasia menapia, Argyrotaenia velutinana, Schizura concinna,' Sibine stimulea, Heterocampa guttivitta,' Estigmene acrea,' Crambus sp.; Ennomos subsignaria, Alsophila pometar ia, Choristoneura fumiferana, Lasiocampidae sp.; Theda basilides,' Ephestia elutella, Platynota idaeusalis,' Anarsia lineatella,' Peridroma saucia, Platynota flavedana, Anticarsia gemmatalis, Datana integerrima,' Hyphantria cunecr, Orgyia vetusta,' Southern Diatraea crambidoides,' Cylas formicarius, Anthonomus eugenii,' Diaprepes abbreviates,' 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 gossypielkr, Plodia interpunctella,' Plutella xylostella,'Pseudoplusia inchidens,' Spodoptera exigua, Spodopter frugiperda, Spodoptera littoralip Trichoplusia ni; and Xanthogaleruca hiteola.

23. The method of claim 22, wherein the pest is selected from the group consisting of: Aedes aegypti, Busseola fusca, Chdo suppressalis,' Culex pipiens, Culex qu inquefasciatus,' Diabrotica virgifera, Diatraea saccharalis', Helicoverpa armigera, Helicoverpa zea, Heliothis virescens,' Leptinotarsa decemlineata,' Ostrinia furnacalis,' Ostrinia mibilalis,' Pectinophora gossypiella, Plodia interpunctella, Plutella xylostella, Pseudoplusia includens, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis,' Trichoplusia ni, and Xanthogaleruca luteola.

24. An Omega Variant Peptide (OVP) having pesticidal activity against one or more insect species, said OVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to any one OVP as provided in Formulas (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (II), (III), Table 1, or any one of of SEQ ID NOs: 3-16, 19-35, or 38-40, wherein the OVP has a pesticidal activity greater than SEQ ID NO: 1 as measured via a pesticidal activity assay; with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

25. The Omega Variant Peptide (OVP) of claim 24, wherein the OVP is deglycosylated.

26. A host cell comprising the vector of claim 10.

27. The host cell of claim 26, wherein the host cell is a bacterium, a yeast cell, an insect cell, or a mammalian cell.

28. A method of combating, controlling, or inhibiting a Varroa mite comprising: applying a pesticidally-effective amount of:(1) an OVP comprising, consisting essentially of, or consisting of an OVP as provided in any one of claims 1-5, or an agriculturally acceptable salt thereof;(2) an OVP comprising, consisting essentially of, or consisting of, an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof;(3) a wild-type Omega ACTX peptide comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NOs: 2 or 69; or(4) an Omega+2 ACTX peptide comprising, consisting essentially of, or consisting of, an amino sequence as set forth in SEQ ID NO: 1; wherein the OVP, or the agriculturally acceptable salt thereof, or the wild-type Omega ACTX peptide, or the Omega+2 ACTX peptide, are applied to: the Varroa mite, a locus of the Varroa mite, a habitat of the Varroa mite or a breeding ground of the Varroa mite; a plant, a seed, a plant part, or a locus of a plant; an animal, a locus of an animal, or an environment of an animal, susceptible to an attack by the Varroa mite; or any combination thereof.

29. The method of claim 28, wherein the OVP comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 3-16, 19-35, or 38-40, or an agriculturally acceptable salt thereof.

30. The method of claim 28, wherein the wild-type Omega ACTX peptide, is a wild-type Omega-ACTX-Hvla peptide (SEQ ID NO:2) or a wild-type Omega-ACTX-Hv2a peptide (SEQ ID NO: 69).

31. The method of any one of claims 28-30, wherein the Varroa mite is any species belonging to genus: Varroa.

32. The method of claim 31, wherein the Varroa mite is Varroa destructor, Varroa jacobsoni, Varroa rindereri, or Varroa underwoodi.

33. The method of claim 32, wherein the Varroa mite is Varroa destructor.

34. The method of any one of claims 28-33, wherein the plant, the seed, the plant part, or the locus of the plant, are any plant, seed, plant part, or locus thereof that is contacted or pollinated by a beneficial insect.

35. The method of claim 34, wherein the plant, the seed, or the plant part, are selected from: Abelmoschus esculentus,' Averrhoa carambola, Actinidia deliciosa, Allium cepa, Anacardium occidentale,' Apium graveolens,' Arbutus unedo,' Bertholletia excelsa,' Beta vulgaris,' Brassica alba, Brassica hirta, Brassica nigra, Brassica napus, Brassica oleracea Botrytis Group, Brassica oleracea Capitata Group, Brassica oleracea cultivar, Brassica oleracea Gemmifera Group,' Brassica rapa, Cajanus cajan, Canavalia spp. Capsicum annuum, Capsicum frutescens, Carica papaya, Carthamus tinctorius,' Carum carvi,' Castanea sativa, Citrullus lanatus, Citrus limetta, Citrus limon,' Citrus spp., ' Citrus tangerina, Cocos nucif era, Coffea spp., ' Coriandrum sativum,' Coronilla varia L. Crataegus azarolus,' Cucumis melo L. Cucumis sativus', Cucurbita spp. , Cyamopsis tetragonoloba, Cydonia oblonga Mill. ; Daucus carota, Dimocarpus longan, Diospyros kaki, Diospyros virginiana,' Dolichos spp., ' Elettaria cardamomunr, Eriobotrya japonica, Fagopyrum esculentum, Feijoa sellowiana, Foeniculum vulgare,' Fragaria spp., ' Gossypium spp. ; Helianthus annuus,' Linum usitatissimum,' Litchi chinensis,' Lupinus angustifolius, Macadamia ternifolia, Malpighia glabra, Malus domestica or Malus sylvestris,' Mammea americana, Mangifera indica, Medicago sativa, Nepheli um lappaceum, Onobrychis spp., ' Opuntia spp. ; Passiflora edulis,' Per sea americana,' Phaseolus coccineus L. Phaseolus spp. ; Pimenta dioica, Prunus armeniaca, Prunus avium spp. Prunus cerasus,' Prunus domestica, Prunus spinosa, Prunus dulcis, Prunus amygdalus, or Amygdalus communis,' Prunus persica, Psidium guajava,' Punica granatimr, Pyrus communis,' Ribes nigrum, Ribes rubrum,' Rosa spp. Rubus fruticosus,' Rubus idaeus,' Rubus spp. ; Sambucus nigra,' Sesamum indicunr, Solanum lycopersicum, Solanum melongena, Solanum quitoense,' Solanum tuberosum, Sorbus aucuparia, Sorbus domestica,' Spondias spp. ; Tamarindus indica, Trifolium alba,' Trifolium hybridum L. Trifolium incarnatum, Trifolium pratense,' Trifolium spp. , Trifolium vesiculosum, Vaccinium oxycoccus, Vaccinium macrocar pon: Vaccinium spp. , Viciafaba, Vicia spp., ' Vigna unguiculata, Vitellaria paradoxa,' Vitis spp. or Ziziphus jujuba.

36. The method of any one of claims 28-32, wherein the animal susceptible to an attack by the Varroa mite is any species belonging to the genus: Apis.

1. The method of any one of claims 28-32, wherein the animal susceptible to an attack by the Varroa mite is a bee.

38. The method of claim 37, wherein the animal susceptible to an attack by the Varroa mite is a species selected from: Apis mellifera, Apis ceratia, Apis dorsata, Apis florea. Apis labor iosa Apis andreniformis, or Apis koschevnikovi .

39. The method of any one of claims 28-32, wherein the locus of the animal, or the environment of the animal, susceptible to an attack by the Varroa mite, is: an apiary, a natural beehive, a man-made beehive, or a nucleus box.

40. The method of any one of claims 28-39, wherein the pesticidally-effective amount of the OVP, or the wild-type Omega ACTX peptide or the homolog thereof, is formulated as a composition further comprising an agriculturally acceptable excipient.

41. The method of claim 41, wherein the composition is formulated as a powder, a dust, a pellet, a granule, a spray, an emulsion, a colloid, a solution, or combinations thereof.

42. The method of any one of claims 28-39, wherein the OVP, or the wild-type Omega ACTX peptide or the homolog thereof, is applied as a combination with a secondary pesticidal agent, wherein the secondary pesticidal agent comprises an essential oil, formic acid, or a chemical pesticide.

43. The method of claim 42, wherein the essential oil is thymol, eucalyptol, menthol, or any combination thereof.

44. The method of claim 42, wherein the chemical pesticide is a pyrethroid, amitraz, an organophosphate, or any combination thereof.

45. The method of claim 44, wherein the organophosphate is Coumaphos.

46. The method of claim 42, wherein the combination of the pesticidally-effective amount of the OVP, or the wild-type Omega ACTX peptide or the homolog thereof, and the secondary pesticidal agent, are formulated in separate compositions.

47. The method of claim 46, wherein the separate compositions are formulated as powders, dusts, pellets, granules, sprays, emulsions, colloids, solutions, or combinations thereof.

48. The method of claim 47, wherein the separate compositions are formulated using the same excipients or different excipients.

49. The method of claim 42, wherein the combination is formulated in a single composition.

50. The method of claim 49, wherein the single composition is formulated as a powder, a dust, a pellet, a granule, a spray, an emulsion, a colloid, a solution, or combinations thereof.

51. The method of claim 50, wherein the separate compositions are formulated using the same excipients or different excipients.

52. The method of claim 42, wherein the combination comprises the OVP, or the wild-type Omega ACTX peptide or the homolog thereof, integrally expressed in a plant; and the secondary pesticidal agent formulated as a composition comprising at least one excipient.

53. The method of any one of claims 28-52, wherein the OVP, or the wild-type Omega ACTX peptide or homolog thereof, is present in an amount between about 0.1 pg to about 1000 pg per 5 pL of the composition.

54. The method of claim 53, wherein the OVP, or the wild-type Omega ACTX peptide or homolog thereof, is present in in an amount of about 10 pg per 5 pL of the composition.

55. The method of any one of claims 28-54, wherein the OVP, or the wild-type Omega ACTX peptide or homolog thereof, ranges from about 0.02% to about 200% w / w of the total weight of the composition.

56. The method of claim 55, wherein the OVP, or the wild-type Omega ACTX peptide or homolog thereof, is about 2.0% w / w of the total weight of the composition.

57. The method of any one of claims 28-56, wherein the pesticidally-effective amount of the OVP, or the wild-type Omega ACTX peptide or the homolog thereof, is pesticidal to the Varroa mite, but is not pesticidal the animal susceptible to an attack by the Varroa mite.

58. An Omega Variant Peptide (OVP) having pesticidal activity, the OVP comprising, consisting essentially of, or consisting of an amino acid sequence according to Formula (ID): Xi- X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 74); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NOs: 1 and 2; wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; wherein X3 is D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof; wherein the OVP further comprises 0, 1, 2, 3, 4, or 5 conservative amino acid substitutions.

59. The OVP of claim 58, wherein:(1) Xi is any amino acid, and X2 is serine (S);(2) Xi is glycine (G) and X2 is any amino acid; or(3) Xi is glycine (G) and X2 is serine (S).

60. The Omega Variant Peptide (OVP) of any one of claims 1-6 and 58-59, wherein the OVP is a deglycosylated peptide.

61. An Omega Variant Peptide (OVP) having pesticidal activity, said OVP comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 95%, 96%,97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (I): X1-X2-S-P-T-C-I-P-X3-G-Q-P-C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C- D (SEQ ID NO: 70); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2, wherein Xi and X2 are each independently any amino acid, or each are independently optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

62. The Omega Variant Peptide (OVP) of claim 61, wherein the OVP comprises or consists of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IB): X1-X2-S-P-T-C-I-P-X3-G-Q-P- C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 72); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2, wherein Xi is glycine (G) and X2 is any amino acid, or each independently are optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

63. The Omega Variant Peptide (OVP) of claim 61, wherein the OVP comprises or consists of an amino acid sequence that is at least 90%, 95%, 96%, 97%, 97%, 98%, 99%, or 100% identical to the amino acid sequence according to Formula (IA): X1-X2-S-P-T-C-I-P-X3-G-Q-P- C-P-Y-N-E-N-C-C-S-Q-S-C-T-F-K-E-N-E-N-G-N-T-V-K-R-C-D (SEQ ID NO: 71); wherein the OVP comprises at least one amino acid substitution relative to the amino acid sequence set forth in SEQ ID NO: 1 and 2; wherein Xi is any amino acid and X2 is serine (S), or each independently are optionally absent; and wherein X3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; or an agriculturally acceptable salt thereof, with the proviso that the OVP does not comprise the amino acid sequence of SEQ ID NOs: 1 or 2 in its entirety.

64. The Omega Variant Peptide (OVP) of claim 6, wherein the OVP comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 15, 16, 19, 34, 35 and 38, or an agriculturally acceptable salt thereof.

65. The Omega Variant Peptide (OVP) of claim 6, wherein the OVP consists of an amino acid sequence according to any one of SEQ ID NOs: 15, 16, 19, 34, 35 and 38, or an agriculturally acceptable salt thereof, and wherein the OVP is deglycosylated.

66. The Omega Variant Peptide (OVP) of claim 61, wherein if Xi and X2 are each absent, then X3 is not alanine (A); or an agriculturally acceptable salt thereof.

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