METHODS FOR PRODUCING Vpb4Da2 OR Mpp75Aa1.1 RESISTANT COLONIES AND METHODS OF USE THEREOF

Abstract

The present disclosure is directed to a method for producing a Vpb4Da2 resistant insect pest species colony and / or a Mpp75Aa1.1 resistant insect pest species colony from an insect pest species isolated from an agricultural field. The present disclosure is also directed to methods for determining the inheritance of resistance to Vpb4Da2 and / or Mpp75Aa1.1, determining the allele frequency of resistance in an agricultural field, and / or mapping the locus of disease resistant genes or loci in the resistant insect pest species.

Description

PCT / US25 / 53499 31 October 2025 (31.10.2025)TITLE OF THE INVENTIONMETHODS FOR PRODUCING Vpb4Da2 OR Mpp75Aal.l RESISTANT COLONIES AND METHODS OF USE THEREOFREFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefit of United States Provisional Application No. 63 / 715,288, filed November 1, 2024, which is incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The file named “MONS:603WO_ST26.xml” containing a computer-readable form of the Sequence Listing was created on October 30, 2025, and has a fde size of 10.3 kb (measured in MS-Windows®). This Sequence Listing is filed herewith and incorporated herein by reference in its entirety.FIELD

[0003] The invention generally relates to methods of using field-derived colonies of an insect pest species to produce colonies resistant to pesticidal insect toxins, Vpb4Da2 and Mpp75Aal.l, and methods of using such resistant colonies, such as to determine the inheritance of the resistance and assess the development of resistance in an agricultural field of maize plants expressing Vpb4Da2 and / or Mpp75Aal.l.BACKGROUND

[0004] Improving crop yield from agriculturally significant plants including, among others, corn, soybean, sugarcane, rice, wheat, vegetables, and cotton, has become increasingly important. In addition to the growing need for agricultural products to feed, clothe and provide energy for a growing human population, climate-related effects and pressure from the growing population to use land other than for agricultural practices are predicted to reduce the amount of arable land available for farming. These factors have led to grim forecasts of food security, particularly in the absence of major improvements in plant biotechnology and agronomic practices. In light of these pressures, environmentally sustainable improvements in technology, agricultural techniques, andPCT / US25 / 53499 31 October 2025 (31.10.2025) pest management are vital tools to expand crop production on the limited amount of arable land available for farming.

[0005] Insect pest species, particularly insect pest species within the Coleoptera order, are considered a major cause of damage to field crops, thereby decreasing crop yields in over infested areas. For example, Coleopteran pests often associated with crop damage and a reduction of yield include, but are not limited to, Western Corn Rootworm (Diabrotica virgifera virgifera, WCR), Northern Corn Rootworm (Diabrotica barberi, NCR), Mexican Com Rootworm (Diabrotica virgifera zeae, MCR), Banded Cucumber Beetle (Diabrotica balteata, BCR), Southern Corn Rootworm (Diabrotica undecimpunctata howardii, SCR), Brazilian Corn Rootworm complex (BCR) consisting of Diabrotica viridula and Diabrotica speciosa), Crucifer Flea Beetle (Phyllotreta cruciferae), Striped Flea Beetle (Phyllotreta striolata), Western Black Flea Beetle (Phyllotreta pusilia), Maize Billbug (Sphenophorus maidis), and Southern Com Billbug (Sphenophorus callosus).

[0006] Historically, the intensive application of synthetic chemical insecticides was relied upon as the pest control agent in agriculture. Concerns for the environment and human health, in addition to emerging resistance issues, stimulated the research and development of biological pesticides. This research effort led to the progressive discovery and use of various entomopathogenic microbial species, including bacteria.

[0007] The biological control paradigm shifted when the potential of entomopathogenic bacteria, especially bacteria belonging to the genus Bacillus, was discovered and developed as a biological pest control agent. Strains of the bacterium Bacillus thuringiensis (Bt) have been used as a source for pesticidal proteins since it was discovered that Bt strains show a high toxicity against specific insect pest species. Bt strains are known to produce delta-endotoxins that are localized within parasporal crystalline inclusion bodies at the onset of sporulation and during the stationary growth phase (e.g., Cry proteins), and are also known to produce secreted insecticidal protein. Upon ingestion by a susceptible insect pest, delta-endotoxins as well as secreted toxins exert their effects at the surface of the midgut epithelium, disrupting the cell membrane, leading to cell disruption and death. Genes encoding insecticidal proteins have also been identified in bacterial species other than Bt, including other Bacillus and a diversity of additional bacterial species, such asPCT / US25 / 53499 31 October 2025 (31.10.2025)Brevibacillus laterosporus, Lysinibacillus sphaericus (^ s'' formerly known as Bacillus sphaericus) and Paenibacillus popilliae.

[0008] Three Bacillus thuringiensis- nve toxins have been used in transgenic maize for management of Western Com Rootworm: Cry3Bbl, mCry3A, and Cry34 / 35Abl. In 2009, farmers in Iowa observed severe injury to Cry3Bbl maize by larval Western Com Rootworm in the field, and subsequent laboratory assays revealed that this injury was associated with Cry3Bbl resistance. Injury to Cry3Bbl maize in the field has persisted through 2011 and expanded to include mCry3A maize. Analysis of Western Corn Rootworm collected in 2011 revealed that (i) severe injury to Cry3Bbl maize and mCry3A maize in the field was associated with resistance, and (ii) cross-resistance between Cry3Bbl and mCry3A was present (Gassmann et al. (2013) Current Issue, 111(14): 5141-5146, doi: 10.1073 / pnas.l317179111).

[0009] The pesticidal insect toxins comprised within corn event MON95275, Vpb4Da2 (United States Patent No. 10,100,330, SEQ ID NO:2, coding sequence, SEQ ID NO:3) and Mpp75Aal.l (Cry75Aal; United States Patent No. 10,662,439, SEQ ID NO:25, coding sequence, SEQ ID NO:37) provide new modes of action (MOA) against Western Com Rootworm. While resistance to Vpb4Da2 and Mpp75Aal.l has not been reported in field studies in which Vpb4Da2 and Mpp75Aal.l expressing plants are used to control an insect pest species, there is always the potential for resistance to develop to Vpb4Da2 and / or Mpp75Aal.l under the right selection pressures. As com event MON95275 becomes more widely adapted in the coming years, greater exposure of populations of insect pest species to Vpb4Da2 and Mpp75Aal.l may increase the probability of colonies of specific insect pest species developing resistance to Vpb4Da2 and / or Mpp75Aal.l. It would be of great advantage to develop under laboratory conditions a Vpb4Da2 and / or Mpp75Aal.l resistant colony / colonies of an insect species to better understand the likelihood of resistance development, the selective pressures that would permit such resistance to occur, and the mechanism of resistance.

[0010] Therefore, what is needed in the art is the development of colonies of an insect pest species (WCR) isolated and developed from agricultural fields having plants that express Vpb4Da2 and / or Mpp75Aal.l and agricultural fields that comprise plants that do not express either of the toxins.PCT / US25 / 53499 31 October 2025 (31.10.2025)SUMMARY

[0011] Disclosed herein are methods for producing a Vpb4Da2 resistant colony and a Mpp75Aal.l resistant colony, or colonies of an insect pest species. The resultant Vpb4Da2 and Mpp75Aal.l resistant colonies can be used in breeding experiments with a Vpb4Da2 and / or Mpp75Aal.l susceptible colony to map the locus or loci responsible for resistance to Vpb4Da2 or Mpp75Aal.l. The Vpb4Da2 resistant colony and the Mpp75Aal.l resistant colony, or colonies can be used to determine the inheritance, allele frequency in a field, and to discover the mechanism for resistance to Vpb4Da2 and Mpp75Aal.l. In addition, the Vpb4Da2 resistant colony and the Mpp75Aal.l resistant colony can be used to understand the potential or probability for Vpb4Da2 and Mpp75Aal.l resistance to develop in an agricultural field comprising plants expressing Vpb4Da2 and Mpp75Aal.l.

[0012] Disclosed herein are methods for producing a resistant colony of an insect pest species exhibiting increased resistance or decreased susceptibility to a pesticidal insect toxin, said method comprising: (a) collecting one or more insects of an insect pest species from an agricultural field site comprising plants that express a pesticidal insect toxin; (b) crossing one or more male or female insects collected in step (a) with females or males, respectively, of said insect pest species that are susceptible to the pesticidal insect toxin, and obtaining Fi progeny from such cross; (c) feeding said Fi progeny of step (b) on a diet lacking the pesticidal insect toxin and obtaining F2 progeny by selfing the Fi progeny of step (b) or crossing the Fi progeny of step (b) with insects susceptible to the pesticidal insect toxin; (d) feeding F2 progeny of step (c) on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin; (e) selecting insect survivors from step (d) as individuals exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin; and (f) producing the resistant colony of the insect pest species exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin relative to susceptible control insects, and descended from the insects collected in step (a), by selfing the insect survivors selected in step (e) or their progeny or descendant(s) or crossing the insect survivors selected in step (e) or their progeny or descendant(s) with insects susceptible to the pesticidal insect toxin, through one or more generations. According to present embodiments, the pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.PCT / US25 / 53499 31 October 2025 (31.10.2025)

[0013] Disclosed herein are methods for producing a resistant colony of an insect pest species exhibiting increased resistance or decreased susceptibility to a pesticidal insect toxin, said method comprising: (a) collecting one or more insects of an insect pest species from an agricultural field site comprising plants that express a pesticidal insect toxin; (b) crossing one or more male or female insects collected in step (a) with females or males, respectively, of said insect pest species that are susceptible to the pesticidal insect toxin and obtaining Fi progeny from such cross; (c) feeding said Fi progeny of step (b) on a diet lacking the pesticidal insect toxin and obtaining F2 progeny by selfing the Fi progeny of step (b) or crossing the Fi progeny of step (b) with insects susceptible to the pesticidal insect toxin; (d) feeding said F2 progeny of step (c) on a diet lacking the pesticidal insect toxin and obtaining F? progeny by selfing the F2 progeny of step (c) or crossing the F2 progeny of step (c) with insects susceptible to the pesticidal insect toxin; (e) feeding F3 progeny of step (d) on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin; (f) selecting insect survivors from step (e) as individuals exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin; and (g) producing the resistant colony of the insect pest species exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin relative to susceptible control insects, and descended from the insects collected in step (a), by selfing the insect survivors selected in step (f) or their progeny or descendant(s) or crossing the insect survivors selected in step (f) or their progeny or descendant(s) with insects susceptible to the pesticidal insect toxin, through one or more generations. According to present embodiments, the pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

[0014] According to some embodiments, methods for producing a resistant colony may further comprise: obtaining F3 progeny of F2 progeny survivors by selfing the F2 progeny or crossing F2 progeny with insects susceptible to the pesticidal insect toxin, or obtaining F4 progeny of F3 progeny survivors by selfing the F3 progeny or crossing the F3 progeny with insects susceptible to the pesticidal insect toxin; feeding said obtained F3 progeny or said F4 progeny on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin; selecting insect survivors as individuals exhibiting further increased resistance or decreased susceptibility to the pesticidal insect toxin; and obtaining a resistant colony of said insect pest species.PCT / US25 / 53499 31 October 2025 (31.10.2025)

[0015] According to some embodiments, methods for producing the resistant colony may further comprise: obtaining F3 progeny of F2 progeny survivors by selfing the F2 progeny or crossing the F2 progeny with insects susceptible to the pesticidal insect toxin, or obtaining F4 progeny of F3 progeny survivors by selfing the F3 progeny or crossing the F3 progeny with insects susceptible to the pesticidal insect toxin; feeding said F3 progeny or said F4 progeny on a diet lacking the pesticidal insect toxin and obtaining a next generation progeny of said F3 progeny or said F4 progeny by selfing said F3 progeny or said F4 progeny or crossing said F3 progeny or said F4 progeny with insects susceptible to the pesticidal insect toxin; feeding said next generation progeny on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin; selecting insect survivors as individuals exhibiting further increased resistance or decreased susceptibility to the pesticidal insect toxin; and obtaining a resistant colony of said insect pest species.

[0016] According to some embodiments, methods may further comprise: collecting one or more insects of an insect pest species from an agricultural field site comprising plants that do not express the pesticidal insect toxin; crossing one or more male or female insects collected in step (m) with females or males, respectively, of said insect pest species that exhibit susceptibility to the pesticidal insect toxin and obtaining Fi progeny from such cross; feeding said Fi progeny obtained in step (n) on a diet lacking the pesticidal insect toxin and obtaining F2 progeny by selfing the Fi progeny of step (n) or crossing the Fi progeny of step (n) with insects susceptible to the pesticidal insect toxin; feeding F2 progeny obtained in step (0) on a diet lacking the pesticidal insect toxin; selecting insect members of said insect pest species susceptible to the pesticidal insect toxin from the F2 progeny of step (p) that, when fed on a diet comprising an ordinarily effective amount or concentration of the pesticidal insect toxin, or on plants expressing an effective amount of the pesticidal insect toxin, would inhibit or result in mortality of said insect pest species members; and producing a susceptible colony of the insect pest species exhibiting susceptibility to the pesticidal insect toxin, and descended from the insects collected in step (m), by selfing the insect members selected in step (q) or their progeny or descendant(s) or crossing the insect members selected in step (q) or their progeny or descendant(s) with insects susceptible to the pesticidal insect toxin, through one or more generations. According to some embodiments, insects susceptible to the pesticidal insect toxin are insects from a susceptible colony.PCT / US25 / 53499 31 October 2025 (31.10.2025)

[0017] Disclosed herein are methods for determining the frequency of resistance among a plurality of insects of an insect pest species comprising: crossing a plurality of insect survivors, or a plurality of insects of a resistant colony, or a plurality of insects of a field-derived collection or colony of the insect pest species, with one or more individuals of said insect pest species susceptible to the pesticidal insect toxin and obtaining test progeny of said crossing; feeding said test progeny with a diet comprising an effective amount or concentration of said pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin; and analyzing the rate(s) of inhibition, mortality and / or stunting of said test progeny to determine the frequency of resistance to the pesticidal insect toxin in or among said plurality of insects or said plurality of insect survivors. According to present embodiments, said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

[0018] Disclosed herein are methods for determining the inheritance of resistance among a plurality of insects of an insect pest species comprising: crossing one or more insect survivors, or one or more insects of a resistant colony, or one or more insects of a field-derived collection or colony of the insect pest species, with one or more individuals of said insect pest species susceptible to the pesticidal insect toxin and obtaining a first progeny of said crossing; optionally backcrossing through one or more generations the first progeny with one or more individuals of said insect pest species susceptible to said pesticidal insect toxin and obtaining a descendant progeny; and determining the inheritance of resistance to the pesticidal insect toxin among the test progeny and / or the descendant progeny. According to present embodiments, said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

[0019] Disclosed herein are methods for mapping the genetic locus or identity of one or more resistance alleles of an insect pest species comprising: crossing one or more insect survivors, or one or more insects of a resistant colony, or one or more insects of a field-derived collection or colony of the insect pest species, with one or more individuals of said insect pest species susceptible to the pesticidal insect toxin and obtaining a first progeny of said crossing; optionally backcrossing through one or more generations the first progeny with one or more individuals of said insect pest species susceptible to said pesticidal insect toxin and obtaining a descendant progeny; and determining the genetic locus of one or more resistance alleles in the genome of said insect pest species among the test progeny and / or the descendant progeny. According to present embodiments, said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l. According to somePCT / US25 / 53499 31 October 2025 (31.10.2025) embodiments, a genetic locus or gene of one or more resistance alleles in the genome of said insect pest species is determined by association with one or more genetic markers and / or genetic linkage with one or more traits, phenotypes or genetic markers.

[0020] Disclosed herein are methods for producing a colony of an insect pest species exhibiting decreased susceptibility to a pesticidal insect toxin, said method comprising: (a) collecting an insect pest species from an agricultural field site comprising plants that express a pesticidal insect toxin; (b) collecting an insect pest species from an agricultural field site comprising plants devoid of the pesticidal insect toxin; (c) crossing male insects obtained from each of steps (a) and (b), separately with females of said insect pest species that exhibit susceptibility to the pesticidal insect toxin and obtaining Fi progeny from each such cross; (d) feeding said Fi progeny on a diet lacking the pesticidal insect toxin and obtaining F2 and successive generations of progeny; (e) feeding F2 and selected successive generations of progeny derived from the insects of step (a) on a diet comprising the pesticidal insect toxin sufficient to inhibit said insect pest species members that are susceptible to the pesticidal insect toxin; (f) selecting survivors from step (e) as individuals exhibiting decreased susceptibility to the pesticidal insect toxin; (g) feeding F2 and successive generations of progeny derived from insects of step (b) on a diet devoid of the pesticidal insect toxin; and (h) selecting insects susceptible to the pesticidal insect toxin from the progeny of (g) that, when fed on a diet comprising an ordinarily effective amount of the pesticidal insect toxin, or on plants expressing an ordinarily pest inhibitory effective amount of the pesticidal insect toxin, results in mortality of the insect pest species, wherein the pest inhibitory effective amount of the pesticidal insect toxin is sufficient to kill a susceptible insect pest species. According to present embodiments, the pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

[0021] In another embodiment, the method further comprises: (a) mating the pesticidal insect toxin resistant insect pest species derived from step (f) with individuals susceptible to the pesticidal insect toxin; wherein progeny are produced; (b) feeding said progeny with a diet comprising an amount of said pesticidal insect toxin that is ordinarily a pest inhibitory effective amount to a susceptible individual; (c) analyzing the mortality rates of said progeny; and (d) selecting individuals that are lest impacted by said pesticidal insect toxin. Further, the progeny of (a) can be backcrossed with an insect pest species colony exhibiting resistance to said pesticidal insect toxin.PCT / US25 / 53499 31 October 2025 (31.10.2025)

[0022] The method can be used for determining the inheritance of resistant to the pesticidal insect toxin in a field-derived colony of an insect pest species that comprises field-evolved resistance to said pesticidal insect toxin. According to some embodiments, the female insects used in any of the method steps herein are non-diapausing.

[0023] Disclosed herein are methods for determining the frequency of resistance alleles for resistance to at least one pesticidal insect toxin in populations of an insect pest species in which resistance to the pesticidal insect toxin has not evolved, the method comprising: (a) providing an insect pest species collected from a field; (b) mating virgin adults of said insect pest species from the collected insect pest species with virgin adults from the insect pest species resistant to the pesticidal insect toxin from a field-derived colony, whereby progeny larvae are produced, and wherein the resistant insect pest species comprise resistance to the pesticidal insect toxin; (c) allowing the larvae to feed on a diet comprising a pest inhibitory effective amount of the pesticidal insect toxin, or upon plants expressing a pest inhibitory effective amount of the pesticidal insect toxin, wherein the pest inhibitory effective amount of the pesticidal insect toxin is sufficient to kill the insect pest species susceptible to the pesticidal insect toxin but does not kill the insect pest species resistant to the pesticidal insect toxin; and (d) determining mortality of the larvae of step (c). wherein said pesticidal insect toxin is selected from the group consisting of: Vpb4Da2 and Mpp75Aal.l.

[0024] According to present embodiments, an insect pest species is from the order of Coleoptera. The Coleopteran insect pest species may be selected from the group consisting of: Agriotes spp., Anthonomus spp., Atomaria linearis, Cerotoma spp., Chaetocnema tibialis, Chrysomelidae, Cosmopolites spp., Curculionidae, Curculio spp., Cylas spp. Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., , Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phyllophaga spp., Phlyctinus spp., Popillia spp., Japanese beetle (Popillia japonica), Psylliodes spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp, Leptinotarsa decemlineata, Diabrotica balteata, Diabrotica barberi, Diabrotica beniensis, Diabrotica cristata, Diabrotica curvipustulata, Diabrotica dissimilis, Diabrotica elegantual, Diabrotica emorsitans, Diabrotica graminea, hispanolae, lemniscata, Diabrotica linsleyi, Diabrotica longicornis, Diabrotica milleri, Diabrotica nummularis, Diabrotica occlusa, Diabrotica porracea, Diabrotica scutellata, Diabrotica speciosa, Diabrotica tibialis, Diabrotica trifasciata, Diabrotica undecimpunctata,PCT / US25 / 53499 31 October 2025 (31.10.2025)Diabrotica virgifera, Diabrotica viridula, Diabrotica virgifera virgifera, Diabrotica barberi, Diabrotica virgifera zeae, Diabrotica balteata, Diabrotica undecimpunctata howardii, Diabrotica viridula, Diabrotica speciosa, Phyllotreta cruciferae, Phyllotreta striolata, Phyllotreta pusilia, Sphenophorus maidis, Sphenophorus callosus, Oulema melanoplus, Colaspis brunei, Carpophilus dimidiatus, Carpophilus lugubris, Glischrochilus quadrisignatus, Stenolophus lecontei, Clivina impressifrons, Myochrous denticollis, Euetheola humiis rugiceps, Cyclocephala spp. and Phyllophaga spp, Aeolus spp. Conderus spp. Limonius, Hemicrepidius spp. Agriotes spp., and Melanotus spp. According to some embodiments, said Coleoptera insect pest species is a Diabrotica or a Leptinotarsa species. According to some embodiments, a Diabrotica species is a Northern Corn Rootworm, a Southern Corn Rootworm, a Western Corn Rootworm, a Mexican Corn Rootworm, a Brazilian Corn Rootworm, or a South American Com Rootworm Complex. According to some embodiments, a Leptinotarsa species is a Colorado Potato Beetle. According to some embodiments, the insect pest species is Western Corn Rootworm (Diabrotica virgifera virgifera or WCR).

[0025] According to some embodiments, plants expressing a pesticidal insect toxin at an agricultural field site are monocot or dicot plants. According to some embodiments, plants expressing the pesticidal insect toxin at the agricultural field site are selected from the group consisting of: an alfalfa, banana, barley, bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, com, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat. According to some embodiments, the plants are com plants.

[0026] According to some embodiments, plants used in the method for producing a colony of an insect pest species exhibiting decreased susceptibility to Vpb4Da2 and / or Mpp75Aal.l is a monocot plant or dicot plant. In a further embodiment, the plant is selected from the group consisting of: an alfalfa, banana, barley, bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, com, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce,PCT / US25 / 53499 31 October 2025 (31.10.2025)Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat. In yet a further embodiment, the plant is com.

[0027] Disclosed herein are isolated resistant colonies of a Coleopteran insect pest species that is resistant to an pesticidal insect toxin produced by the methods described herein.

[0028] According to some embodiments, purified, selected and / or isolated colony of a Coleopteran insect pest species is provided that is insensitive to the pesticidal insect toxin, wherein said race of Coleopteran species is selected from the group of insect genus consisting of a Diabrotica and a Leptinotarsa, and wherein said Diabrotica is further selected from the group consisting of a Northern Corn Rootworm, a Southern Corn Rootworm, a Western Corn Rootworm, a Mexican Com Rootworm, a Brazilian Com Rootworm, and a South American Corn Rootworm Complex, and said Leptinotarsa is further characterized as a Colorado Potato Beetle, for use optionally: (a) in assessing frequency of a resistance allele in a field; (b) to map the resistance allele or alleles on the chromosome of said Coleopteran insect pest species; and (c) to develop compositions and methods for mediating or overcoming the resistance mechanism; wherein said pesticidal insect toxin is selected from the group consisting of: Vpb4Da2 and Mpp75Aal.l.DESCRIPTION OF THE SEQUENCES

[0029] SEQ ID NO: 1 is an artificial sequence encoding a Vpb4Da2 pesticidal protein designed for expression in a plant cell.

[0030] SEQ ID NO: 2 is the amino acid sequence of the pesticidal protein Vpb4Da2 encoded by SEQ ID NO: 1.

[0031] SEQ ID NO: 3 is an artificial sequence encoding a Mpp75Aal.l pesticidal protein designed for expression in a plant cell.

[0032] SEQ ID NO: 4 is the amino acid sequence of the pesticidal protein Mpp75Aal.l encoded by SEQ ID NO: 3.PCT / US25 / 53499 31 October 2025 (31.10.2025)DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows a selection process used to develop a Vpb4Da2 resistant Western Corn Rootworm colony (Diabrolica virgifera virgifera).

[0034] FIG. 2 shows a selection process used to develop a Mpp75Aal.l resistant Western Com Rootworm colony (Diabrolica virgifera virgifera).

[0035] FIG. 3 shows a graph depicting the mean percent mortality of Western Corn Rootworm in both Vpb4Da2 resistant and susceptible colonies with increasing concentrations of Vpb4Da2.

[0036] FIG. 4 shows a graph depicting the mean percent mortality of Western Corn Rootworm in both Mpp75Aal.l resistant and susceptible colonies with increasing concentrations of Mpp75Aal.l.DETAILED DESCRIPTION

[0037] The following is a description of the invention provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention.

[0038] Disclosed herein is a method for producing Vpb4Da2 and Mpp75Aal.l resistant colonies of an insect pest species. The resistant colonies are produced using colonies of an insect pest species isolated from agricultural fields comprising plants which express Vpb4Da2 or Mpp75Aal.l and agricultural fields that comprise plants that do not express Vpb4Da2 and Mpp75Aal.1. A Vpb4Da2 and / or Mpp75Aal.1 resistant colony (or colonies) can be used through breeding or crossing experiments to assess the inheritance of the Vpb4Da2 and / or Mpp75Aal.l trait(s). In addition, a Vpb4Da2 and / or Mpp75Aal.l resistant colony (or colonies) can be used to map any resistance allele(s) of gene(s) on a chromosome of an insect pest species, to assess the frequency of the resistance alleles in a field or insect population or colony, and / or to develop compositions and methods for mediating or overcoming the resistance mechanism. A Vpb4Da2 and / or Mpp75Aal.1 resistant colony (or colonies) can be used in breeding or crossing experiments with a Vpb4Da2 and / or Mpp75Aal.l susceptible colony to map the locus or loci responsible for resistance to Vpb4Da2 and / or Mpp75Aal.l. Knowledge of the gene or genes involved inPCT / US25 / 53499 31 October 2025 (31.10.2025) conferring such resistance can lead to new strategies of insect management and resistance management. In addition, the Vpb4Da2 and / or Mpp75Aal.l resistant colonies can be used to understand the potential or probability for Vpb4Da2 and / or Mpp75Aal.l resistance to develop in an agricultural field comprising plants expressing Vpb4Da2 and / or Mpp75Aal.l.

[0039] Insect pest species have the ability to adapt to insecticides and other control tactics. The evolution or development of resistance by the pests can be a threat to the success of insect resistant crops such as those expressing bacterial derived insect toxin proteins. Two strategies have been adopted to control the development of resistance to a toxin protein in the field. One is to employ a refuge of plants that do not express the insect toxin protein transgene(s). The concept underlying this strategy is that most of the rare resistant pests surviving on the toxin protein expressing plants will mate with the relatively abundant susceptible pests from a nearby refuge. If the inheritance of resistance is recessive, the progeny from such mating will die on the toxin protein expressing plants, delaying the evolution of resistance. A second strategy is to provide in the transgenic plant multiple genes with different modes of action (MOA) against an insect pest species. Two or more toxin proteins having activity against a specific target insect pest species are expressed in the transgenic plant. By providing multiple genes with different MOA, one can significantly increase the time until resistance develops, and extend the durability of the product. Insect pest species have been observed to develop resistance to certain bacterial derived insect toxin proteins (see for example Tabashnik, B., et al. (2013) Insect resistance to BT crops: lessons from the first billion acres. Nature Biotechnology 31(6): 510-521).

[0040] The two pesticidal insect toxins Vpb4Da2 and Mpp75Aal.l provide new modes of action (MOA) against Western corn rootworm (WCR). Vpb4Da2 (Protein presented as SEQ ID NO: 2) derived from a Bacillus thuringiensis strain EG6657 sourced from grain dust samples collected in the eastern and central regions of the US, controls both Cry3Bbl and Cry 34 Ab 1 / 35 Ab 1 -resistant Western corn rootworm colonies. Amino acid sequence analysis of Vpb4Da2 designates this toxin as a new beta-pore forming proteins (P-PFP) member of the Bacterial-exotoxin_B protein family (IPR035088) with Vpb4Da2 domain architecture composed of PAU (PF07691) and BinarytoxB (PF03495) protein family (Pfam) domains. Under field conditions, transgenic maize expressing Vpb4Da2 demonstrates commercial-level root protection against WCR (at or below Node- Injury Scale of 0.25) and reduces populations that are resistant to WCR-active transgenic maizePCT / US25 / 53499 31 October 2025 (31.10.2025) expressing Cry3Bbl and Cry34Abl / 35Abl (Kouadio J-L, Zheng M, Aikins M, Duda D, Duff S, Chen D, et al. (2021) Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4fold, active against western corn rootworm. PLoS ONE 16(12): e0260532. and Yin Y, Flasinski S, Moar W, Bowen D, Chay C, Milligan J, et al. (2020) A new Bacillus thuringiensis protein for Western com rootworm control. PLoS ONE 15(11): e0242791.)

[0041] Mpp75Aal.l (Protein presented as SEQ ID NO: 4) isolated from Brevibacillus laterosporus strain EG5553 is a member of the ETX_MTX2 sub-family of beta-pore forming proteins ((3-PFPs) and is composed of three distinct domains and rich in b-strands arranged in a similar conformation as the conserved structural core of the [3-PFPs from the ETX_MTX2 family. Mpp75Aal.l is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Transgenic plants expressing Mpp75Aal.l provides protection from feeding damage and showed a significant reduction in adult emergence from infested plants by both susceptible Cry3Bbl and Cry34Abl / 35Abl -resistant WCR Kouadio J-L, Duff S, Aikins M, Zheng M, Rydel T, Chen D, et al. (2021) Structural and functional characterization of Mpp75Aal.l, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm. PLoS ONE 16(10): e0258052. and Bowen etal. (2021) Cry75Aa (Mpp75Aa) Insecticidal Proteins for Controlling the Western Com Rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), Isolated from the Insect-Pathogenic Bactereium Brevibacillus laterosporus. Applied and Environmental Microbiology, 87(5) 1-17 e20507-20.)

[0042] To date, no study has documented the development of resistance to Vpb4Da2 and Mpp75Aal.l. Therefore, there is a need for a method of producing Vpb4Da2 and Mpp75Aal.l resistant colonies of an insect pest species, particularly WCR, with decreased susceptibility to Vpb4Da2 and / or Mpp75Aal.l, and / or to Vpb4Da2 and / or Mpp75Aal.l expressing plants, to better understand the potential for developing resistance to Vpb4Da2 and / or Mpp75Aal.l. Such a Vpb4Da2 resistant insect pest species and / or a Mpp75Aal.l resistant insect pest species could then be used to determine the inheritance (dominant, recessive, or partially dominant or recessive), allele frequency in a field, and mechanism by which resistance may occur or evolve. In addition,PCT / US25 / 53499 31 October 2025 (31.10.2025) the Vpb4Da2 and / or Mpp75Aal.l resistance pest species colonies can also be used to determine if there is a fitness cost to the resistance phenotypes. Fitness costs occur when fitness of insects on host plants that do not express an insecticidal component is lower for resistant insects than susceptible insects. Such fitness costs can be in the form of lower survival rates, lower copulation rates, lower fecundity, and longer developmental durations. Knowledge gained using the Vpb4Da2 and / or Mpp75Aal.l resistant insect pest species colonies can be used to develop better strategies of insect resistance management.

[0043] The present inventors disclose herein a method for developing a Vpb4Da2 and / or Mpp75Aal.l resistant insect pest species colonies from insects grown in an agricultural field in which transgenic plants expressing Vpb4Da2 and / or Mpp75Aal.1 are grown. This method can be applied to many insect pest species, particularly those belonging to the order of Coleoptera, such as WCR. Provided in the examples below is a demonstration of a method in which the inventors produce a Vpb4Da2 resistant colony of Western Com Rootworm (Diabrotica virgifera virgifera, WCR) and a Mpp75Aal.l resistant colony of Western Corn Rootworm (Diabrotica virgifera virgifera) through a series of generations in field wherein transgenic corn expressing Vpb4Da2 and / or Mpp75Aal.l was grown. The resultant Vpb4Da2 and / or Mpp75Aal.l resistant colonies are highly resistant to Vpb4Da2 and / or Mpp75Aal.l, respectively.

[0044] A normal rate of mutation in the absence of selection may provide a very small amount of the insect pest species in the field carrying an advantageous mutation that provides resistance to Vpb4Da2 and / or Mpp75Aal.l. Under high selection, wherein transgenic plants expressing Vpb4Da2 or Mpp75Aal.l are grown and fed on by the insect pest species, the frequency of the resistance allele would be expected to increase. Therefore, collection of an insect pest species to produce a Vpb4Da2 or Mpp75Aal.l resistant colony may prove successful if a sufficient number of the insect pest species is present in the field and subjected to selection using plants expressing either Vpb4Da2 or Mpp75Aal.l to develop resistance for the respective toxin.

[0045] To monitor resistance development in an agricultural field, Vpb4Da2 or Mpp75Aal.l resistant insect pest species will typically be collected from one or more agricultural fields in which the occurrence or evolution of resistance is suspected to have occurred because of an increased number of the insect pest species surviving in the presence of the expressed Vpb4Da2 or Mpp75Aal.1. The Vpb4Da2 or Mpp75Aal.1 resistant insect pest species colony can then be usedPCT / US25 / 53499 31 October 2025 (31.10.2025) in reciprocal crosses with insect pest species obtained from each agricultural field to determine the allele frequency which occurs in each agricultural field for the resistance allele. In addition, the Vpb4Da2 or Mpp75Aal.l resistant insect pest species colony can be used to build predictive models for resistance development by using such information as the heredity of resistance, the allele frequency from a sample of agricultural fields, and any fitness costs determined to be associated with the resistance phenotype of the insect pest species. These predictive models can then be used to develop more effective resistance management programs.

[0046] The larvae, nymphs, or adults of a particular insect pest species can be collected at any life stage. Eggs can also be collected from sites. The specific life stage collected will depend upon the particular insect pest species. For example, provided in the examples below is a demonstration of the method using Western Com Rootworm adults collected from a tented plot of an agricultural field having high Corn Rootworm pressure, in which corn plants expressing Vpb4Da2 or Mpp75Aal.l was expressed; and then used in subsequent matings and exposure to Vpb4Da2 or Mpp75Aal.l to select for a resistant WCR colony. A susceptible colony was also derived from a third plot of the same agricultural field in which non-transgenic isoline corn was grown and maintained on non-transgenic isoline com plants. Collection of adult beetles was necessitated since the WCR first feeds underground as larvae and then emerges as adult beetles after pupation. Those beetles emerging after feeding on Vpb4Da2 or Mpp75Aal.l expressing com would presumably be resistant, or at least a portion of the collected population of the emerged beetles would be resistant, to Vpb4Da2 or Mpp75Aal.l. Further selection in the laboratory by presenting Vpb4Da2 or Mpp75Aal.l in the diet of the WCR would further select for resistant WCR.

[0047] With respect to other insect pests, the life stage used for collection will largely depend upon the stage at which the insect feeds upon plants expressing Vpb4Da2 or Mpp75Aal.l and non- transgenic plants to obtain two separate colonies used to create the resistant and susceptible colonies, as well as the ease with which to obtain the insect pest.

[0048] As used herein, “effective amount” or “effective concentration” is the amount or concentration, either in quantity of Vpb4Da2 and / or Mpp75Aal.l or concentration of Vpb4Da2 and / or Mpp75Aal.l, that is required to cause significant stunting or mortality to the insect pest species. Concentration of a pesticidal insect toxin in a transgenic plant can be defined in terms of its amount per dry weight of plant material, sample, plant part or plant tissue of the transgenicPCT / US25 / 53499 31 October 2025 (31.10.2025) plant expressing the pesticidal insect toxin. Significant stunting is stunting that does not permit the insect pest species to advance to the next stage of development and therefore renders the insect functionally dead since it cannot reach adulthood. With respect to Vpb4Da2, a diet-overlay bioassay using a concentration of 31.25 pg / cm2was found to be sufficient to cause potent growth inhibition and high mortality against WCR larvae. Plants expressing Vpb4Da2 with a mean root expression of 45.4 pg / g dry weight was found to demonstrate a commercial level of control of WCR with an NIS of 0.18. A diet overlay bioassay using Mpp75Aal.l at a concentration of 15.2 pg / cm2was found to be sufficient to cause potent growth inhibition and high mortality against WCR larvae. Plants expressing Mpp75Aal.l at 4 to 8 pg / g dry weight in the roots was found to provide commercial level of control of WCR.

[0049] The insect pest species larvae or nymphs can be allowed to feed on Vpb4Da2 or Mpp75Aal.l for a period of time sufficient to cause significant stunting or mortality and the surviving insect pest species are selected.

[0050] It is intended that reference to a pest, particularly a pest of a crop plant, means insect pest species of crop plants, particularly those that are controlled by Vpb4Da2 and / or Mpp75Aal.l. However, reference to a pest can also include Coleopteran, Hemipteran, Dipteran, and Thysanopteran insect pest species of plants and animals, as well as nematodes and fungi when toxic agents targeting these pests are co-localized or present together with Vpb4Da2 and / or Mpp75Aal.l.

[0051] As used herein, “resistant colony” or “resistant insect pest species colony” is a colony of an insect pest species that does not demonstrate significant stunting or mortality when fed an effective amount or concentration of a pesticidal insect toxin(s), such as Vpb4Da2 or Mpp75 Aal .1 , in an insect or artificial diet containing the pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l, or from plants expressing the pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l. As used herein in reference to an insect or an insect colony, collection, progeny, etc., of an insect pest species and a pesticidal insect toxin(s), “resistant” means that the insect or the insect colony, collection, progeny, etc., (i) do / does not demonstrate significant stunting or mortality when fed an effective amount or concentration of the pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l, in an insect or artificial diet containing, or from plants expressing, the pesticidal insect toxin(s), such as Vpb4Da2 or Mpp75Aal.l, or (ii) demonstrate(s) reducedPCT / US25 / 53499 31 October 2025 (31.10.2025) stunting or mortality when fed an effective amount or concentration of the pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l, in an insect or artificial diet containing, or from plants expressing, the pesticidal insect toxin(s), such as Vpb4Da2 or Mpp75Aal.l, relative to a susceptible control insects. As used herein, “susceptible control insects” refer to insects of an insect pest species that are susceptible to a pesticidal insect toxin and can be used for comparison to define a resistant colony of the insect pest species exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin relative to the susceptible control insects.

[0052] As used herein, “susceptible colony” or “susceptible insect pest species colony” is a colony of an insect pest species that demonstrates significant stunting or mortality when fed an effective amount or concentration of a pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l, in an insect or artificial diet containing the pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l, or from plants expressing the pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l. As used herein in reference to an insect or an insect colony, collection, progeny, etc., of an insect pest species and a pesticidal insect toxin(s), “susceptible” means that the insect or the insect colony, collection, progeny, etc., demonstrate(s) significant stunting or mortality when fed an effective amount or concentration of the pesticidal insect toxin(s), such as Vpb4Da2 and / or Mpp75Aal.l, in an insect or artificial diet containing, or from plants expressing, the pesticidal insect toxin(s), such as Vpb4Da2 or Mpp75Aal.l.

[0053] As used herein, “non-diapausing” refers to insects of an insect species wherein the normal diapause does not occur. Diapause is a period of suspended or arrested development during an insect’s life cycle, and can occur during any stage of development, depending upon the insect species. Each insect species will exhibit diapause at a specific phase of development at a genetically predetermined stage of life. Insect diapause is usually triggered by environmental cues, like changes in daylight, temperature, or food availability. Therefore, environmental cues may control when diapause begins and ends. Diapause can be either obligatory or facultative. Insect species with obligatory diapause will undergo a period of arrested development at the predetermined point in their life cycle, regardless of the environmental conditions. Diapause occurs in every generation. Obligatory diapause is most often associated with insects that have one generation per year. Insects with facultative diapause undergo a period of suspended development only when conditions require it for survival. Facultative diapause is found in most insects which have two or more generations per year. Using wild type insect pest species that arePCT / US25 / 53499 31 October 2025 (31.10.2025) subject to obligatory diapause makes measuring their evolutionary response to selection pressures a very slow process. By using a non-diapausing strain of the insect pest species, breeding and assessment of many generations can be accomplished per year. With respect to insect pest species that are subject to facultative diapause, one can manipulate the environmental conditions such as temperature, light cycle, and nutrition to prevent the occurrence of diapause.

[0054] The present inventors used female WCR derived from the Waterman (WMND) colony originally obtained from the USDA laboratory (Brookings, SD). WCR undergo obligatory diapause and have one generation per year. In late summer, mated females deposit small egg clutches near the base of corn stalks, where they remain unhatched for the winter. The eggs must go through a cold period before hatching in late spring. The newly hatched larvae move down into the soil and begin feeding on secondary com roots. The larvae go through three instars and eventually start feeding on and in the primary corn roots. In mid-summer, the larvae pupate in the soil, emerge as adults in five to ten days; and begin feeding on com silks. The non-diapausing WCR females were mated with WCR males collected from an agricultural field comprising three separate plots, a first plot wherein only transgenic com plants expressing the Vpb4Da2 were grown, a second plot wherein only transgenic corn plants expressing the Mpp75Aal.l were grown, and a third plot wherein only non-transgenic isoline corn plants were grown. The WCR males from each plot were mated with non-diapausing WCR females to create non-diapausing resistant and susceptible WCR colonies. The development of the resistant colony was accelerated as a result of not having to wait for the WCR to come out of diapause.

[0055] According to some embodiments, methods disclosed herein can further involve transferring the surviving Vpb4Da2 or Mpp75Aal.l resistant insect pest species to a diet of plant not expressing Vpb4Da2 or Mpp75Aal.l or an artificial diet lacking Vpb4Da2 orMpp75Aal.l to allow the survivors to complete development. Further, the present disclosure can also comprise allowing the larvae or neonates to feed for one or more generations on plants not expressing Vpb4Da2 or Mpp75Aal.l or an artificial diet lacking Vpb4Da2 or Mpp75Aal.l. This relaxation of selection pressure may be necessary to increase the number of insects over one or more generations to offset mortality that can occur for reasons other than exposure to Vpb4Da2 or Mpp75Aal.l such as crowding effects, heavy soil water saturation, temperature fluctuations, and disease. By relaxing the selection pressure, a larger population of insects carrying the resistance allele can be produced and are available to continue selection without a complete loss of thePCT / US25 / 53499 31 October 2025 (31.10.2025) resistant colony due to effects other than ingestion of Vpb4Da2 or Mpp75Aal.l.

[0056] The present disclosure further provides a method for determining the inheritance of resistance in a field-derived colony of an insect pest species that has resistance to Vpb4Da2 or Mpp75Aal.l. This is accomplished by mating a Vpb4Da2 or Mpp75Aal.l resistant insect pest species colony from a field-derived colony of the insect pest species that is susceptible to the Vpb4Da2 or Mpp75Aal.l, preferably in reciprocal crosses, and analyzing the mortality rates of the progeny from each mating when grown in the presence of Vpb4Da2 or Mpp75Aal.l. The invention can also comprise backcrossing the progeny of an insect species from each mating to Vpb4Da2 or Mpp75Aal.l resistant insects from the resistant insect pest species colony. Such methods can be used to determine if the resistance to Vpb4Da2 or Mpp75Aal.l is dominant, semidominant, recessive, or if sex-linkage is involved, and to determine the number of resistance genes that may be involved.

[0057] In some embodiments of the present disclosure, the methods for determining the frequency of resistance alleles in a population in which resistance has not evolved comprise collecting an insect pest species from a field, mating virgin adults from the field with adults from Vpb4Da2 or Mpp75Aal.l resistant insect pest species colony, allowing the larvae or nymphs to feed on a diet comprising Vpb4Da2 or Mpp75Aal.1 at a concentration that is lethal to the susceptible insect pest species, but not lethal to the resistant insect pest species, and determining mortality. Such methods find use, for example, in the development of resistance management strategies.

[0058] Methods of the present disclosure may include, for example, using such a field-derived colony of insect pest species in methods: for understanding the mechanism of the insect resistance to Vpb4Da2 and / or Mpp75Aal.l; for evaluating cross-resistance potential of Vpb4Da2 and / or Mpp75Aal.l with any pesticidal insect toxin with activity against the insect pest species; to improve resistance monitoring strategies for the insect pest of interest in geographic locations where crop plants expressing Vpb4Da2 and / or Mpp75Aal.l have been commercialized or are planned to be commercialized; of validating assumptions used in known resistance-risk computer simulation models for crop plants expressing Vpb4Da2 and / or Mpp75Aal.l; for evaluating alternative refuge deployment strategies for crop plant, such as, for example, seed mixes or refuge- in-a-bag strategies; of investigating whether or not existing insect control tactics will affect the rate at which the insect pest species may develop resistance to transgenic crop plants expressingPCT / US25 / 53499 31 October 2025 (31.10.2025)Vpb4Da2 and / or Mpp75Aal.l under field conditions; to develop molecular marker technology to monitor for the development of resistance (change in resistant alleles’ frequency) to the insecticidal toxin in field populations of the inset pest of interest; and to provide a better understanding on the mode of action of the toxin(s) in the control of the insect pest species.

[0059] The Vpb4Da2 and / or Mpp75Aal.l resistant insect pest species colonies can be derived from insect pest species isolated from agricultural fields in which plants expressing the insect toxin(s) are grown. Insect pest species which have been shown to have negative impacts upon agricultural crops and ornamental plants, and which have the potential to be controlled using Vpb4Da2 and / or Mpp75Aal.l expression strategy, are insect pest species from the orders of Coleoptera.

[0060] Coleopteran pest species which negatively impact agriculture include, but are not limited to, Agriotes spp., Anthonomus spp., Atomaria linearis, Cerotoma spp., Chaetocnema tibialis, Chrysomelidae, Cosmopolites spp., Curculionidae, Curculio spp., Cylas spp. Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., , Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phyllophaga spp., Phlyctinus spp., Popillia spp., Japanese beetle (Popillia japonica), Psylliodes spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp, Colorado potato beetle (Leptinotarsa decemlineata), Diabrotica balteata, Diabrotica barberi, Diabrotica beniensis, Diabrotica cristata, Diabrotica curvipustulata, Diabrotica dissimilis, Diabrotica elegantual, Diabrotica emorsitans, Diabrotica graminea, hispanolae, lemniscata, Diabrotica linsleyi, Diabrotica longicomis, Diabrotica milleri, Diabrotica nummularis, Diabrotica occlusa, Diabrotica porracea, Diabrotica scutellata, Diabrotica speciosa, Diabrotica tibialis, Diabrotica trifasciata, Diabrotica undecimpunctata, Diabrotica virgifera, Diabrotica viridula, Western Corn Rootworm (Diabrotica virgifera virgifera, WCR), Northern Com Rootworm (Diabrotica barberi, NCR), Mexican Com Rootworm (Diabrotica virgifera zeae, MCR), Banded Cucumber Beetle (Diabrotica balteata, BCR), Southern Com Rootworm (Diabrotica undecimpunctata howardii, SCR), Brazilian Corn Rootworm complex (BCR) consisting of Diabrotica viridula and Diabrotica speciosa), Crucifer Flea Beetle (Phyllotreta cruciferae), Striped Flea Beetle (Phyllotreta striolata), Western Black Flea Beetle (Phyllotreta pusilia), Maize Billbug (Sphenophorus maidis), Southern Corn Billbug (Sphenophorus callosus), Cereal Leaf Beetle (Oulema melanoplus), Grape Colaspis (Colaspis brunei), Sap Beetles (Carpophilus dimidiatus, C. lugubris, and GlischrochilusPCT / US25 / 53499 31 October 2025 (31.10.2025) quadrisignatus), Seedcorn Beetles (Stenolophus lecontei and Clivina impressifrons), Southern Com Leaf Beetle (Myochrous denticollis), Sugarcane Beetle (Euetheola humiis rugiceps), White Grubs (Cyclocephala spp. and Phyllophaga spp.), and Wireworms (Aeolus spp. Conderus spp. Limonius, Hemicrepidius spp. Agriotes spp., and Melanotus spp.).

[0061] As commonly understood in the art, “crossing” as used herein means mating one or more male insects of one group, collection, population, colony, selection or progeny of insects with one or more female insects of another group, collection, population, colony, selection or progeny of insects to produce progeny insects of the cross. As commonly understood in the art, “selfing” as used herein means mating one or more male insects of one group, collection, population, colony, selection or progeny of insects with one or more female insects of the same group, collection, population, colony, selection or progeny of insects to produce progeny insects of the selfing cross. As commonly understood in the art, “backcrossing” as used herein means mating one or more male or female insects of the progeny of a cross between two different insect parents with one or more female or male insects, respectively of one of the two parents to produce progeny insects of the backcross that are more genetically similar to the parent used in the backcross. As used herein in reference to an insect pest species, “inhibit” or “inhibition” means the significant stunting or mortality of the insect pest species when fed a pesticidal insect toxin, such as Vpb4Da2 and / or Mpp75Aal.l.EXAMPLESExample 1Rearing of resistant and control Western Corn Rootworm (Diabrotica virgifera virgifera)

[0062] This example describes a rearing process used to develop Vpb4Da2 and Mpp75Aal.l resistant Western Corn Rootworm colonies (Diabrotica virgifera virgifera) as further described in Examples 2 and 3 below, respectively.

[0063] In this example, non-diapausing Western Corn Rootworm (WCR) colonies were derived from crossing field collected male WCR adults with non-diapausing female adult WCR from the Waterman (WMND) colony, which was originally obtained from the USDA laboratoryPCT / US25 / 53499 31 October 2025 (31.10.2025)(Brookings, S. Dak.). Non-diapausing WCR colony-derived eggs require no extended periods of cold storage prior to incubation to induce hatching. The entire rearing timeline in this example takes approximately sixty days to complete a full life cycle.

[0064] Eggs were collected in oviposition (OP) dishes. OP dishes were made by mixing water with milled soil (Crop Characteristics, Farmington, Minn.) until solid, but moist, in consistency. The soil mixture was aliquoted into 100 mm x 15 mm Petri dishes, and the OPs were stored at 10 °C. To collect WCR eggs, OP dishes were added to cages in which the adult WCR emerge. The OP dishes are each covered with a fluted metal lid or aluminum foil that has been folded to resemble a fan, which shields the OP dish from light but allows the female adults to easily access the OP dish and oviposit their eggs. The WCR adults usually emerge about forty-four to fortyseven days into their life cycle. The adults were fed water and a diet comprised of premixed Bio- Serv diet (Product No. F9760B, Frenchtown, NJ), com meal and honey. Water was presented in bottles with a dental wick stuck through the cap. Ten days after emergence of the first adult, the covered OP dishes were added to the cage, and the female WCR adults were allowed to oviposit eggs. The OP dishes were replaced about twice or three times per week. After collection, each OP dish was sprayed with de-ionized water, dusted with a layer of milled soil, and then sprayed an additional time to moisten the new soil. The OP dishes were wrapped in parafilm and then stored at 12 °C to prevent hatching of the eggs until needed.

[0065] After all of the OP dishes were collected from each colony, the OP dishes were incubated at 25 °C. After approximately ten to thirteen days of incubation at 25 °C, the OP dishes were placed inside two, eight-inch coffee filters. The soil was sprayed with deionized (DI) water if the soil was dry to the touch. The coffee filters and OP dishes were each placed in a clear plastic food container (~16oz / 473ml), and the lid of the container was snapped onto the container and around ten holes were punched through the lid to allow air to enter and prevent the accumulation of excess moisture.

[0066] The neonates spent their first, and part of their second, instar in a “primary box” (e.g., Newspring VERSAtainer NC-888-B 38 oz., which is about 8.5 inches long, about 6 inches wide, and about 2 inches high). Seeds of com plants that express Vpd4Da2 or Mpp75Aal.l, or non- transgenic isoline plants, were germinated with water in trays about five to seven days prior to the expected day of infest. Approximately sixty seeds were germinated in each tray. A thin layer ofPCT / US25 / 53499 31 October 2025 (31.10.2025) steam sterilized soil, approximately three quarters of an inch was spread out in the trays. A small hole was dug into the soil, and WCR neonates were placed in the holes and covered with soil. The trays were watered and stored in growth chambers set to a twelve-hour day length and 25 °C. For the control colony, the seed was washed in a bleach solution and then soaked for 24 hours prior to planting. The control colony plants were infested with CRW eggs. Water was added daily as needed.

[0067] Ten to fourteen days after the initial day of incubation of the OP dishes at 25°C, the eggs were washed from the soil and used to infest the emerging corn seedlings. Each primary box was infested with five hundred to six hundred eggs per primary box . For the control colony, the eggs were washed right out of cold storage, and then pipetted into cups of soil. The eggs were allowed to incubate in the soil for approximately ten to fourteen days and then placed on the trays. At around day twenty-one, the eggs usually hatch and the larvae will drill into the soil to feed on the com seedling roots. The larvae are immediately attracted to com roots by the emission of CO2 from the root tips and begin feeding.

[0068] Twenty-eight days from the initial egg incubation, the soil mats from the primary boxes were transferred to secondary boxes. The larvae, if alive, would be in the second instar of development at the time of transfer. Secondary boxes were made using large clear plastic boxes that measure twelve and one-half inches in length, ten and one sixteenth inches wide, and three and thirteen sixteenths inches high. Six days after infestation, more seeds were pre- germinated as described above. The following day, the seeds were drained and rinsed three times. A layer of sterile soil was put into the bottom of the box. The entire soil with seedlings, which is now a mat from the primary box due to the root mass, was transferred to the secondary box. Two primary box soil mats were moved into each secondary box. The primary boxes were tapped over the secondary box to transfer any larvae that were not within the soil mats.

[0069] Forty-three days after the initial egg incubation, the corn seedlings are cut down, leaving the roots intact in the soil. The secondary box was covered with the lid supplied with the box and loaded into emergence chambers. Adults WCR began to emerge between five to ten days afterwards.

[0070] As WCR beetles emerge, they were transferred to cages. Male WCR beetles usually emerged before females. No more than five hundred beetles should be placed in each cage afterPCT / US25 / 53499 31 October 2025 (31.10.2025) collection on the first day and no more than seven hundred beetles on the fourth day of collection. This helps maximize egg production by ensuring a good sex ratio in each cage. The total number of beetles added to a cage over time did not exceed one thousand. OP dishes were added to the cage as described above ten days after the first emergence. Food was added every first day and fourth day as described above. OP dishes are replaced every first, third, and fourth day as described above. Egg production begins to fall off after three to four weeks of OP collection. The cages are removed and frozen. Remaining live beetles are collected and stored at -80°C for use in molecular assays.Example 2 Development of a Vpb4Da2-resistant Western Corn Rootworm colony

[0071] This example describes the method used to create a Vpb4Da2 -resistant colony of WCR using high selective pressure to select for WCR beetles resistant to Vpb4Da2.

[0072] Adult WCR beetles were collected from field plots in Nebraska (NE) and Illinois (IL) experiencing high WCR pressure which exceeded the economic threshold for application of insecticides. Each plot was tented to prevent beetle escape and transgenic plants expressing the Vpb4Da2 pesticidal protein (coding sequence SEQ ID NO: 1, protein sequence SEQ ID NO: 2) were planted. Large tents (40 ft x 90 ft) and small tents (12 ft x 12 ft) were erected over the plots, depending upon the plot size. In total, approximately 3,400 and 2,800 Vpb4Da2 expressing plants were grown under tents in NE and IL, respectively. Additional plots were used to grow a non- transgenic isoline of corn. The isoline plots consisted of non-transgenic isoline com plants. Beetles were collected from the plots at least once per week. In total, 838 WCR beetles were collected from the Vpb4Da2 expressing plants.

[0073] FIG.l shows the selection process over multiple generations of WCR used to develop the Vpb4Da2 -resistant colony. In the initial mating, 41 diapausing male WCR were mated with 151 non-diapausing virgin female susceptible WCR. Eggs were collected from all cages two times per week. The offspring of this cross were fed on a diet of isoline corn roots. The Fl offspring from this cross were fed isoline com roots and beetles were sib mated to produce F2 eggs. Neonates from F2 were then selected on Vpd4Da2 expressing corn roots. After this first round of selection, the resulting F2 adults were allowed to mate and the offspring was then fed on isoline com rootsPCT / US25 / 53499 31 October 2025 (31.10.2025) to increase the number of adults for continued selection. Feeding on isoline roots, relaxed selection to assure there would be a large enough population to continue driving selection. Table 1 below shows the approximate number of eggs collected from subsequent matings and the number emerging adults with each generation on either isoline or Vpb4Da2 expressing plants, where in “EBRA” indicates “Estimate Below Emerging Adults” and “TBD” indicates “To Be Determined.” In some instances, it was difficult to estimate the number of eggs due to a number of factors such as identifying eggs within the soil from oviposition. In those cases, it was better to not disturb the eggs to prevent loss due to damage to the eggs while picking through the soil.Table 1. Number of eggs and emerging adults for each generation of Vpd4Da2-resistant WCR.PCT / US25 / 53499 31 October 2025 (31.10.2025)

[0074] As can be seen in Table 1 above, each successive generation was fed on either an isoline of com or transgenic com expressing Vpb4Da2. The resulting emerging adults from later generations were used to assess the level of resistance using increasing doses of Vpd4Da2 in diet overlay assays. The Vpd4Da2-resistant colonies will be used to assess the resistance allele frequency present in com fields, characterize the resistance mechanism, and predict the durability of the Vpd4Da2 trait under insect pressure in the com field.Example 3Development of a Mpp75Aal.l-resistant Western Corn Rootworm colony

[0075] This example describes the method used to create a Mpp75Aal.l-resistant colony of WCR using high selective pressure to select for WCR beetles resistant to Mpp75Aal.1.

[0076] Adult WCR beetles were collected from field plots in Nebraska (NE) and Illinois (IL) experiencing high WCR pressure which exceeded the economic threshold for application of insecticides. Each plot was tented to prevent beetle escape and transgenic plants expressing the Mpp75Aal.l pesticidal protein (coding sequence SEQ ID NO: 3, protein sequence SEQ ID NO: 4) were planted. In total, approximately 3,300 and 2,700 Mpp75Aal.l expressing plants were grown under tents in NE and IL, respectively.

[0077] The first attempt to produce an Mpp75Aal.l-resistant colony used an initial mating of diapausing male CRW with non-diapausing virgin female susceptible WCR as was performed similarly to the initial mating to make a Vpb4Da2-resistant colony. However, this was unsuccessful in that a generation of larvae exposed to the Mpp75Aal.l expressing com roots resulted in no emerging adults due to the high level of toxicity of Mpp75 Aal .1 to WCR. Therefore, eggs left over from the field collection were allowed to mate and feed for several generations on isoline com roots to relax the selection and increase the numbers of offspring available to select for resistance WCR.

[0078] FIG. 2 shows the selection process over multiple generations of WCR used to develop the Mpp75Aa 1.1 -resistant colony. After the first attempt to make a Mpp75Aal.1 -resistant colony was unsuccessful, a remaining collection of 12,432 field-harvested WCR eggs were allowed to hatch, and the neonates fed a diet of isoline roots. After emergence, 1,753 diapausing adult male WCR from the Mpp75Aal.l expressing com were mated with 2,145 non-diapausing virgin femalePCT / US25 / 53499 31 October 2025 (31.10.2025) susceptible WCR. The resulting eggs were harvested and after hatch, the hatched neonates were fed a diet of isoline corn roots to increase the amount of WCR subsequent selection steps. Both Fi and F2 generation WCR were fed on the isoline diet. Following these steps, the neonates were fed a diet of Mpp75Aal.l expressing corn roots. Table 1 below shows the approximate number of eggs collected from subsequent matings and the number emerging adults with each generation on either isoline or Mpp75Aal.lexpressing plants, where in “EBEA” indicates “Estimate Below Emerging Adults.” As can be seen in Table 2 below, only 10 adults emerged in the F3 generation out of an initial 138,250 eggs from the previous generation. Therefore, the F4 generation was again fed on an isoline diet to increase the number of WCR for the next rounds of selection using Mpp55Aal.l expressing plants.Table 2. Number of eggs and emerging adults for each generation of Vpd4Da2-resistant WCR.

[0079] As can be seen in Table 2 above, each successive generation was fed on either an isoline of corn or transgenic com expressing Mpp75Aal.l. The resulting emerging adults from later generations were used to assess the level of resistance using increasing doses of Mpp75Aal.l in diet overlay assays. The Mpp75Aal.1-resistant colonies will be used to assess the resistance allele frequency present in com fields, characterize the resistance mechanism, and predict the durability of the Mpp75Aal.l trait under insect pressure in the com field.PCT / US25 / 53499 31 October 2025 (31.10.2025)Example 4Effect of increasing concentrations of Vpb4Da2 on susceptible and resistant Vpb4Da2 colonies

[0080] This example describes the effect of increasing concentrations of Vpb4Da2 on the survival of Vpb4Da2 susceptible and resistant colonies using a diet overlay assay.

[0081] To evaluate the level of resistance in the Vpb4Da2 colony to Vpb4Da2, a diet overlay bioassay was performed to compare the Vpb4Da2 resistant and susceptible CRW when presented with increasing concentrations of Vpb4Da2 in an artificial diet. For each insect bioassay, the eggs of both Vpb4Da2 resistant and susceptible WCR were stored in total darkness at 10° C with 60% relative humidity until needed for the assay. To hatch the eggs, the eggs were incubated in total darkness for 13 days at 25° C with 70% relative humidity. On the 14thday, the eggs were examined to determine how many eggs had hatched. If there was sufficient hatch for a particular batch of eggs, then the dilution plates were prepared.

[0082] The artificial diet plates consisted of a 96-well plate in which a standard solid artificial diet similar to those known in the art for maintaining WCR was added to each well. Prior to use, the plates were dried in a bench top drier to remove excess moisture. A negative and positive control were also provided in each of the plates to use for comparison and to assure consistency in the analysis. To perform the assay, 20 ml of the protein sample was applied to each well containing 200 ml of artificial diet. A protein preparation of Vpb4Da2 was used to make serial dilutions which ranged from 0 to 1,800 pg / ml in concentration. Each sample used one column of 8 wells per plate for 4 plates to provide replicates for statistical accuracy. One WCR neonate was used to infest each well using a fine paint brush to transfer the neonates to the wells of the bioassay plate. The assay plates were incubated at 27° C with 70% relative humidity in complete darkness for seven days. After seven days, the plates were examined to assess mortality and survival of both the Vpb4Da2 exposed larvae and the controls. Contamination of any wells was recorded. The resulting data was analyzed using JMP ® 12 statistical software (SAS Institute, 2015).

[0083] FIG. 3 shows the mean mortality of the Vpb4Da2 resistant and susceptible WCR to increasing concentrations of Vpb4Da2. As can be seen in FIG. 3, mortality of the susceptible WCR increased until 100% mortality was observed while little to no mortality was observed in thePCT / US25 / 53499 31 October 2025 (31.10.2025)Vpb4Da2 resistant WCR. This data demonstrates that the Vpb4Da2 WCR colony is resistant to the Vpb4Da2 toxin.Example 5Effect of increasing concentrations of Mpp75Aal.l on susceptible and resistant Mpp75Aal.l colonies

[0084] This example describes the effect of increasing concentrations of Mpp75Aal.l on the survival of Mpp75Aal.l susceptible and resistant colonies using a diet overlay assay.

[0085] The diet overlay bioassay to determine the level of resistance to Mpp75Aal.l in the Mpp75Aal.l resistant WCR colony was performed in a similar manner as that described in Example 4. Serial dilutions of Mpp75Aal.l ranged from 0 to 400 pg / ml in concentration. FIG. 4 shows the mean mortality of the Mpp75Aal.l resistant and susceptible WCR to increasing concentrations of Mpp75Aal.l.

[0086] As can be seen in FIG.4, less than 20% mortality was observed in the resistant WCR colony with dilutions ranging from 0 to 100 [tg / ml, while at 100 pg / ml of Mpp75Aal.l resulted in over 84% mortality in the susceptible colony. The Mpp75Aal.l resistant colony of WCR demonstrates resistance to Mpp75Aal.l.Example 6Determination of inheritance of the Vpb4Da2 and Mpp75Aal.l resistance phenotype

[0087] This example describes the determination of inheritance of the resistance phenotype to Vpb4Da2 and Mpp75Aal.l.

[0088] WCR Fl larvae resulting from four crosses with either an Mpp75Aal- or Vpb4Da2- resistant colony and a susceptible colony (SS - Susceptible? x Susceptible?1; SR - Susceptible? x Resistant?; RS - Resistant? x Susceptible?; RR - Resistant? x Resistant?) were used to initiate concentration-response diet-overlay bioassays to assess the inheritance of Mpp75Aal.l- or Vpb4Da2-resistance, respectively. Because dominance can vary by dose (Bourguet et al., (2000) Insect Resistance and Dominance Levels. J. Econ. Entomol. 93(6): 1588-1595) it was importantPCT / US25 / 53499 31 October 2025 (31.10.2025) to estimate the dominance at the relevant field dose for each protein. The relevant dose for dominance calculations was estimated for Mpp75Aal.l and Vpb4Da2 single traited com to be 98.9% and 97.0%, respectively, using the average reduction in WCR adult emergence of the Mpp75Aal.l and Vpb4Da2 single traited com compared to the non-CRW traited com from field trials. In this study, 98.9% and 97.0% mortality in the Mpp75Aal.l- and Vpb4Da2-suspectible (SS) colonies was estimated to occur at 234.49 pg Mpp75Aal.l / mL and 857.03 pg Vpb4Da2 / mL, respectively, in the diet bioassay. Percent mortality at 234.49 pg Mpp75Aal.l / mL and 857.03 pg Vpb4Da2 / mL was then calculated for the corresponding RS, SR, and RR colonies. Dominance was estimated using DML = MLRS+ MLSR-2MLSS,) I 2(MLRR- MLSS,), wherein MLSS, MLRS, MLSR, and MLRRwere the mortality level of SS, RS, SR, and RR colonies at the field-relevant dose for each protein. DML varies between 0 and 1, wherein a DML of 0 indicates survival is recessive and a DML of 1 indicates survival is dominant. Dominance was estimated at 0.23 and 0.26 for Mpp75Aal.l and Vpb4Da2, respectively, indicating that resistance is partially recessive for both proteins.Example 7 Determination of allele frequency Vpb4Da2 and Mpp75Aal.l resistance in a field infested with WCR

[0089] This example describes the determination of the Vpb4Da2 and Mpp75Aal.l resistant allele frequency in a field infested with WCR.

[0090] To determine the resistance allele frequency, single pair mating families of WCR can be produced by crossing field collected male adult beetles with unmated females from the Vpb4Da2 or Mpp75Aal.l resistant colonies. The resulting neonates can be exposed to a diet overlay containing an insecticidally effective concentration of toxin. A second group of the resulting neonates can be reared on a diet without the toxin. The percent survival is calculated in the presence or absence of the toxin in the diet. A Bayesian statistical analysis can be performed to determine the resistant allele frequency in the field collected populations.PCT / US25 / 53499 31 October 2025 (31.10.2025)Example 8 Determination of Vpb4Da2 and Mpp75Aal.l resistance loci

[0091] This example describes the determination of the resistance loci that contribute to the resistance phenotype of WCR to the toxins Vpb4Da2 and Mpp75Aal.l.

[0092] To determine the location of potential Vpb4Da2 or Mpp75Aal.l resistance gene(s), reciprocal single parent crosses can be made between WCR resistant and susceptible beetles. The resulting progeny can then be fed on Vpb4Da2 or Mpp75Aal.l expressing plant roots and isoline plant roots. The surviving WCR can be genotyped by either whole genome sequencing or using the WCR SNP genotyping platform (Flagel LE, Swarup S, Chen M, Bauer C, Wanjugi H, et al. (2015) Genetic markers for western com rootworm resistance to Bt toxin. G3: Genes\ Genomes\ Genetics 5: 399-405). Chi-square tests can be performed on genotype counts for the survivors and plotted along the genetic map.

[0093] Having described the present disclosure in detail, it will be apparent to those skilled in the art that modifications, variations, and equivalent embodiments are possible without departing from the spirit and scope of the present disclosure as described herein and in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are illustrative and provided as non-limiting examples. Thus, specific structural and functional details disclosed herein are not to be interpreted as limiting. It should be further understood that the entire disclosure of any reference cited herein is incorporated within the disclosure of this application.

Claims

CLAIMS1. A method for producing a resistant colony of an insect pest species exhibiting increased resistance or decreased susceptibility to a pesticidal insect toxin, said method comprising:(a) collecting one or more insects of an insect pest species from an agricultural field site comprising plants that express a pesticidal insect toxin;(b) crossing one or more male or female insects collected in step (a) with females or males, respectively, of said insect pest species that are susceptible to the pesticidal insect toxin, and obtaining Fi progeny from such cross;(c) feeding said Fi progeny of step (b) on a diet lacking the pesticidal insect toxin and obtaining F2 progeny by selfing the Fi progeny of step (b) or crossing the Fi progeny of step (b) with insects susceptible to the pesticidal insect toxin;(d) feeding F2 progeny of step (c) on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin;(e) selecting insect survivors from step (d) as individuals exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin; and(f) producing the resistant colony of the insect pest species exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin relative to susceptible control insects, and descended from the insects collected in step (a), by selfing the insect survivors selected in step (e) or their progeny or descendant(s) or crossing the insect survivors selected in step (e) or their progeny or descendant(s) with insects susceptible to the pesticidal insect toxin, through one or more generations. wherein said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

2. A method for producing a resistant colony of an insect pest species exhibiting increased resistance or decreased susceptibility to a pesticidal insect toxin, said method comprising:(a) collecting one or more insects of an insect pest species from an agricultural field site comprising plants that express a pesticidal insect toxin;(b) crossing one or more male or female insects collected in step (a) with females or males, respectively, of said insect pest species that are susceptible to the pesticidal insect toxin and obtaining Fi progeny from such cross;(c) feeding said Fi progeny of step (b) on a diet lacking the pesticidal insect toxin and obtaining F2 progeny by selfing the Fi progeny of step (b) or crossing the Fi progeny of step (b) with insects susceptible to the pesticidal insect toxin;(d) feeding said F2 progeny of step (c) on a diet lacking the pesticidal insect toxin and obtaining F3 progeny by selfing the F2 progeny of step (c) or crossing the F2 progeny of step (c) with insects susceptible to the pesticidal insect toxin;(e) feeding F3 progeny of step (d) on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin;(f) selecting insect survivors from step (e) as individuals exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin; and(g) producing the resistant colony of the insect pest species exhibiting increased resistance or decreased susceptibility to the pesticidal insect toxin relative to susceptible control insects, and descended from the insects collected in step (a), by selfing the insect survivors selected in step (f) or their progeny or descendant(s) or crossing the insect survivors selected in step (f) or their progeny or descendant(s) with insects susceptible to the pesticidal insect toxin, through one or more generations. wherein said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

3. The method of claim 1 or 2, wherein step (f) of claim 1 or step (g) of claim 2 for producing the resistant colony further comprises:(h) obtaining F3 progeny of the F2 progeny survivors of step (e) of claim 1 by selfing the F2 progeny of step (d) or crossing the F2 progeny of step (d) with insectssusceptible to the pesticidal insect toxin, or F4 progeny of the F3 progeny survivors of step (f) of claim 2 by selfing the F3 progeny of step (e) or crossing the F3 progeny of step (e) with insects susceptible to the pesticidal insect toxin;(i) feeding said F3 progeny or said F4 progeny obtained in step (h) on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin; and(j) selecting insect survivors from step (j) as individuals exhibiting further increased resistance or decreased susceptibility to the pesticidal insect toxin; and(k) obtaining the resistant colony of said insect pest species.

4. The method of claim 1 or 2, wherein step (f) of claim 1 or step (g) of claim 2 for producing the resistant colony further comprises:(h) obtaining F3 progeny of the F2 progeny survivors of step (e) of claim 1 by selfing the F2 progeny of step (d) or crossing the F2 progeny of step (d) with insects susceptible to the pesticidal insect toxin, or the F4 progeny of the F progeny survivors of step (f) of claim 2 by selfing the F3 progeny of step (e) or crossing the F3 progeny of step (e) with insects susceptible to the pesticidal insect toxin;(i) feeding said F3 progeny or said F4 progeny obtained in step (h) on a diet lacking the pesticidal insect toxin and obtaining a next generation progeny of said F3 progeny or said F4 progeny by selfing said F3 progeny or said F4 progeny or crossing said F3 progeny or said F4 progeny with insects susceptible to the pesticidal insect toxin;(j) feeding said next generation progeny of step (i) on a diet comprising an effective amount or concentration of the pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin;(k) selecting insect survivors from step (j) as individuals exhibiting further increased resistance or decreased susceptibility to the pesticidal insect toxin; and(1) obtaining the resistant colony of said insect pest species.

5. The method of any one of claims 1-4, further comprising:(m) collecting one or more insects of an insect pest species from an agricultural field site comprising plants that do not express the pesticidal insect toxin;(n) crossing one or more male or female insects collected in step (m) with females or males, respectively, of said insect pest species that exhibit susceptibility to the pesticidal insect toxin and obtaining Fi progeny from such cross;(o) feeding said Fi progeny obtained in step (n) on a diet lacking the pesticidal insect toxin and obtaining F2 progeny by selfing the Fi progeny of step (n) or crossing the Fi progeny of step (n) with insects susceptible to the pesticidal insect toxin;(p) feeding F2 progeny obtained in step (0) on a diet lacking the pesticidal insect toxin;(q) selecting insect members of said insect pest species susceptible to the pesticidal insect toxin from the F2 progeny of step (p) that, when fed on a diet comprising an ordinarily effective amount or concentration of the pesticidal insect toxin, or on plants expressing an effective amount of the pesticidal insect toxin, would inhibit or result in mortality of said insect pest species members; and(r) producing a susceptible colony of the insect pest species exhibiting susceptibility to the pesticidal insect toxin, and descended from the insects collected in step (m), by selfing the insect members selected in step (q) or their progeny or descendant(s) or crossing the insect members selected in step (q) or their progeny or descendant(s) with insects susceptible to the pesticidal insect toxin, through one or more generations.

6. The method of claim 5, wherein the insects susceptible to the pesticidal insect toxin are insects from the susceptible colony of claim 5.

7. A method of determining the frequency of resistance among a plurality of insects of an insect pest species comprising:(a) crossing a plurality of insect survivors of step (e) of claim 1 or step (f) of claim (2), or a plurality of insects of the resistant colony of any one of claims 1-6, or a plurality of insects of a field-derived collection or colony of the insect pest species, with one or more individuals of said insect pest species susceptible to the pesticidal insect toxin and obtaining test progeny of said crossing;(b) feeding said test progeny with a diet comprising an effective amount or concentration of said pesticidal insect toxin sufficient to inhibit members of said insect pest species that are susceptible to the pesticidal insect toxin; and(c) analyzing the rate(s) of inhibition, mortality and / or stunting of said test progeny to determine the frequency of resistance to the pesticidal insect toxin in or among said plurality of insects or said plurality of insect survivors, wherein said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

8. A method of determining the inheritance of resistance among insects of an insect pest species comprising:(a) crossing one or more insect survivors of step (e) of claim 1 or step (f) of claim (2), or one or more insects of the resistant colony of any one of claims 1-6, or one or more insects of a field-derived collection or colony of the insect pest species, with one or more individuals of said insect pest species susceptible to the pesticidal insect toxin and obtaining a first progeny of said crossing;(b) optionally backcrossing through one or more generations the first progeny of (a) with one or more individuals of said insect pest species susceptible to said pesticidal insect toxin and obtaining a descendant progeny; and(c) determining the inheritance of resistance to the pesticidal insect toxin among the test progeny and / or the descendant progeny, wherein said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

9. The method of any one of claims 1-6, wherein the one or more females of any one of the crossing steps are non-diapausing.

10. A method for mapping the genetic locus or identity of one or more resistance alleles of an insect pest species comprising:(a) crossing one or more insect survivors of step (e) of claim 1 or step (f) of claim (2), or one or more insects of the resistant colony of any one of claims 1-6, or one or more insects of a field-derived collection or colony of the insect pest species, with one or more individuals of said insect pest species susceptible to the pesticidal insect toxin and obtaining a first progeny of said crossing;(b) optionally backcrossing through one or more generations the first progeny of (a) with one or more individuals of said insect pest species susceptible to said pesticidal insect toxin and obtaining a descendant progeny; and(c) determining the genetic locus of one or more resistance alleles in the genome of said insect pest species among the test progeny and / or the descendant progeny, wherein said pesticidal insect toxin is Vpb4Da2 or Mpp75Aal.l.

11. The method of claim 10, wherein the genetic locus of the one or more resistance alleles in the genome of said insect pest species is determined by association with one or more genetic markers or genetic linkage with one or more traits, phenotypes or genetic markers.

12. The method of any one of claims 1-11, wherein the insect pest species is in the order of Coleoptera.

13. The method of claim 12, wherein said Coleoptera insect pest species is selected from the group consisting of: Agriotes spp., Anthonomus spp., Atomaria linearis, Cerotoma spp., Chaetocnema tibialis, Chrysomelidae, Cosmopolites spp., Curculionidae, Curculio spp., Cylas spp. Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., , Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phyllophaga spp., Phlyctinus spp., Popillia spp., Japanese beetle (Popillia japonica), Psylliodes spp., Rhizopertha spp., Scarabeidae. Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp, Eeptinotarsa decemlineata, Diabrotica balteata, Diabrotica barberi, Diabrotica beniensis, Diabrotica cristata, Diabrotica curvipustulata, Diabroticadissimilis, Diabrotica elegantual, Diabrotica emorsitans, Diabrotica graminea, hispanolae, lemniscata, Diabrotica linsleyi, Diabrotica longicomis, Diabrotica milleri, Diabrotica nummularis, Diabrotica occlusa, Diabrotica porracea, Diabrotica scutellata, Diabrotica speciosa, Diabrotica tibialis, Diabrotica trifasciata, Diabrotica undecimpunctata, Diabrotica virgifera, Diabrotica viridula, Diabrotica virgifera virgifera, Diabrotica barberi, Diabrotica virgifera zeae, Diabrotica balteata, Diabrotica undecimpunctata howardii, Diabrotica viridula, Diabrotica speciosa, Phyllotreta cruciferae, Phyllotreta striolata, Phyllotreta pusilia, Sphenophorus maidis, Sphenophorus callosus, Oulema melanoplus, Colaspis brunei. Carpophilus dimidiatus, Carpophilus lugubris, Glischrochilus quadrisignatus, Stenolophus lecontei, Clivina impressifrons, Myochrous denticollis, Euetheola humiis rugiceps, Cyclocephala spp. and Phyllophaga spp, Aeolus spp. Conderus spp. Limonius, Hemicrepidius spp. Agriotes spp., and Melanotus spp.

14. The method of claim 12 or 13, wherein said Coleoptera insect pest species is a Diabrotica or a Leptinotarsa species.

15. The method of claim 14, wherein the Diabrotica species is a Northern Corn Rootworm, a Southern Corn Rootworm, a Western Com Rootworm, a Mexican Com Rootworm, a Brazilian Corn Rootworm, or a South American Com Rootworm Complex.

16. The method of claim 14, wherein the Leptinotarsa species is a Colorado Potato Beetle.

17. The method of claim 12, 13 or 14, wherein the insect pest species is Western Corn Rootworm (Diabrotica virgifera virgifera or WCR).

18. The method of any one of claims 1-6, wherein the plants expressing the pesticidal insect toxin at the agricultural field site are monocot or dicot plants.

19. The method of claim 18, wherein the plants expressing the pesticidal insect toxin at the agricultural field site are selected from the group consisting of: an alfalfa, banana, barley,bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, com, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye. safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat.

20. The method of claim 18 or 19, wherein the plants are corn plants.

21. An isolated resistant colony of a Coleopteran insect pest species that is resistant to an pesticidal insect toxin produced by the method of any one of claims 1-6.

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