WO2024201384A1

WO2024201384A1 - Recombinant cry protein for controlling insect pests

Info

Publication number: WO2024201384A1
Application number: PCT/IB2024/053053
Authority: WO
Inventors: Keini DRESSANO; Paulo Henrique de Oliveira CECILIATO; Antonio Adalberto Kaupert NETO; Alinne Lozano SAONCELLA
Original assignee: Centro De Tecnologia Canavieira SA
Current assignee: Centro De Tecnologia Canavieira SA
Priority date: 2023-03-31
Filing date: 2024-03-28
Publication date: 2024-10-03
Anticipated expiration: 2025-09-30
Also published as: AU2024245952A1; AR132289A1

Abstract

The present disclosure relates to recombinant and modified Cry proteins for use in methods and compositions for controlling insect pests, in particular Coleopteran pests, and more particularly, the Sphenophorus levis pest of the crop plant sugarcane. More specifically, the disclosure relates to recombinant and modified Cry proteins based on and derived from the Cry protein designated SCW123. The present disclosure further relates to the production of pesticidal compositions using these proteins. In addition, the present disclosure relates to methods for controlling insect pests, more specifically Coleopteran pests such as S. levis, using this protein.

Description

RECOMBINANT CRY PROTEIN FOR CONTROLLING INSECT PESTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/493,458, filed March 31, 2023, hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (207422000840seqlist.xml; Size: 10,494 bytes; and Date of Creation: March 26, 2024) is herein incorporated by reference in its entirety.

TECHNICAL FIELD

[0003] The present disclosure relates to recombinant and modified Cry proteins for use in methods and compositions for controlling insect pests, in particular Coleopteran pests, and more particularly, the Sphenophorus levis pest of the crop plant sugarcane. More specifically, the disclosure relates to recombinant and modified Cry proteins based on and derived from the Cry protein designated SCW123. The present disclosure further relates to the production of pesticidal compositions using these proteins. In addition, the present disclosure relates to methods for controlling insect pests, more specifically Coleopteran pests such as S. levis, using this protein.

BACKGROUND

[0004] Invertebrate pests cause hundreds of billions of dollars of damage yearly to the global economy, consuming and destroying cultivated crop plants that are critical for the health and sustenance of human populations. Alongside these monetary consequences, the damage caused by invertebrate pests wastes a substantial portion of the potential crop food.

[0005] One important crop plant is sugarcane (genus Saccharum), which is used both in the production of many foods as well as in the production of ethanol. Sugarcane is a vegetatively propagated crop in the same family as maize, rice, and wheat, and it is cultivated as the world’s largest source of sugar. Alongside culinary importance, sugarcane provides a source of biofuel in the form of ethanol, which has a global market of about 50 billion dollars. Pests of sugarcane include boring pests (e.g., sugarcane borer), sucking pests (e.g., whiteflies), soil pests (e.g., termites), and others. The most important sugarcane pests are from the orders Lepidoptera, Coleoptera, Hemiptera, Hymenoptera and Isoptera; examples of pests from each order are the sugarcane borer, boll weevil, red-leafhopper, leaf-cutting ant, and termite, respectively. One particularly damaging insect pest is the sugarcane weevil or sugarcane billbug (Sphenophorus levis), which is a Coleopteran pest in the family Curculionidae. S. levis damages sugarcane plants through an initial boring process into stem tissue of the plant and laying of eggs, followed by intense feeding damage from larvae that hatch from those eggs. This destruction can lead to 60% loss of young stems, and an overall crop loss of 30%.

[0006] To combat insects affecting sugarcane, the most common solutions are treatment with chemical mixtures that contain at least one insecticidal and/or nematocidal component, or biological insecticides. The chemical mixtures work by inhibiting various stages of the insect’s life cycle, impacting insect behaviors necessary for survival, or directly causing lethality. However, these chemicals often affect much broader ranges of taxa than the target pest, and this can harm the crop’s beneficial symbiotic organisms, which can result in reduced yields. Additional concerns surrounding these chemical mixtures include potential environmental buildup of the mixtures’ toxic compounds in the environment surrounding the crop. Further, chemical insecticides may not be effective to control insect pests on sugarcane due to the feeding behavior of the larva in the stalk, which prevents the insecticide from effectively contacting the insect. Some insect pests, such as S. levis, feed at the base of the stalk. This feeding behavior increases the challenge of applying chemicals over large areas, as effective application would be terrestrial application instead of aerial application. For biological insecticides, there are a few options, including parasitoid wasps (e.g., Cotesia flavipes, Trichogramma galloi, and Tetrastichus howardil). entomopathogenic fungi (e.g., Metarhizium anisopliae and Beauveria bassiand) and bacteria (e.g., Bacillus ihuringiensis). In comparison to chemical mixtures, biological insecticides have a high cost. Moreover, biological insecticides have stringent requirements for the method of application, and there are limited conditions under which application will be effective. In order to obtain some efficacy of treatment, often more than one approach is required to control the insect pest.

[0007] The use of heterologous Bacillus thuringiensis (Bt) proteins (e.g., 6-endotoxin proteins) presents an alternative to chemical mixtures and biological insecticides, as these proteins offer more accurate targeting of pests, higher rates of degradation in the surrounding soil, and lower levels of bioaccumulation than many chemical insecticidal mixtures. Additionally, these proteins allow alternative delivery approaches, including through compositions and/or transgenic plants. These active 6-endotoxin proteins are toxic to the targeted insect pest by inducing selective stomach poisoning in the insect gut. The proteins attach to the interior of the insect gut and cause the gut’s cell membranes to deteriorate, ultimately creating holes in the gut that kill the insect. In contrast to the insect gut, a human gut is able to quickly and harmlessly breaks down any Bt proteins that contact it.

[0008] Many Bt proteins are species-selective, and so their applicability is limited to specific pest species. While the species- selectivity of Bt proteins makes them safer for human consumption than some chemical insecticides, this also means that targeted Bt proteins are needed for addressing specific pests. Extensive research is therefore required in identifying specific Bt proteins that are effective against specific pests of interest. Additionally, there are multiple instances of insects growing resistant to specific Bt proteins. This is especially true for those Bt proteins that have been consistently applied over many generations of crops.

[0009] There exists a clear need for identifying novel and useful proteins that are toxic to the insecticidal pests of plants, especially sugarcane pests, and more particularly, the sugarcane weevil or sugarcane billbug, Sphenophorus levis. This damaging pest has not yet been successfully targeted by Bt proteins, but the advantages of Bt proteins over chemical mixtures make it an ideal tool for controlling this pest.

BRIEF SUMMARY

[0010] In order to meet these needs, the present disclosure provides methods of using an isolated protein designated SCW123, as well as recombinant, modified, truncated, and/or mutated forms of SCW123, for controlling insect pests, specifically Coleopteran pests. The present disclosure further provides pesticidal compositions including these proteins, as well as expression cassettes encoding these proteins, and plants including them. In particular, the present disclosure relates to the use of isolated, recombinant, modified, truncated, and/or mutated SCW123 proteins that are toxic to the Coleopteran pest Sphenophorus levis.

[0011] In some aspects, the present disclosure relates to a recombinant polypeptide including at least one amino acid substitution, deletion, and/or insertion compared to SEQ ID NO: 1; at least one addition at the N-terminus or C-terminus compared to SEQ ID NO: 1; at least one domain swap compared to SEQ ID NO: 1; at least one truncation compared to SEQ ID NO: 1; and/or at least one other alteration compared to SEQ ID NO: 1. In some embodiments of this aspect, the recombinant polypeptide is a fusion protein including at least a portion of the polypeptide of claim 1, and/or variants or fragments thereof. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the recombinant polypeptide has insecticidal activity against at least one agricultural insect pest. In some embodiments of this aspect, the at least one insect pest is a Coleopteran pest. In some embodiments of this aspect, the Coleopteran pest is selected from the group consisting of Sphenophorus levis, Sphenophorus maidis, Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus , and Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis.

[0012] Some aspects of the disclosure relate to a recombinant polynucleotide encoding the polypeptide of any of the preceding embodiments. In some embodiments of this aspect, the recombinant polynucleotide includes the sequence of SEQ ID NO: 2. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the polynucleotide has codons optimized for expression in an agriculturally important crop. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the polynucleotide is a non-genomic polynucleotide. In some embodiments of this aspect, the polynucleotide is a synthetic polynucleotide, and/or the polynucleotide is a cDNA.

[0013] Some aspects of the disclosure relate to an isolated construct or expression cassette including a nucleotide encoding the polypeptide of any one of the preceding embodiments or the polynucleotide of any one of the preceding embodiments, wherein the nucleotide or the polynucleotide is operably linked to a promoter, and optionally operably linked to a heterologous regulatory element. In some embodiments of this aspect, the polypeptide includes SEQ ID NO: 1. In some embodiments of this aspect, the polynucleotide includes SEQ ID NO: 2. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the promoter is selected from the group of a constitutive promoter, an inducible promoter, and a tissue- specific promoter.

[0014] In some aspects, the present disclosure relates to a transgenic plant, plant part, propagule, seed, tissue, organ, embryo, or plant cell including the polypeptide of any one of the preceding embodiments, the polynucleotide of any one of the preceding embodiments, or the isolated construct or expression cassette of any one of the preceding embodiments. In some embodiments of this aspect, the polypeptide comprises the sequence of SEQ ID NO: 1. In some embodiments of this aspect, the polynucleotide comprises the sequence of SEQ ID NO: 2. [0015] A further aspect of the disclosure relates to a pesticidal composition including: (i) one or more polypeptides of any one of the preceding embodiments, wherein the one or more polypeptides are present at a concentration sufficient to control at least one agricultural insect pest; (ii) one or more polynucleotides of any one of the preceding embodiments, wherein the polynucleotide has codons optimized for expression in an agriculturally important crop; and/or (iii) one or more isolated constructs or expression cassettes of any one of the preceding embodiments. In some embodiments of this aspect, at least one of the one or more polypeptides includes the sequence of SEQ ID NO: 1. In some embodiments of this aspect, at least one of the one or more polynucleotides includes the sequence of SEQ ID NO: 2. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the at least one agricultural insect pest is a Coleopteran pest. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the one or more polypeptides are present at a concentration sufficient to control at least one agricultural insect pest in or on a sugarcane plant when the composition is applied to the sugarcane plant or to a sugarcane plantation. Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include one or more inert ingredients, acceptable carriers, surfactants, or adjuvants customarily employed in the art of formulation, or other components to facilitate product handling and application for particular target pests. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition further includes one or more inert ingredients and/or acceptable carriers. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste (e.g., for coating or spreading), a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier (e.g., a polymer substance). In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as an orally acceptable, orally administrable, or orally ingestible diet intended for consumption by the insect pest. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated for direct soil application and/or direct plant pot substrate application. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a controlled release formulation. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, control of the Coleopteran pest includes: (a) decreasing pest infestation by 40%, 50%, 60%, 70%, 80%, 90%, or 100%; or (b) increasing pest mortality by 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the Coleopteran pest is selected from the group consisting of Sphenophorus levis, Sphenophorus maidis, Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus , and Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis. Such formulated compositions may be prepared by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide.

[0016] An additional aspect of the disclosure relates to methods for controlling an insect pest population, including: (a) providing a composition including at least one polypeptide of any one of the preceding embodiments or providing the composition of any one of the preceding embodiments; and (b) contacting an insect pest population with an effective amount of the composition. In some embodiments of this aspect, the at least one polypeptide is SEQ ID NO: 1. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the contacting of step (b) includes one or more of: providing the pest with the composition formulated as an insect diet; feeding the composition to the pest; applying the composition to the exterior surface of the pest; applying the composition to a plant; applying the composition to a part of a plant where the pest feeds; applying the composition to a soil area where the pest may be present; applying the composition to an area where the pest population may be present; providing the composition formulated as a controlled release formulation to an area where the pest is expected to be; applying the composition to a trap for the insect pest; injecting the composition into a plant; or injecting the composition into the pest. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste, a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the pest is contacted with an effective amount of the composition by feeding, spraying, dusting, coating, or wetting with the composition, or any combination thereof. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the contacting of step (b) includes applying the composition to a plant or an area to be planted. In some embodiments of this aspect, the composition is applied to the plant as at least one of a foliar treatment, a seed coating, an injection treatment, a preemergence treatment, and/or a post-emergence treatment. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is prepared through desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, cryopreservation, or concentration. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest population is decreased by 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to an insect pest population not contacted with the composition. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest or insect pest population is resistant to at least one Bt toxin. Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include providing a chemical mixture, a pesticidal protein, and/or a biological control agent, and contacting the insect pest population with an effective amount of the chemical mixture, the pesticidal protein, and/or the biological control agent before step (b), in step (b), or after step (b). In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest is a Coleopteran pest. In some embodiments of this aspect, the Coleopteran pest is selected from Sphenophorus levis, Sphenophorus maidis, Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus, and Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis.

[0017] Yet another aspect of the disclosure relates to methods of controlling an insect pest population, including: (a) providing a pesticidal composition including SEQ ID NO: 1; (b) introducing the pesticidal composition to the insect pest population, wherein introducing is through providing the composition in or on a food source for the insect pest; and wherein the insect pest population is decreased. In some embodiments of this aspect, the introducing of step (b) includes one or more of: providing the pest with the composition formulated as an insect diet; feeding the composition to the pest; applying the composition to a plant; applying the composition to a part of a plant where the pest feeds; applying the composition to a trap for the insect pest; or injecting the composition into a plant. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste, a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is applied to the plant as at least one of a foliar treatment, a seed coating, an injection treatment, a preemergence treatment, and/or a post-emergence treatment. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is prepared through desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, cryopreservation, or concentration. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest population is decreased by 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to an insect pest population not contacted with the composition. Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include providing a chemical mixture, a pesticidal protein, and/or a biological control agent, and contacting the insect pest population with an effective amount of the chemical mixture, the pesticidal protein, and/or the biological control agent before step (b), in step (b), or after step (b). In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest or insect pest population is resistant to at least one Bt toxin. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest is a Coleopteran pest. In some embodiments of this aspect, the Coleopteran pest is selected from the group consisting of Sphenophorus levis, Sphenophorus maidis , Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus , and Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis.

[0018] Further aspects of the present disclosure relate to use of the polypeptide of any one of the preceding embodiments to inhibit growth of an insect, control or kill an insect, and/or control or kill an insect population. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows results of protein toxicity bioassay screening against sugarcane weevil (or sugarcane billbug, S. levis) larvae. The vertical axis displays the relative corrected mortality rate percentage from each of the tested proteins, which are listed along the horizontal axis. The dark grey bars provide the mortality rate percentages for proteins of the present disclosure; from left to right: SCW123 (SEQ ID NO: 1) and SCW19 (SEQ ID NO: 3). Additional experimental data related to these results are presented in Table 1.

DETAILED DESCRIPTION

[0020] The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

Methods of controlling insect pests

[0021] An aspect of the disclosure relates to methods for controlling an insect pest population, including: (a) providing a composition including at least one polypeptide of any one of the preceding embodiments or providing the composition of any one of the preceding embodiments; and (b) contacting an insect pest population with an effective amount of the composition. In some embodiments of this aspect, the at least one polypeptide is SEQ ID NO: 1, and/or variants or fragments thereof. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the contacting of step (b) includes one or more of: providing the pest with the composition formulated as an insect diet; feeding the composition to the pest; applying the composition to the exterior surface of the pest; applying the composition to a plant; applying the composition to a part of a plant where the pest feeds (e.g., the stalk of sugarcane); applying the composition to a soil area where the pest may be present; applying the composition to an area where the pest population may be present; providing the composition formulated as a controlled release formulation to an area where the pest is expected to be; applying the composition to a trap for the insect pest; injecting the composition into a plant; or injecting the composition into the pest. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste (e.g., for coating or spreading), a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the pest is contacted with an effective amount of the composition by feeding, spraying, dusting, coating, or wetting with the composition, or any combination thereof. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the contacting of step (b) includes applying the composition to a plant or an area to be planted. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is applied to the plant as at least one of a foliar treatment, a seed coating, an injection treatment, a pre-emergence treatment, and/or a postemergence treatment. The method of plant treatment may vary based on factors known in the art, such as the timing of planting, the resilience of various plant parts or plant organs, the timing of the insect pest emergence, the extent and location of the insect pest infestation, and/or effective amount of the composition included in the treatment. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is prepared through desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, cryopreservation, or concentration. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest population is decreased by 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to an insect pest population not contacted with the composition. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest or insect pest population is resistant to at least one Bt toxin. Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include providing a chemical mixture, a pesticidal protein (e.g., a Bt protein), and/or a biological control agent, and contacting the insect pest population with an effective amount of the chemical mixture, the pesticidal protein, and/or the biological control agent before step (b), in step (b), or after step (b). In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest is a Coleopteran pest. In some embodiments of this aspect, the Coleopteran pest is selected from Sphenophorus levis, Sphenophorus maidis, Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus, and Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis. [0022] A further aspect of the disclosure relates to methods of controlling an insect pest population, including: (a) providing a pesticidal composition including SEQ ID NO: 1, or variants or fragments thereof; (b) introducing the pesticidal composition to the insect pest population, wherein introducing is through providing the composition in or on a food source for the insect pest; and wherein the insect pest population is decreased. In some embodiments of this aspect, the introducing of step (b) includes one or more of: providing the pest with the composition formulated as an insect diet; feeding the composition to the pest; applying the composition to a plant; applying the composition to a part of a plant where the pest feeds; applying the composition to a trap for the insect pest; or injecting the composition into a plant. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste (e.g., for coating or spreading), a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is applied to the plant as at least one of a foliar treatment, a seed coating, an injection treatment, a preemergence treatment, and/or a post-emergence treatment. The method of plant treatment may vary based on factors known in the art, such as the timing of planting, the resilience of various plant parts or plant organs, the timing of the insect pest emergence, the extent and location of the insect pest infestation, and/or effective amount of the composition included in the treatment. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is prepared through desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, cryopreservation, or concentration. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest population is decreased by 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to an insect pest population not contacted with the composition. Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include providing a chemical mixture, a pesticidal protein (e.g., a Bt protein), and/or a biological control agent, and contacting the insect pest population with an effective amount of the chemical mixture, the pesticidal protein, and/or the biological control agent before step (b), in step (b), or after step (b). In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the insect pest or insect pest population is resistant to at least one Bt toxin. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the Coleopteran pest is selected from Sphenophorus levis, Sphenophorus maidis , Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus , and Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis.

[0023] Further aspects of the present disclosure relate to use of the polypeptide of any one of the preceding embodiments to inhibit growth of an insect, control or kill an insect, and/or control or kill an insect population. In some embodiments, bringing one or more of the polypeptides of the preceding embodiments into contact with the insects (larvae) results in stunted development of the insect, cessation of destruction of the plant, or insect mortality.

[0024] The active ingredients (i.e., recombinant polypeptides) of the embodiments are generally in a composition, and can be applied to the crop area, plant, or seed to be treated. The compositions of the embodiments may be applied simultaneously or in succession with other compounds. The number of applications and the rate of application depend on the intensity of infestation by the insect pest.

[0025] By “controlling the population of an insect pest,” limiting or eliminating insect pest related damage to a plant by, for example, inhibiting the ability of the insect pest to grow, feed, and/or reproduce or by killing the insect pest is intended. The methods include producing a compound or mixture including insecticidal recombinant polypeptides, then bringing that compound or mixture into contact with an insect pest. In some embodiments, this contact may include delivering insecticidal polypeptides in an insect diet to an insect pest. Another embodiment includes the insecticidal polypeptides coating a plant seed, followed by subsequent insect interaction with the seed or resultant plant that triggers the insecticidal activity of the insecticidal polypeptides. In some embodiments, contacting a pest with the insecticidal compositions of the present disclosure includes the insect pest being exposed to the insecticidal polypeptides at the site of insect attack. “Contacting” also includes applying the composition to the exterior surface of the pest, applying the composition to a plant where the pest feeds, applying the composition to the soil where the pest may be present, applying the composition to the general area of the pest population, applying the composition to a trap for the insect pest, injecting the composition into a plant or the pest, and any combination thereof. [0026] The presence of the pesticidal polypeptide therein protects the plant from the insect pest, and the insect pest is controlled. In one aspect, the use of an expression cassette for the production of active pesticidal polypeptides can be used in preparing compositions of the present disclosure. In an additional embodiment, a recombinant microorganism, a transgenic plant, and/or any other animal organism expressing the active pesticidal polypeptides are provided. The methods include transforming organisms with nucleic acid sequences encoding insecticidal polypeptides. In particular, the nucleic acid sequences of the present disclosure are useful for preparing plants and microorganism that possess pesticidal activity. Thus, transformed bacteria, yeasts, plants, plant cells, plant tissues, plant parts, propagules, organs, tissues, embryos, and seeds are provided. The compositions are pesticidal nucleic acids and proteins of bacterial species. The embodiments herein find use in agriculture in methods for protecting plants from insect pests and for impacting insect pests.

[0027] Compositions and formulations including a pesticidal polypeptide, or variant or fragment thereof, are useful in methods for controlling or impacting an insect pest. “Impact an insect pest” or “impacting an insect pest” is intended to mean, for example, deterring the insect pest from feeding further on the plant, harming the insect pest, or killing the insect pest. In this use, “impacting an insect pest” is a form of controlling an insect pest. Certain aspects and embodiments of the present disclosure further provide methods for impacting an insect pest of a plant including the application, for example, of a composition or formulation including a pesticidal polypeptide to the environment of the insect pest. In one embodiment, the pesticidal polypeptide is combined with a carrier for subsequent application to the environment of the insect pest. While the embodiments are not bound by any theory of operation, in one embodiment, an insect pest ingests the pesticidal polypeptide, thereby impacting the insect pest. Insect pests can be contacted with an effective amount of the pesticidal compositions by feeding, spraying, dusting, coating, wetting, and/or a combination thereof. The pesticidal compositions can be applied to a plant where the insect pest is and/or will be feeding through a foliar treatment, a seed coating, an injection treatment, a preemergence treatment, a post-emergence treatment, and/or any combination thereof.

[0028] Compositions of the embodiments find use in protecting plants, plant parts, propagule, embryos, tissue, organ, embryo, seeds, and plant products in a variety of ways. For example, the compositions can be used in a method that involves placing an effective amount of the pesticidal composition in the environment of the pest by a procedure including spraying, dusting, or seed coating. In a specific embodiment, such a plant is a sugarcane plant. Before plant propagation material (fruit, tuber, bulb, stalk, plantlets, corm, grains, seed, artificial seed) is sold as a commercial product, it is customarily treated with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these preparations, if desired together with further carriers, surfactants, or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal, or animal pests. The protective coating may be applied either by impregnating the plant materials with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, e.g., treatment directed at the buds or the fruit. The plant material of the embodiments coated with the pesticidal polypeptides of the present disclosure may be treated with a protective coating comprising a treatment compound, such as, for example, captan, carboxin, thiram, methalaxyl, pirimiphos- methyl, and others that are commonly used in seed treatment. Alternatively, a material of the embodiments includes a protective coating including a pesticidal composition of the embodiments used alone or in combination with one of the protective coatings customarily used in seed treatment.

[0029] In other embodiments, the pesticidal compositions of the present disclosure may be applied to an area wherein a plant is planted or will be planted, for example, directly to the soil or in the plant pot substrate where the materials are prepared to be planted. This method of treatment may be particularly suitable for sugarcane, as sugarcane is generally propagated using plantlets or stalk pieces (not seeds). Further, Sphenophorus levis lays eggs close to sugarcane roots, and the larvae feed on the sugarcane stalk, meaning that treatment of the soil or planting substrate is likely to be effective.

[0030] One of skill in the art would recognize that the compositions and methods of the embodiments can be used alone or in combination with other compositions and methods for controlling insect pests that impact plants. For example, the embodiments may be used in conjunction with other pesticidal proteins, chemical mixtures, and/or biological control agents (e.g., biological insecticides). In a further embodiment, the pesticidal polypeptides of the present disclosure, variants of the pesticidal peptides, and/or fragments thereof may be used in integrated pest management practices.

[0031] The terms “polypeptide,” “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0032] The methods described herein can provided control by decreasing pest population by 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a pest population not contacted with the composition or formulation.

Coleopteran pests and other insect pests

[0033] The recombinant proteins of the present disclosure may be insecticidally effective against many species of insect pests. Specifically, the recombinant proteins of the present disclosure may be insecticidally effective against many species of Coleopteran insect pests. Nonlimiting examples of Coleopteran insect pests according to the present disclosure include Eeptinotarsa spp. such as E. decemlineata (Colorado potato beetle); Chrysomela spp. such as C. scripta (cottonwood leaf beetle); Hypothenemus spp. such as H. hampei (coffee berry borer); Rhynchophorus ferrugineus (red palm weevil); Sitophilus spp. such as S. zeamais (maize weevil); Epitrix spp. such as E. hirtipennis (tobacco flea beetle) and E. cucumeris (potato flea beetle); Phyllotreta spp. such as P. cruciferae (crucifer flea beetle) and P. pusilia (western black flea beetle); Anthonomus spp. such as A. eugenii (pepper weevil);

Hemicrepidus spp. such as El. memnonius (wireworms); Melanotus spp. such as M. communis (wireworm); Ceutorhychus spp. such as C. assimilis (cabbage seedpod weevil); Phyllotreta spp. such as P. cruciferae (crucifer flea beetle); Aeolus spp. such as A. mellillus (wireworm); Aeolus spp. such as A. mancus (wheat wireworm); Horistonotus spp. such as H. uhlerii (sand wireworm); Phyllophaga spp. (White grubs); Chaetocnema spp. such as C. pulicaria (corn flea beetle); Popillia spp. such as P. japonica (Japanese beetle); Epilachna spp. such as E. varivestis (Mexican bean beetle); Cerotoma spp. such as C. trifurcate (Bean leaf beetle); Epicauta spp. such as E. pestifera and E. lemniscata (Blister beetles); Sphenophorus spp., such as S. levis and S. maidis', Diabrotica spp. such as Diabrotica speciosa, Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Cosmopolites spp., such as C. sordidus (banana borer); and Scarabaeidae.

[0034] In some embodiments, the insecticidal proteins of the present disclosure are active against Sphenophorus spp. Sphenophorus is a genus of beetles of the order Coleoptera and the family Curculionidae commonly referred to as “billbugs”. While the genus possesses over 60 species, exemplary Sphenophorus species include without limitation Sphenophorus spp. such as S. levis (sugarcane weevil or sugarcane billbug), S. maidis (maize billbug), S. zeae (timothy billbug), S. parvulus (bluegrass billbug), and S. callosus (southern com billbug). [0035] Of particular interest is Sphenophorus levis, the sugarcane weevil or sugarcane billbug (S. levis). The adults of this insect bore a hole into the near- ground internode of the sugarcane plant and lay their eggs close to sugarcane roots or place their eggs inside the sugarcane stalk. S. levis larvae hatch after 7-12 days and feed directly on the sugarcane stalk. While the female adults’ boring causes some amount of initial damage, the majority of the damage comes from the feeding of the larvae. Larval movement is limited to the first and second internodes of the sugarcane plant. The larval feeding phase takes 26 to 50 days and is followed by a pupal phase of 5 to 13 days. S. levis adults live in the soil for up to 250 days, according to Degaspari et al. (Degaspari, N., et al. Biologia de Sphenopherus levis Vaurie, 1978 (Col.: Curculionidade), em dieta artificial e no campo. Pesquisa Agropecuaria Brasileira, Brasilia, DF, v. 22, n. 3, p. 553-558, 1987).

[0036] S. levis causes 1% loss of sugarcane for every 1% of rhizome biomass it damages (Casteliani, A. et al. Crop Protection, 137, 105262, 2020). The effect of S. levis on the sugarcane plant can cause the death of up to 60% of sugarcane tillers, and this damage can result in up to 30% of lost sugarcane product, alongside greatly shortening the lifespan of sugarcane fields (Precetti and Arrigoni, Vaurie, 1978 (Coleoptera: Curculionidae).

Copersucar, Sao Paulo, Brazil. 1990). Because S. levis is incapable of very long-range flight, it is believed that infection spreads between fields through the transport of infested seedlings (Vinha, F. et al., Sci. Agrar. Parana., 280-288. 2020).

[0037] The rearing of Sphenophorus levis larvae presents challenges for experimental design, as they possess a larval phase lasting from 26 up to 50 days and larval viability is only 35.8%. Of the portion of larvae that reach a pupal stage, the viability is a much higher 93% and the pupal period lasts from 5 to 13 days (Degaspari, N., et al. Biologia de Sphenopherus levis Vaurie, 1978 (Col.: Curculionidade), em dieta artificial e no campo. Pesquisa Agropecuaria Brasileira, Brasilia, DF, v. 22, n. 3, p. 553-558, 1987). These viability challenges and long duration of metamorphic stage result in slow growth of the insect population to suitable, sustainable sample sizes.

[0038] Contemplation is given to the potential activity of the presented proteins’ insecticidal activity against Hemipteran, Dipteran, Lygus spp., and/or other piercing and sucking insects, for example of the order Orthoptera or Thysanoptera. Insects in the order Diptera include but are not limited to Liriomyza spp. such as L. trifolii (leafminer) and L. sativae (vegetable leafminer); Scrobipalpula spp. such as S. absoluta (tomato leafminer); Delia spp. such as D. platura (seedcorn maggot), D. brassicae (cabbage maggot) and D. radicum (cabbage root fly); Psilia spp. such as P. rosae (carrot rust fly); and Tetanops spp. such as T. myopaeformis (sugarbeet root maggot).

[0039] Further contemplated is the potential activity of the present disclosure’s insecticidal proteins against Lepidopteran insects. Insects in the order Lepidoptera include without limitation any insect now known or later identified that is classified as a Lepidopteran, including those insect species within suborders Zeugloptera, Glossata, and Heterobathmiina, and any combination thereof. Exemplary Lepidopteran insects include, but are not limited to, Ostrinia spp. such as O. nubilalis (European com borer); Plutella spp. such as P. xylostella (diamondback moth); Spodoptera spp. such as S. frugiperda (fall armyworm), S. ornithogalli (yellowstriped armyworm), S. praefica (western yellowstriped armyworm), S. eridania (southern army worm) and S. exigua (beet army worm); Agrotis spp. such as A. ipsilon (black cut worm), A. segetum (common cutworm), A. gladiaria (claybacked cutworm), and A. orthogonia (pale western cutworm); Striacosta spp. such as S. albicosta (western bean cutworm); Helicoverpa spp. such as H. zea (corn earworm), H. punctigera (native budworm), S. littoralis (Egyptian cotton leafworm) and H. armigera (cotton bollworm); Heliothis spp. such as H. virescens (tobacco budworm); Diatraea spp. such as D. grandiosella (southwestern com borer) and D. saccharalis (sugarcane borer); Trichoplusia spp. such as T. ni (cabbage looper); Sesamia spp. such as S. nonagroides (Mediterranean corn borer); Pectinophora spp. such as P. gossypiella (pink bollworm); Cochylis spp. such as C. hospes (banded sunflower moth); Manduca spp. such as M. sexta (tobacco hornworm) and M. quinquemaculata (tomato hornworm); Elasmopalpus spp. such as E. lignosellus (lesser cornstalk borer); Pseudoplusia spp. such as P. includens (soybean looper); Anticar sia spp. such as A. gemmatalis (velvetbean caterpillar); Plathypena spp. such as P. scabra (green cloverworm); Pieris spp. such as P. brassicae (cabbage butterfly), Papaipema spp. such as P. nebris (stalk borer); Pseudaletia spp. such as P. unipuncta (common armyworm); Peridroma spp. such as P. saucia (variegated cutworm); Keiferia spp. such as K. lycopersicella (tomato pinworm); Artogeia spp. such as A. rapae (imported cabbageworm); Phthorimaea spp. such as P. operculella (potato tuberworm); Crymodes spp. such as C. devastator (glassy cutworm); Feltia spp. such as F. ducens (dingy cutworm); and Telchin spp., such as T. licus. In one aspect of this embodiment, the insecticidal proteins of the present disclosure are active against black cutworm, sugarcane borer, and/or southwestern com borer.

[0040] The preferred developmental stage for testing for pesticidal activity is larvae or immature forms of these above-mentioned insect pests. The insects may be reared in total darkness at temperatures from about 20° C. to about 25° C, and relative humidity from about 30% to about 70%. Bioassays may be performed as described in Czapla and Lang (1990) J. Econ. Entomol. 83(6):2480-2485. Methods of rearing insect larvae and performing bioassays are well known to one of ordinary skill in the art.

[0041] Various bioassay techniques for assessing pesticidal activity and efficacy would be known to one skilled in the art. Common protocols involve adding the experimental compound to the pest’s food source in an enclosed container, and common measurements of pesticidal activity and efficacy include changes in mortality or other behaviors. Pesticidal activity can be measured by, but is not limited to, changes in mortality, weight loss, attraction, repellency and other behavioral and physical changes after feeding and exposure for an appropriate length of time.

Cry proteins from Bacillus thuringiensis

[0042] Strains of Bacillus thuringiensis (Bt) expressing pesticidal toxins are used as biological pest control agents. This is due to their production of 6-endotoxins (deltaendotoxins), also called crystalline toxins or Cry proteins. The Bt toxins are a family of insecticidal proteins that are synthesized as protoxins and crystallize as parasporal inclusions. When ingested by an insect pest, the microcrystal structure is dissolved by the alkaline pH of the insect midgut, and the protoxin is cleaved by insect gut proteases to generate the active toxin. The activated Bt toxin binds to receptors in the gut epithelium of the insect, causing membrane lesions and associated swelling and lysis of the insect gut. Insect death results from starvation and septicemia. See, e.g., Li et al. (1991) Nature 353:815-821. Any bacterial host cell expressing the novel nucleic acid sequences disclosed herein and producing a crystal protein is contemplated to be useful, such as B. thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.

[0043] The classification of various 6-endotoxins was performed based on their activity spectrum and sequence homology. Until 1990, the main classes were determined by their activity spectrum, with Cryl proteins having activity against Lepidoptera (moths and butterflies), Cry2 proteins having activity against both Lepidoptera and Diptera (flies and mosquitoes), Cry3 proteins having activity against Coleoptera (beetles), and Cry4 proteins having activity against Diptera (Hofte and Whitely, 1989, Microbiol. Rev. 53: 242-255). In 1998, a new nomenclature was developed that provided a systematic classification of Cry proteins based on amino acid sequence homology, rather than activity against certain insect species (Crickmore et al. 1998, Microbiol. Molec. Biol. Rev. 62: 807 -813). A database of Cry protein nomenclature and believed cladistic relationships of the various protein groups is maintained by the Bacillus thuringiensis Delta-Endotoxin Nomenclature Committee and can be accessed at http://www[dot]lifesci[dot]sussex[dot]ac[dot]uk/home/Neil_Crickmore/Bt/.

[0044] A comparison of the amino acid sequences of active Cry toxins of different specificities further reveals five highly-conserved sequence blocks. Structurally, the Cry toxins include three distinct domains, which are, from the N- to C-terminus: a cluster of seven alpha-helices implicated in pore formation (referred to as “domain I” or “domain 1”), three anti-parallel beta sheets implicated in cell binding (referred to as “domain II” or “domain 2”), and a beta sandwich (referred to as “domain III” or “domain 3”). The location and properties of these domains are known to those of skill in the art. See, for example, Li et al. (1991) supra and Morse et al. (2001) Structure 9:409-417.

Bt (Bacillus thuringiensis) Proteins

[0045] Certain aspects of the present disclosure related to an isolated, recombinant, modified, and/or mutated Bt protein, including a Bt protein that is isolated from nature, as well as a Bt protein that contains one or more point mutations, domain swaps, truncations and/or any other structural and/or sequence changes. In particular, the present disclosure relates to the SCW123 protein, which is an isolated protein from B. thuringiensis disclosed by Agarwal et al. and designated AXMI068 (Agarwal, A., et al. 2009 W02009158470A2). AXMI068 is very similar to the B. thuringiensis protein designated CrylMa2 (GenBank accession number: AGU13824.1, deposited November 14, 2013), which was primarily described by Kimberly S. Sampson as an isolate from the B. thuringiensis strain ARP080. Cry lMa2 is part of the Cry 1 group of Bt proteins. The Cry 1 group of Bt proteins have been shown to have insecticidal effects on some Coleopteran pests. For instance, Grossi de Sa et al. disclosed Crylla activity against some insects, such as the Coleopteran pest Anthonomus grandis (cotton boll weevil) (Grossi de Sa et al. (2014) W02014100880), and Seguin et al. disclosed Cryllal2 activity against the Coleopteran pest Diabrotica virgifera. To date, nearly 300 Cryl genes have been reported (Crickmore, N. “Full list of delta-endotoxins”; accessed March 23, 2023; available at: www [dot] lifesci [dot] S u s sex [dot] ac [dot] uk/home/N eil_Crickmore/B t/toxins2 [dot]html) .

[0046] Isolated AXMI068 protein has been shown to have a moderate effect against C. elegans in prior work (W02009158470A2). However, activity against insects generally, or against Coleopteran insects specifically, such as, for example against Sphenophorus levis, has not been demonstrated. Similarly, an absence of toxicity has previously been found against S. levis of such known Coleopteran toxic proteins from the Cry3 family, the Cry7 family, and the Cry 8 family (data not shown).

[0047] In some aspects, the present disclosure relates to a recombinant polypeptide having at least 40%, at least 60%, or at least 90% sequence identity to SEQ ID NO: 1, and/or variants or fragments thereof. In some embodiments of this aspect, the recombinant polypeptide includes SEQ ID NO: 1.

[0048] In additional aspects, the present disclosure relates to a recombinant polypeptide including at least one amino acid substitution, deletion, and/or insertion compared to SEQ ID NO: 1; and/or at least one addition at the N-terminus or C-terminus compared to SEQ ID NO: 1; and/or at least one domain swap compared to SEQ ID NO: 1; and/or at least one truncation compared to SEQ ID NO: 1; and/or at least one other alteration compared to SEQ ID NO: 1. In further embodiments of this aspect, which may be combined with any of the preceding embodiments, the recombinant polypeptide is a fusion protein including at least a portion of the polypeptide of any one of the preceding embodiments, variants, and fragments thereof. In some embodiments of this aspect, the polypeptide has insecticidal activity against at least one agricultural insect pest. In some embodiments of this aspect, the at least one insect pest is a Coleopteran pest. In some embodiments of this aspect, the Coleopteran pest is selected from the group of Sphenophorus levis, Sphenophorus maidis, Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus , or Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis.

[0049] Some aspects of the disclosure relate to a recombinant polynucleotide encoding the polypeptide of any of the preceding embodiments. In some embodiments of this aspect, the recombinant polynucleotide includes the sequence of SEQ ID NO: 2. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the polynucleotide has codons optimized for expression in an agriculturally important crop. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the polynucleotide is a non-genomic polynucleotide. In some embodiments of this aspect, the polynucleotide is a synthetic polynucleotide, and/or the polynucleotide is a cDNA. [0050] The present disclosure provides a B. thuringiensis-derived protein designated as SCW123 (SEQ ID NO: 1) and a nucleic acid sequence coding therefor (SEQ ID NO: 2). SCW123 (SEQ ID NO: 1) is 597 amino acids in length with an estimated molecular weight of 66,495 kDa, and is encoded by the nucleic acid sequence of SEQ ID NO: 2, which is 1,791 residues in length.

[0051] In some embodiments, which may be combined with of any of the above embodiments, the insecticidal Bt protein is a polypeptide with at least 40% sequence identity to SEQ ID NO: 1. In further embodiments, which may be combined with of any of the above embodiments, the insecticidal Bt protein is a polypeptide at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0052] Without wishing to be bound by theory, it is thought that insecticidal activity will be maintained if at least domain I and domain II of a protein of the present disclosure (e.g., SCW123) are present, and combined with domain III of a different Bt protein. Chimeras incorporating one or more or two or more domains of proteins of the present disclosure are considered in the present disclosure. Variants and/or fragments from the proteins disclosed herein are also considered to be part of the present disclosure, and include, without limitation, homologous (or partially homologous) sequences and polypeptides based on SEQ ID NO: 1, and/or derived from polynucleotides altered by site directed mutagenesis, domain swapping, DNA shuffling, or any other methods known in the art.

[0053] In other embodiments, the isolated and/or recombinant proteins disclosed herein are useful as recombinant proteins expressed by transgenic plants, microrganisms and fungi. Thus, a recombinant microorganism or fungi as well as a transgenic plant expressing the active pesticidal protein are provided. The methods involve transforming organisms with nucleic acid sequences encoding insecticidal polypeptides. In particular, the pesticidal polynucleotides are useful for preparing plants, microorganisms, fungi, and other animal cells that possess pesticidal activity. Thus, transformed bacteria, yeasts, fungi, plants, plant cells and animal cells, plant tissues, plant parts, propagule, organs, tissues, embryo, and seeds are provided. A further embodiment of the present disclosure includes compositions including pesticidal nucleic acids and/or pesticidal proteins incorporated or expressed in microorganism, fungi, or plants. The embodiments herein find use in agriculture in methods for protecting plants from insect pests and for controlling insect pests or insect pest populations. [0054] As used herein, the terms “pesticidal activity,” “pesticidal gene,” or “pesticidal polynucleotide” refers to a nucleotide sequence that encodes a polypeptide that exhibits pesticidal activity. As used herein, the term "pesticidal activity” refers to the ability of a substance, such as a polypeptide, to inhibit the growth, feeding, or reproduction of an insect pest and/or to kill the insect pest. A "pesticidal polypeptide,” “pesticidal protein”, or “insect toxin” is intended to mean a protein having pesticidal activity.

[0055] As used herein, the terms “pesticidal activity” and “insecticidal activity” are used synonymously to refer to activity of an organism or a substance (such as, for example, a protein) that can be measured by, but is not limited to, pest mortality, pest weight loss, pest repellency, and other behavioral and physical changes of a pest after feeding and exposure for an appropriate length of time. In this manner, pesticidal activity impacts at least one measurable parameter of pest fitness. Assays for assessing pesticidal activity are well known in the art. See, e.g., U.S. Pat. Nos. 6,570,005 and 6,339,144. As used herein, “pesticidal efficacy” refers to the level of pesticidal or insecticidal activity an organism or substance displays. In general, higher pesticidal efficacy results in lower pest fitness and higher pest mortality.

[0056] The isolated and/or recombinant proteins disclosed herein include many embodiments that may be readily useful as exogenously applied insecticidal compositions, for example, for topical and/or systemic application to field crops, grasses, fruits and vegetables, and ornamental plants, and/or as recombinant proteins expressed by transgenic plants or microorganisms. A more detailed description about the insecticidal compositions of the present invention is disclosed herein, in the following section (“Compositions including SCW proteins”). In one embodiment, the biological insecticidal composition includes a water dispersible granule. This granule includes one or more of the SCW proteins disclosed herein. In another embodiment, the insecticidal composition includes an oil flowable suspension of one or more of the SCW proteins disclosed herein.

[0057] The orally acceptable insect diet or orally administrable diet into which the insecticidal proteins of the present disclosure may be incorporated are well known in the art as described herein. These can be included in any composition which can be orally ingested by the target insect pest taking the form of, for example, when the proteins or fusions of the present disclosure are expressed from within a host cell such as a plant, fungal, or bacterial cell, consisting of a cell extract, a cell suspension, a cell homogenate, a cell lysate, a cell supernatant, a cell filtrate, a cell pellet, and/or a protein extract or a purified protein or fusion of the present disclosure. In one embodiment, the composition containing the insecticidal polypeptides of the present disclosure can be formulated into a powder, a dust, a pellet, a granule, a spray, an emulsion, a colloid, or a solution, any of which can be topically applied to a substrate which is or can become an orally ingestible, orally acceptable, or an orally administrable diet for a target insect pest.

[0058] In some embodiments, controlling the insect pest includes feeding the composition to the pest, applying the composition to the exterior surface of the pest, applying the composition to a plant where the pest feeds, applying the composition to the soil where the pest may be present, applying the composition to the general area of the pest population, or injecting the composition into a plant or pest. In a specific embodiment, the plant is a sugarcane plant. In some embodiments, the insect pest population is decreased by 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In some embodiments, insect infestation is decreased by 50% to 100% as compared to a method of treating insect infestation that does not use an exogenously applied SCW protein of the present disclosure.

SCW proteins in expression cassettes

[0059] Some aspects of the disclosure relate to an isolated construct or expression cassette including a nucleotide encoding the polypeptide of any one of the preceding embodiments or the polynucleotide of any one of the preceding embodiments, wherein the nucleotide or the polynucleotide is operably linked to a promoter, and optionally operably linked to a heterologous regulatory element. In some embodiments of this aspect, the polypeptide includes SEQ ID NO: 1. In some embodiments of this aspect, the polynucleotide includes at least one of SEQ ID NO: 2. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the promoter is selected from the group of a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0060] In yet another aspect, the present disclosure provides methods for producing an expression cassette which includes a nucleic acid sequence encoding one of the SCW proteins of the present disclosure. The process of producing expression cassettes is well- known in the art. Expression cassettes are typically designed with a promoter at the 5' end of the cassette, upstream of a desired polynucleotide segment encoding a protein of the present disclosure, including SEQ ID NO: 2, and/or variants, fragments, and combinations thereof. [0061] A promoter can consist of multiple different promoter elements operably linked to provide for the initiation of transcription of the sequences encoding a protein of the present disclosure. The DNA sequence consisting of the promoter-protein-encoding DNA can be operably linked at its 3' end to a transcriptional termination signal sequence functional in microorganisms, fungi, animals, and/or plants to produce the recombinant DNA construct.

[0062] In one aspect, the aforementioned recombinant DNA construct is in an expression cassette for expression in a cell. Expression cassettes are designed with a promoter at the 5' end of the cassette, upstream of a desired polynucleotide segment encoding a protein of the present disclosure. 5' untranscribed DNA can include a promoter which can consist of multiple different promoter and enhancer elements operably linked to provide for the initiation of transcription of downstream sequences including sequences encoding the polypeptides of the present disclosure. One or more transcribed but non-translated DNA sequence(s) can be operably linked 3' to the promoter in the expression cassette, including leader and/or intron sequence(s). An intron sequence is optionally provided 3' to the leader sequence or in some cases within the open reading frame encoding the desired protein. A polynucleotide segment encoding an optional translocation polypeptide (a signal peptide or a chloroplast transit peptide, for example) may be inserted 5' to the coding sequence of the protein of the present disclosure for localizing the protein of the disclosure to a particular subcellular position. The nucleotide sequence encoding the protein of the present disclosure is optionally operably positioned within the aforementioned expression cassette, along with any requisite operably linked polyadenylation (poly A) and/or transcriptional termination sequence functional in microorganism, fungal, animal, and/or plant cells.

[0063] As used herein, an “expression cassette” is a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically includes sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may have at least one of its components heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.

[0064] An expression cassette including a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. An expression cassette may also be one that includes a native promoter driving its native gene; however, it has been obtained in a recombinant form useful for heterologous expression. Such usage of an expression cassette makes it so it is not naturally occurring in the cell into which it has been introduced. As used herein, the term “isolated nucleic acid molecule” refers to a nucleic acid molecule that, by the hand of Man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule may exist in a purified form or may exist in a non-native environment such as, for example without limitation, a recombinant microbial cell, plant cell, plant tissue, or plant.

[0065] The aforementioned elements are arranged contiguously and can be used in various combinations depending on the desired expression outcome. Additional aspects of the disclosure include an isolated nucleic acid sequence or construct containing a promoter operatively linked to a coding region that encodes, for example, a SCW123 protein or a modified or recombinant SCW123 protein. Additional aspects of the disclosure include an isolated nucleic acid sequence or construct containing a promoter operatively linked to a coding region that encodes, for example, a protein with sequence SEQ ID NO: 1. Such a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region, and hence providing the cell the ability to produce the recombinant protein in vivo.

[0066] In one embodiment, the disclosure provides an isolated or recombinant polynucleotide encoding the insecticidal polypeptides disclosed herein, wherein the polynucleotide has codons optimized for expression in an agricultural crop. In one aspect, the agricultural crop is sugarcane.

[0067] The recombinant polynucleotide finds use in the construction of expression vectors for subsequent transformation into organisms of interest, as well as in the development of probes for the isolation of other homologous (or partially homologous) genes, and for the generation of altered SCW polypeptides by methods known in the art, such as site directed mutagenesis, domain swapping or DNA shuffling. In a particular embodiment, the polynucleotide sequence is SEQ ID NO: 2.

[0068] In some embodiments the SCW polypeptides include amino acid sequences modified from the full-length nucleic acid sequences disclosed herein and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism the protein is expressed in or in the pest after ingestion of the protein. Thus, provided herein are isolated or recombinant nucleic acid sequences that confer pesticidal activity. Also provided are the amino acid sequences of SCW polypeptides. The protein resulting from translation of these genes allows cells to control or kill pests that ingest it.

Molecular biological and biotechnological methods

[0069] Any methodology known in the art to modify the cellular DNA (e.g., genomic DNA and organelle DNA) and produce proteins can be used in practicing what is disclosed herein.

[0070] The term “recombinant” or “modified nucleic acids” refers to polynucleotides which are made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.

[0071] In some embodiments, a non-integrated expression system can be used to induce expression of one or more introduced genes. Expression systems (expression vectors) can include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer, and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides can also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, cell wall, or be secreted from the cell. [0072] Screening and molecular analysis of recombinant strains of the present disclosure can be performed utilizing nucleic acid hybridization techniques. Hybridization procedures are useful for identifying polynucleotides, such as those modified using the techniques described herein, with sufficient homology to the subject regulatory sequences to be useful as taught herein. The particular hybridization techniques are not essential to the subject disclosure. As improvements are made in hybridization techniques, they can be readily applied by one of skill in the art. Hybridization probes can be labeled with any appropriate label known to those of skill in the art. Hybridization conditions and washing conditions, for example temperature and salt concentration, can be altered to change the stringency of the detection threshold. See, e.g., Sambrook et al. (1989) vide infra or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y., for further guidance on hybridization conditions.

[0073] Similarly, screening can be performed using polypeptide-based techniques including enzyme-linked immunosorbent assays (ELISAs), fluorescence detection (if a fluorescent marker was used), or Western blots. One of skill in the art will recognize that any polypeptide-based techniques available can be utilized in screening aspects or embodiments disclosed herein.

[0074] Additionally, screening and molecular analysis of genetically altered strains and/or plants or plant cells and materials, as well as creation of desired isolated nucleic acids can be performed using Polymerase Chain Reaction (PCR). PCR is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science 230: 1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3 ' ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5’ ends of the PCR primers. Because the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as the Taq polymerase, which is isolated from the thermophilic bacterium Thermits aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.

[0075] Nucleic acids and proteins of the present disclosure can also encompass homologues of the specifically disclosed sequences. Homology or genetic identity can be 40% -100%. In some instances, such homology or genetic identity is greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. The degree of homology or identity needed for any intended use of the sequence(s) is readily identified by one of skill in the art. As used herein percent sequence identity of two nucleic acids is determined using an algorithm known in the art, such as that disclosed by Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264- 2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389- 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See www.ncbi.nih.gov.

[0076] Plant cells and all forms of plants are also preferred targets for coatings and other applications of insecticidal compositions disclosed herein. Plant cells from monocot species, especially including sugarcane (e.g., Saccharum spp.) may be used. Cells may be derived from tissue types including embryo, callus, leaf disk, and other explants. Plant cells targeted by the insecticidal composition can be differentiated or undifferentiated (e.g. callus, undifferentiated callus, immature and mature embryos, immature zygotic embryo, immature cotyledon, embryonic axis, suspension culture cells, protoplasts, leaf, leaf cells, root cells, phloem cells and pollen). Plant cells include, without limitation, cells from seeds, suspension cultures, explants, immature embryos, embryos, zygotic embryos, somatic embryos, embryogenic callus, meristem, somatic meristems, organogenic callus, protoplasts, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, cotyledons, immature cotyledons, embryonic axes, meristematic regions, callus tissue, cells from leaves, cells from stems, cells from roots, cells from shoots, gametophytes, sporophytes, pollen and microspores. Plant cells further include various forms of cells in culture (e.g., single cells, protoplasts, embryos, and callus tissue), wherein the protoplasts or cells are produced from a plant part selected from the group of leaf, stem, anther, pistil, root, fruit, flower, seed, cotyledon, hypocotyl, embryo, or meristematic cell. Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated or recombinant nucleic acid molecule, or contain one or more genes to produce at least one recombinant protein. The nucleic acid(s) encoding the SCW proteins of the present disclosure can be introduced by any means known to the art which is appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or any other methodology known by those skilled in the art.

[0077] A method of detecting any nucleotide or polypeptide of the preceding embodiments from a genetically modified plant material is provided, comprising the steps of: a) obtaining a plant material sample for analysis; b) extracting DNA from the sample; c) providing primer pairs comprising at least a forward and a reverse primer; d) amplifying a region between the primer pair; and e) detecting the presence of a product from amplification; or comprising the steps of

(a) obtaining a plant material sample for analysis;

(b) extracting DNA or RNA from the sample;

(c) providing a probe or a combination of probes designed to bind to a polynucleotide comprising a polynucleotide according to the preceding embodiments;

(d) hybridizing said probe with the sample; and

(e) detecting the actual hybridization of the probe.

Antibodies

[0078] Antibodies to a SCW polypeptides of the embodiments or to variants or fragments thereof are also encompassed. The antibodies of the disclosure include polyclonal and monoclonal antibodies as well as fragments thereof which retain their ability to bind to SCW polypeptides. An antibody, monoclonal antibody, or fragment thereof is said to be capable of binding a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody, monoclonal antibody, or fragment thereof.

[0079] A kit for detecting the presence of a SCW polypeptide or detecting the presence of a nucleotide sequence encoding a SCW polypeptide in a sample is provided. In one embodiment, the kit provides antibody-based reagents for detecting the presence of a SCW polypeptide in a sample. In another embodiment, the kit provides labeled nucleic acid probes useful for detecting the presence of one or more polynucleotides encoding SCW polypeptides. The kit is provided along with appropriate reagents and controls for carrying out a detection method, as well as instructions for use of the kit.

[0080] A kit for detecting one or more polynucleotide or polypeptides of the preceding embodiments from a genetically modified plant material is provided. In one embodiment the kit comprises a means to detect the presence of one or more polynucleotides of the preceding embodiments and/or a means to detect one or more polypeptides of the preceding embodiments, wherein the means comprise primer pairs designed to bind to the polynucleotides or wherein the means comprise primer pairs and a probe designed to bind to the polynucleotides, and/or wherein the means comprise an antibody for detection of one or more polypeptides of the preceding embodiments.

Compositions comprising SCW proteins and related nucleotides

[0081] A further aspect of the disclosure relates to a pesticidal composition including: (i) one or more polypeptides of any one of the preceding embodiments, wherein the one or more polypeptides are present at a concentration sufficient to control at least one agricultural insect pest; (ii) one or more polynucleotides of any one of the preceding embodiments, wherein the polynucleotide has codons optimized for expression in an agriculturally important crop; and/or (iii) one or more isolated constructs or expression cassettes of any one of the preceding embodiments. In some embodiments of this aspect, the one or more polypeptides include SEQ ID NO: 1. In some embodiments of this aspect, the one or more polynucleotide are SEQ ID NO: 2. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the at least one insect pest is a Coleopteran pest. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the one or more polypeptides are present at a concentration sufficient to control at least one agricultural insect pest in or on a sugarcane plant when the composition is applied to the sugarcane plant or to a sugarcane plantation. Some embodiments of this aspect, which may be combined with any of the preceding embodiments, further include one or more inert ingredients, acceptable carriers, surfactants, or adjuvants customarily employed in the art of formulation, or other components to facilitate product handling and application for particular target pests. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste (e.g., for coating or spreading), a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier (e.g., a polymer substance). In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as an orally acceptable, orally administrable, or orally ingestible diet intended for consumption by the insect pest. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated for direct soil application and/or direct plant pot substrate application. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the composition is formulated as a controlled release formulation. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, control of the Coleopteran pest includes: (a) decreasing pest infestation by 40%, 50%, 60%, 70%, 80%, 90%, or 100%; or (b) increasing pest mortality by 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments of this aspect, which may be combined with any of the preceding embodiments, the Coleopteran pest is selected from the group of Sphenophorus levis, Sphenophorus maidis, Anthonomus grandis, Diabrotica spp., Trechus subsignatus, Migdolus fry anus, Cerotoma arcuata tingomariana, Leptinotarsa decemlineata, Cosmopolites sordidus, Hypothenemus hampei, Rhynchophorus ferrugineus , or Scarabaeidae spp. In some embodiments of this aspect, the Coleopteran pest is Sphenophorus levis. Such formulated compositions may be prepared by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide.

[0082] Compositions of the present disclosure include at least one SCW 123 protein as disclosed herein, or a mutated, recombinant, or otherwise modified version thereof. In some of the embodiments, the composition further includes one or more additional Bt proteins, such as a Cry8 protein or any additional Cry protein. In some embodiments, compositions of the present disclosure further include additional active agents, such as chemical mixtures (e.g., insecticidal chemicals), insecticidal proteins (e.g., Bt proteins), or biological control agents (e.g., Bacillus thuringiensis'). In some embodiments, compositions of the present disclosure further include agriculturally relevant agents (i.e., agrochemicals). In some embodiments, compositions of the present disclosure include one or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematocides, molluscicides, acaricides, plant growth regulators, harvest aids, and fertilizers.

[0083] In one embodiment, the formulation of the biological insecticidal composition can be prepared in a number of ways well known in the art, including but not to be limited to desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration. In any such orally acceptable, orally administrable, or orally ingestible diet intended for consumption by a target insect pest, the protein of the present disclosure should at least be present in a concentration from about 0.001% of the total weight of the composition to about 99% of the weight of the composition. In one embodiment, the protein of the present disclosure should be present in a concentration of about 1 part protein to 4 parts diet of a target insect pest.

[0084] Such compositions disclosed above may be obtained by the addition of a surfaceactive agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent or fertilizers, micronutrient donors, or other preparations that influence plant growth. Suitable surface-active agents include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; a carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g., the sulfonated condensation product of oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g., the sodium sulfonate of dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alky I- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g., polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate or oleate; or oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

[0085] Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, mica, amorphous silica gel, talc, clay, volcanic ash or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells. Kaolins such as kaolinite, dickite, nacrite, anauxite, halloysite and endellite are useful as carrier materials. Montmorillonites, such as beidellite, nontronite, montmorillonite, hectorite, saponite, sauconite and bentonite are useful as carrier materials. Vermiculites such as biotite are useful as carrier materials.

[0086] The compositions of the embodiments can be in a suitable form for direct application or as a concentrate of a primary composition that requires dilution with a suitable quantity of water or other diluent before application. The pesticidal concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition contains 1 to 98% of a solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of a surfactant. These compositions will be administered at the labeled rate for the commercial product, for example, about 0.01 lb to 5.0 lb. per acre when in dry form and at about 0.01 pts. to 10 pts. per acre when in liquid form.

[0087] Compositions, as disclosed herein, including cell or tissue extract, suspension, homogenate, lysate, supernatant, filtrate, pellet, wherein such cells or tissues express at least one of SCW proteins, and/or purified proteins derived from such cells and/or tissues are also provided.

Sugarcane plants

[0088] Sugarcane plants of the present disclosure include species and hybrids in the genus Saccharum, e.g., Saccharum officinarum, Saccharum sinense, Saccharum barberi, Saccharum robustum, Saccharum spontaneum, Saccharum spp., Saccharum spp. hybrid, etc. Cultivated Saccharum crop plants are generally hybrids of the many sugarcane species that can interbreed. Sugarcane is classified as a monocotyledonous plant, and is in the same plant family (Poaceae) as other important crops, such as maize, rice, and wheat. As one of the main global sources of sugar, sugarcane is a critical crop to the economy of many subtropical and tropical countries that cultivate it, especially Brazil. Additionally, as a source of ethanol, sugarcane also provides an environmentally valuable potential alternative to gasoline for some fuel processes worldwide. [0089] The jointed, prominent stems of sugarcane are the commercially desirable portion of the plant, containing rich quantities of sucrose between the “joints”, or within the internodes, of the stems. These internode portions of the stems are full of vasculature tissue, and in sugarcane, this vasculature contains rich deposits of sucrose.

[0090] Propagation of the sugarcane crop is performed by cutting near the top of mature canes and replanting the removed stem portions. Additionally, plantlets derived from buds and cultivated in greenhouses are also used for propagation. The harvesting of mature sugarcane may be accomplished through either hand-cutting, in a labor-intensive process that provides thousands of jobs, or through mechanical harvesting with heavy machinery. Sucrose is extracted from the cane through a milling process, then prepared for commercial markets through a refinery process.

[0091] In some aspects, the present disclosure relates to treatment of a seed, plant part, or plant tissue with the recombinant proteins, or a composition containing the recombinant proteins, of any of the above embodiments. In some embodiments, the plant part is selected from the group of leaf, stem, anther, pistil, root, fruit, flower, seed, cotyledon, hypocotyl, embryo, somatic embryo, or meristematic cell. Plant parts include differentiated and undifferentiated tissues including, but not limited to, roots, stems, shoots, leaves, pollen, seeds.

[0092] In some aspects, the present disclosure relates to a transgenic plant, plant part, propagule, seed, tissue, organ, embryo, or plant cell including the polypeptide of any one of the preceding embodiments, the polynucleotide of any one of the preceding embodiments, or the isolated construct or expression cassette of any one of the preceding embodiments. In some embodiments of this aspect, the one or more polypeptides include SEQ ID NO: 1 or fragments or variants thereof. In some embodiments of this aspect, the one or more polynucleotides include SEQ ID NO: 2 or fragments or variants thereof. In other aspects, the present disclosure relates to a plant, plant part, propagule, seed, tissue, organ, embryo, or plant cell transformed (e.g., transient or stable transformation) with at least one polynucleotide encoding one of the insecticidal polypeptides.

[0093] As used herein, the term “plant” refers to any plant at any stage of development, particularly a seed plant. The term “plant cell” refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant. The term “plant cell culture” refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development. The term “plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant. The term “plant organ” refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo. The term “plant tissue” refers to a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0094] In some aspects, the present disclosure relates to an insecticidal composition applied to protoplasts or cells from the plant of any of the above embodiments. Plant cells can be differentiated or undifferentiated (e.g., callus, undifferentiated callus, immature and mature embryos, immature zygotic embryo, immature cotyledon, embryonic axis, suspension culture cells, protoplasts, leaf, leaf cells, root cells, phloem cells and pollen). Plant cells include, without limitation, cells from seeds, leaves, stems, roots, or shoots, suspension cultures, explants, immature embryos, embryos, zygotic embryos, somatic embryos, embryogenic callus, meristems, somatic meristems, organogenic callus, protoplasts, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, cotyledons, immature cotyledons, embryonic axes, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, or microspores. Plant cells further include various forms of cells in culture (e. g., single cells, protoplasts, embryos, and callus tissue), wherein the protoplasts or cells are produced from a plant part selected from the group of leaf, stem, anther, pistil, root, fruit, flower, seed, cotyledon, hypocotyl, embryo, or meristematic cell. In addition to sugarcane, as described above, the plant cells or tissue may be derived from plants including, without limitations, corn (e.g., maize, Zea mays), barley (e.g., Hordeum vulgare), millet (e.g., finger millet, fonio millet, foxtail millet, pearl millet, barnyard millets, Eleusine coracana, Panicum sumatrense, Panicum milaceum, Pennisetum glaucum, Digitaria spp., Echinocloa spp.), oat (e.g., Avena sativa), rice (e.g., indica rice, japonica rice, aromatic rice, glutinous rice, Oryza sativa, Oryza glaberrimd), rye (e.g., Secale cereale, Secale cereanum), setaria (e.g., Setaria italica, Setaria viridis), Brachypodium sp., sorghum (e.g., Sorghum bicolor), teff (e.g., Eragrostis tef), triticale (e.g., X Triticosecale Wittmack, Triticosecale schlanstedtense Wittm., Triticosecale neoblaringhemii A. Camus, Triticosecale neoblaringhemii A. Camus), wheat (e.g., common wheat, spelt, durum, einkorn, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.), switchgrass (e.g., Panicum virgatum), Brassica sp., tobacco (e.g., Nicotiana benthamiana, Nicotiana tabacum), peanut (Arachis hypogaea), banana Musa sp.), potato Solarium tuberosum), strawberry (Fragaria ananassa), coffee (Coffea arabica), cotton (Gossypium hirsutum), tomato (Solarium lycopersicum) or any other polyploid and/or vegetatively propagated plant species.

In planta expression

[0095] There are several plant genetic transformation techniques grouped into two main categories: indirect and direct gene transfer. Indirect transfer is when exogenous DNA is inserted into the genome by the action of a biological vector, while direct transfer is based on physical-biochemical processes.

[0096] Different tissues and/or cells could be used according to the genetic transformation technique and according to the species or genotypes to be transformed. Generally, these tissues or cells include, without limitation, embryogenic callus, callus, protoplasts, embryos, somatic embryos, meristematic tissues, and any other part, tissue or cell of plant with regenerative capacity.

[0097] Indirect transformation is based on the bacterium-mediated system of the genus Agrobacterium and has been the most widely used method for obtaining transgenic plants. Advantages to this method include the ability to transfer relatively long DNA segments without rearrangement while maintaining low copy number integration of the transgenes, thus ensuring greater genotypic stability for the generated events. Several Agrobacterium species and strains, plasmids and protocols have been developed and adapted for genetic transformation of several plant species. The advantages of these methods include higher probabilities to single copy events, stable integration, and genetic heritage of the introduced genetic traits, as well as, consistent genic expression through generations and lower rates of gene silencing.

[0098] Agrobacterium tumefaciens and A. rhizogenes are gram negative soil phytopathogenic bacteria belonging to the Rhizobiaceae family that cause diseases in dicotyledons, known as crown and hairy root galls, respectively. In this plant-pathogen interaction there is a process of natural gene transfer between the Agrobacterium and the plant cell wherein fragments of bacterial DNA are transferred into the plant cell (T-DNA), integrating with the nuclear genome. In its natural form, the bacterium transfers T-DNA (“transferred DNA”), which is part of the bacterial plasmid called Ti (“tumor-inducing”) and integrates into the genome of infected plant cells. The T-DNA fragment that is transferred to the plant cell is comprised of genes involved in the constitutive biosynthesis of phytohormones (auxins and cytokinins), which alter the normal developmental program of infected tissue and cause tumor formation. In addition, it also contains oncogenes for the synthesis of sugars and amino acids called opines, which serve as carbon and nitrogen sources for bacteria (Oger et al. 1997). Repeated ends of 25 base pairs (bp) at the right and left borders delimit the T-DNA and are essential for its transfer. Phenolic compounds released by injured plant tissues activate specific regions (vir regions), initiating the process of transfer of T-DNA to the plant cell. Agrobacterium also has chromosomal (chv) genes that promote binding between bacterial and host cells, allowing the formation of the pore passage of the T-DNA-containing complex (Sheng & Citovsky. 1996).

[0099] Since the segment to be transferred is defined by its borders, any sequence flanked by the borders can be transferred to a plant by means of agrobacteria, making it possible to manipulate these sequences in order to transfer coding sequences of interest. The replacement or deletion of the coding regions of wild-type T-DNA (oncogenes) allows for the generation of non-oncogenic (disarmed) Agrobacterium strains, which can carry the sequences of interest. The modified T-DNA is able to transfer the sequences of interest to plants because the virulence genes (vir region) remain intact.

[0100] Additionally, the Agrobacterium indirect transformation system allows for the transfer of artificial plasmid constructs to plants as long as the constructs contain such T- DNA borders, which enables the flexibility to use molecular tools and materials developed for other bacterial strains.

[0101] These artificial plasmid constructs have promoters from different origins, as for example, plant promoters, viral promoters, bacterial and or chimeric promoters, besides genes that confer antibiotic resistance, herbicide resistance or tolerance, or enzymatic activity (phosphomannose isomerase (PMI)/mannose (Man)), so these markers can be used for the selection of transformed cells or plants. [0102] These constructions also can contain auxiliaries genes which interfere with relevant morphogenesis signaling pathways, enhancing the efficiency of the genetic transformation process and regeneration of vegetal tissues. Included, without limitations, LEAFY COTYLEDON1 (Lotan et al., 1998), Led (Lowe et al., 2002), LEAFY COTYLEDON2 (Stone et al., 2001), WUSCHEL (WUS; Zuo et al., 2002), e BABY BOOM (BBM; Boutilier et al., 2002), among others.

[0103] In a first aspect of the present invention, foreign or exogenous DNA to be introduced into the plant is cloned into a binary plasmid between the left and right border consensus sequences (T-DNA). The binary plasmid is transferred to an Agrobacterium cell, which is subsequently used to infect plant tissue. The T-DNA region of the vector comprising the exogenous DNA is inserted into the plant genome. The marker gene expression cassette and the characteristic gene expression cassette may be present in the same region of T-DNA, in different regions of T-DNA on the same plasmid, or in different regions of T-DNA on different plasmids. In one embodiment of the present invention, the cassettes are present in the same region as the T-DNA. One of skill in the art is familiar with the methods of indirect transformation by Agrobacterium.

[0104] Alternatively, direct DNA transfer can be used to directly introduce DNA into a plant cell. One method of direct DNA transfer is to bombard plant cells with a vector comprising DNA for insertion using a particle gun (particle-mediated biolistic transformation). Other methods for transformation of plant cells include protoplast transformation (optionally in the presence of polyethylene glycols); ultrasound treatment of plant tissues, cells, or protoplasts in a medium comprising the polynucleotide or the vector; microinjection of the polynucleotide or vector into plant material; microinjection, vacuum infiltration, sonication, use of silicon carbide, chemical transformation with PEG, electroporation of plant cells and the like. Between the disadvantages of direct transformation are challenges related to regeneration of plant tissue and the low transgene expression.

[0105] In addition, genetic transformation could be performed by site direct insertion through homologous recombination mediated by nucleases (genome editing). In recent years, genome editing technology based on use of engineered or chimeric nucleases has enabling the generation of genetically modified organisms in a more precise and specific way. The introduction of exogenous or foreign genes occur by homologous recombination through introduction of a Homologous recombination template (HR) having the exogeneous DNA linked to a DNA fragment homologous to the genome of the receptor organism. Between the tools available are the chimeric enzymatic system CRISPR (clustered, regularly interspaced, short palindromic repeats) - Cas, the Zinc finger (ZFN)nucleases and TAL effector nucleases (TALENs). Crispr-Cas systems are enzymatic systems comprising two main components: an endonuclease (Cas) and a guide-RNA (single-guide RNA - sgRNA; a guide to the specific cleavage site of Cas endonuclease). The guide RNA may also comprise of two components: a Crispr RNA (crRNA) - a sequence of 17-20 mer complementary to specific DNA genomic sequences and, optionally, of a tracrRNA. The specific cleavage performed by endonuclease and guide by the sgRNA would be repair by homologous recombination, specifically inserting the exogenous DNA flanked by the homologous sequences to the cleavage site. The introduction of this enzymatic system to the cell could occur by several manners, using plasmids, through direct or indirect transformation, or using carriers like proteins and other chemical agents. The expression of the system components would occur in a transient or stable manner, using the cellular machinery of the receptor organism or being realized in a exogeneous way, in vitro, delivering to the target cell or tissue all the components ready to use (endonucleases + sgRNA, in vitro transcribed and combined before cell delivery). The description presented herein is not exhaustive and should not limit the use of different variations, systems and methods of genome editing on scope of the present invention, known in the State of the Art and even the ones not yet discovered.

[0106] Following transformation, transgenic plants are regenerated from the transformed plant tissue and the progeny that have exogenous DNA can be selected using an appropriate marker such as kanamycin, geneticin or ammonium glufosinate resistance. One skilled in the art is familiar with the composition of suitable regeneration media.

[0107] Alternatively, other selection methods could be applied, without the insertion of any gene marker in the host genome (receptor organism) as described before.

[0108] Promoters suitable for plant expression may be isolated from plants or from other organisms. Several promoters have been isolated or developed including constitutive promoters, "on and off" promoters, and promoters that are responsive to tissue- specific abiotic stresses, among others. Many of these promoters have intronic sequences described as relevant for proper gene expression. In a preferred aspect of the invention, promoters are constitutive promoters and may be selected from the non-limiting group consisting of CaMV 35s, CoYMV (Commelina yellow mottle virus), FMV 35s, Ubiquitin, Actin Rice Promoter (Act-1), Act -2, nopaline synthase promoter (NOS), octopine synthase promoter (OCS), com alcohol dehydrogenase promoter (Adh-1), PvUbil, SCBV, among others. [0109] Additional elements such as introns, enhancer sequences and transporters may also be incorporated into the expression cassette for the purpose of enhancing gene expression levels, for example, transcriptional or translation enhancers such as CaMV 35s enhancers, FMV 35s, Nos, supP, non-translated leader sequence from wheat major Chlorophyll a/b-Binding Polypeptide (L-Cab), kosak sequences 5’ upstream from the translational start site, among others.

[0110] Terminator sequences are also contemplated on the expression cassettes. Examples of suitable and functional plant polyadenylation signals include those from the Agrobacterium tumefaciens nopaline synthase gene (nos), proteinase inhibitor II gene rbcS (pea ribulose- 1,5-bisphosphate carboxylase small subunit), Lhcbl (tobacco chlorophyll a/b- binding proteins), CaMV 35s, octopine synthase, alpha-tubulin gene, among others.

[0111] According to the invention, the polynucleotide encoding the protein may have optimized (or otherwise altered) codons to improve expression in plant material. Such codon optimization may be used to alter the predicted secondary structure of the RNA transcription product produced in any transformed cell or to destroy the cryptic RNA instability elements present in the unchanged transcription product, thereby enhancing the stability and/or availability of the transcription product in the transformed cell.

[0112] Several marker genes for plant event selection have already been characterized, including some that confer tolerance to antibiotics and others that confer resistance to herbicides. Examples of marker genes that may be selected for use in the present invention include those that confer resistance or tolerance to hygromycin, kanamycin, gentamicin, geneticin, glyphosate, ammonium glufosinate or resistance to toxins such as eutypine. Other forms of selection are also available such as hormone-based selection systems, visual selection through expression of fluorescent proteins, mannose isomerase, xylose isomerase, among others. In one embodiment of the present invention, the event selection marker gene is one which confers tolerance to kanamycin and geneticin.

[0113] The use of selection marker genes, such as the nptll gene, is important for selecting cells transformed in the process of genetic modification (HORSCH et al., 1985). The objective of inserting the nptll gene in the event of the present invention was, therefore, the selection of cells transformed with the target gene.

[0114] Suitable methods for detecting plant material derived from a genetic modified plant (event) based on antibody binding include (but are not limited to): western blots, ELISA (Enzyme-Linked ImmunoSorbent Assays), and mass spectrometry (e.g. surface-enhanced laser desorption/ionization (SELDI)). One of skill in the art is familiar with these immunological techniques. Typical steps include incubating a sample with an antibody that binds to the protein, washing for removal of unbound antibody, and detecting whether the antibody has bound. Many such detection methods are based on enzymatic reactions: for example, the antibody may be linked with an enzyme such as peroxidase and upon application of a suitable substrate, a color change is detected. Such antibodies may be monoclonal or polyclonal.

[0115] A method of detecting plant material from an event includes, but is not limited to, biological feeding assays where a leaf or other suitable part of the plant of an event, or any plant material derived from event, is infested with one or more insect pests. Measurement of said detection can include assessing leaf or plant damage after adjusted time periods, assessing mortality or assessing other insecticidal effects. Such biological assays may be performed in the field or greenhouses and may entail either natural or artificial insect infestation.

[0116] In another embodiment of the present invention, said kit may comprise antibody binding detection technology such as western blots, ELISAs, mass spectrometry (SELDI) or test strips. In a further embodiment of the present invention, said kit may comprise detection technology by biological insect testing such as leaf feeding biological assays or biological mortality assays. In a further embodiment of the present invention, said kit may comprise any combination of the detection technologies mentioned above.

[0117] Having generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein to further illustrate the disclosure and are not intended to limit the scope of the invention as defined by the claims.

[0118] The following words and phrases have the meanings set forth below.

[0119] As used herein, the term “associated nucleic acids” or “operatively linked nucleic acids” describes at least two nucleic acids that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be “associated with” a DNA sequence that codes for RNA or a protein if the two sequences are operatively linked or situated such that the regulatory DNA sequence will affect the expression level of the coding or structural DNA sequence. [0120] As used herein, the term “biological insecticidal composition” is used in reference to a substance, compound, or mixture that exhibits pesticidal activity against insects and includes at least one aspect that is biologically derived. Many embodiments of the present disclosure are derived from bacteria and/or plants.

[0121] As used herein, “controlling an insect/insect population” means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, and/or reproduce, or to limit insect - related damage or loss in crop plants. To "control” insects may or may not mean killing the insects.

[0122] As used herein, “delivery” refers to bringing a composition (in most preferred embodiments here, the insecticidal protein) into contact with an insect, resulting in a toxic effect and control of the insect. The insecticidal protein may be delivered in many recognized ways, e.g., through a transgenic plant expressing the insecticidal protein, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized toxin delivery system.

[0123] As used herein, the terms “identity” and “percent identity” refer to the degree of similarity between two nucleic acid or protein sequences. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. The phrase "substantially identical,” in the context of two nucleic acids or two amino acid sequences, refers to two or more sequences or subsequences that have at least about 50 % nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, substantially identical sequences have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70 %, 75%, 80%, 85%, or even at least about 90% or 95% nucleotide or amino acid residue identity. In certain embodiments, substantial identity exists over a region of the sequences that is at least about 50 residues in length, or over a region of at least about 100 residues, or the sequences are substantially identical over at least about 150 residues. In further embodiments, the sequences are substantially identical when they are identical over the entire length of the coding regions. [0124] As used herein, the term “insecticidal” is a descriptor attributing pesticidal activity against insects.

[0125] As used herein, the term “isolated toxin” refers to a toxin that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated toxin may exist in a purified form or may exist in a non-native environment such as, for example without limitation, a recombinant microbial cell, plant cell, plant tissue, or plant.

[0126] As used herein, the term “promoter” refers to a recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.

[0127] As used herein, the term “protein” refers to an organic compound consisting of one or more chains of amino acids. These amino acids may be natural, unnatural, or a combination of natural and unnatural amino acids. The terms "protein," "peptide,” and “polypeptide” are used interchangeably herein.

[0128] As used herein, the term “recombination” refers to any modification, alteration or manipulation of a polynucleotide or protein. The terms “recombinant” and “genetic modification” are used interchangeably and are in any modification, alteration or manipulation of a polynucleotide or protein in its native form or structure, or in its native environment or context. For modification, alteration or manipulation of a polynucleotide or protein, one or more nucleotide or amino acid deletions, the production of a fusion protein from two component polypeptides of heterologous origin to each other, whole gene deletions, gene codon optimization, amino acid conservative substitutions, or one or more heterologous polynucleotides insertion may be included, but are not limiting examples.

[0129] As used herein, the term “susceptible insect larva” refers to an insect larva which, upon having orally ingested a sample of diet containing one or more of the proteins of the present disclosure, the diet being either artificially produced or obtained from a plant tissue artificially coated with or expressing one or more of the proteins of the present disclosure from a recombinant gene or genes, is growth inhibited as measured by failure to gain weight, molting cycle frequency inhibition, observed lethargic behavior, reduction in frass production, or death in comparison to either 1) a larva which does not exhibit any of these indications when feeding upon the same diet provided to a susceptible larva, or 2) a larva which is feeding upon a control diet which does not contain the one or more proteins of the present disclosure.

[0130] As used herein, the term “transformation” refers to a process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication. Transformation includes stable or transient transformation of an exogenous DNA sequence.

[0131] As used herein, the term “transformed cell” refers to a cell whose genetic composition, either chromosomal DNA or other naturally occurring intracellular DNA, has been altered by the introduction of an exogenous DNA molecule into the genetic composition of that cell.

[0132] As used herein, the term “treatment” or “treatments” refers to the application of a composition for the purpose of insecticidal activity against a target insect pest. Insecticidal treatments are commonly applied to plants or planting areas for controlling insect pests.

[0133] As used herein, the term “vector” refers to a DNA molecule capable of replication in a host cell and/or to which another DNA sequence can be operatively linked so as to bring about replication of the attached sequence. A plasmid is an exemplary vector.

[0134] Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G) Amino acids are likewise indicated by the following standard abbreviations: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q) , glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0135] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods are well known to those skilled in the art and are described infra.

EXAMPLES

Example 1. Selection of SCW123 for use in bioassays against insects.

[0136] This example describes the selection process for the SCW123 protein, and the production of the SCW123 protein for use in bioassays against insects. Identification of SCW123 as a candidate protein

[0137] In order to identify possible candidate proteins that may be effective against insects and/or specifically against Coleopteran insects such as Sphenophorus levis, protein sequences were analyzed for similarity to known Cry proteins.

[0138] A literature review revealed that SCW123 was disclosed by Agarwal et al. and designated AXMI068 (Agarwal, A., et al. 2009 W02009158470A2), but only had a moderate effect against Caenorhabditis elegans. SCW123 has 71.4% identity to Crylldl, and 70% identity to Crylla and Cryllal2. Grossi de Sa et al. disclosed Crylla activity against some insects, such as the Coleopteran pest Anthonomus grandis (cotton boll weevil) (Grossi de Sa et al. 2014 W02014100880). Seguin et al. disclosed Cryllal2 activity against the Coleopteran pest Diabrotica virgifera.

[0139] Further searches did not reveal any data demonstrating an effect or lack thereof of SCW123 on any insects. Thus, whether SCW123 may have an effect against insects, and more specifically against Coleopteran insects, and in particular against S. levis, was unknown. The potential strength of such effect(s), if existent at all, was further unknown.

Synthesis and cloning of candidate Cry proteins

[0140] The coding sequences of SCW123 (SEQ ID NO: 2) and Crylla (referred to herein as SCW19) as a control were synthesized and cloned into the pD424-CH E. coli expression vector. This vector was IPTG inducible and generated recombinant proteins fused to a C- terminal Hexahistidine (His-6) tag.

Small-scale expression screen

[0141] The vector constructs were then transformed into the E. coli BL-21(DE3) expression strain. A small-scale expression screen followed, which was based on a 4 mL induction, with cell lysis and small-scale Immobilized Metal Affinity Chromatography (IMAC). The presence of proteins expressed in the soluble form was detected by SDS-PAGE, after elution from the IMAC resin with a buffer containing imidazole. The SCW123 protein and SCW19 were detected by SDS-PAGE and thus considered to be positive clones.

Scaled-up expression

[0142] SCW123 and SCW19 expression was then scaled-up in 4 L of autoinduction media (F. W. Studier, Protein Expr. Purif., 41, 207-234. 2005). After 48h of induction at 20°C, the cells were harvested, and lysed by treatment with lysis buffer containing lysozyme. This was followed by freeze/thaw and sonication. The lysate was then centrifuged, and the supernatant was submitted to IMAC purification in an Akta Pure® FPLC System (GE Healthcare). The purified eluted samples were dialyzed against Buffer A (TrisCi 50 mM, NaCl 300 mM, Glycerol 10%), and quantified by BCA assay. This was followed by protein purity evaluation by SDS-PAGE densitometry. The final protein samples were concentrated to 500 pg/mL for further testing in S. levis bioassays.

Results

[0143] When expressed, both proteins demonstrated positive results of solubility. These positive clones were then scaled up for use in bioassay analysis.

Example 2. Bioassays of SCW123 protein compared to SCW19/CrylIa

[0144] This example describes methods of testing the efficacy of SCW123 identified through the methodology of Example 1. More specifically, this example describes oral delivery of soluble recombinant Cry proteins to Sphenophorus levis, the sugarcane weevil (S. levis). SCW123 resulted in significantly higher stunting or mortality in S. levis compared to SCW19.

Materials and Methods

Sphenophorus levis rearing

[0145] Neonate larvae of Sphenophorus levis were required for bioassays. Rearing S. levis was, however, challenging due to the low viability of eggs and larvae. The larval phase of S. levis varies from 26 to 50 days, and has a viability of only 35.8%, while the pupal period lasts from 5 to 13 days and has a viability of 93% (Degaspari, N. et al. Biologia de Sphenopherus levis Vaurie, 1978 (Col.: Curculionidade), em dieta artificial e no campo.

Pesquisa Agropecuaria Brasileira, Brasilia, DF, v. 22, n. 3, p. 553-558, 1987). This resulted in a slow growth of the insect population and required constant collection of adults from the field to supplement the population available for testing. Since Sphenophorus levis has a seasonal cycle, it was only possible to collect a substantial number of adults from October to March. In the beginning of the bioassay screening phase, the larval availability was around 25 larvae/day, but toward the end of September and October, larval availability for bioassays increased.

Bioassay

[0146] The purified recombinant Cry proteins (selected in Example 1) at 500 pg/mL were incorporated into S. levis artificial diet at 45°C (max.) in the proportion of 1 part of protein solution to 4 parts of diet, resulting in 100 pg Bt protein / mL of diet. After incorporation, the diet was dispensed uniformly into 96-well assay plates (300 pL of diet per well), infested with one neonate S. levis larva per well, and incubated at 25 °C for 7 days.

[0147] The Cry protein concentration used in the bioassays (100 pg/mL of diet), was selected on the basis of literature LC50 values of coleopteran- specific Bt proteins, which range from 0.1 to 5.1 pg/mL (G. R. Oliveira et al., BMC Biotechnol. 11, 85. 2011; M. Ekobu et al. J. Econ. Entomol. 103, 1493-1502. 2010; I. O. Oyediran et al. Insect Sci. 23, 913-917. 2016). It was expected that toxic proteins would trigger more than 50% mortality of S. levis larvae. Therefore, the 100 pg/mL of diet protein concentration ensured that Cry proteins that did not result in S. levis mortality were non-toxic to S. levis.

Toxicity screening protocol

[0148] The screening protocol consisted of bioassays using the same concentration of protein (100 pg/mL of diet). After 7 days of incubation, the percentage of mortality was calculated, taking into consideration both dead larvae and practical mortality (low active larvae after touching with a brush). LC50 was also calculated for each purified recombinant Cry protein. As explained above, S. levis have low larval viability and, for this reason, the screening bioassay controls consistently showed 30-40% mortality. Herein, higher mortality rates resulted from higher toxicity of the Cry proteins.

Results

[0149] The results of recombinant Cry protein toxicity screening are displayed in Table 1 and summarized in FIG. 1.

Table 1: Results of Bioassay Against S. levis

[0150] In FIG. 1, relative mortality is reported as a percentage of mortality above the expected control group mortality, i.e., the negative control mortality percentage is subtracted from the mean practical mortality percentage, corresponding to the “corrected” mortality rate displayed on the right-most column in Table 1. FIG. 1 illustrates the superior mortality rates exhibited by SEQ ID NO: 1 compared to SCW19. [0151] These surprising results display especially high toxicity of SCW123 protein against Sphenophorus levis.

Example 3. Bioassays of SCW123 protein variants compared to SCW19

[0152] This example describes methods of testing the efficacy of SCW123 variants. More specifically, this example describes oral delivery of soluble recombinant and/or variant SCW123 proteins to Sphenophorus levis, the sugarcane weevil (SCW). SCW123 variants are expected to result in significantly higher stunting or mortality in S. levis compared to SCW19.

Synthesis and cloning of candidate Cry proteins

[0153] Variants of the SCW123 protein described in the Examples above are constructed, including variants containing one or more of the following modifications, alone or in any combination: point mutation(s), domain swap(s), truncation(s), and other variation(s) in amino acid sequence and/or protein structure. Synthesis and cloning of these variants is done as described in Example 1.

Expression

[0154] The variants are expressed in small-scale expression screens and scaled up expression screens as described in Example 1. The variants are expected to demonstrate positive results of solubility. The positive clones are then be scaled up for use in bioassay analysis.

Sphenophorus levis rearing

[0155] S. levis is reared for use in the bioassays as described in Example 2.

Bioassay

[0156] Bioassays on the SCW123 variants are conducted as described in Example 2.

Toxicity screening protocol

[0157] The screening protocol is conducted as described in Example 2.

Results

[0158] The results of the bioassays are analyzed and displayed as in Example 2. The SCW123 variants are expected to have approximately equal to or higher toxicity against Sphenophorus levis compared to the SCW123 protein tested in Example 2, and significantly higher stunting or mortality in S. levis compared to SCW19.

Example 4. SCW123 in planta expression and efficacy [0159] This example describes the use of SCW123 in planta for controlling S. levis in sugarcane. Specifically, this example describes the production of constructs for expression of SCW123, transformation of the constructs into sugarcane, assessment of SCW123 protein presence in sugarcane, determination of the number of transgene copies inserted, and bioassays of the inserted transgene.

Materials and Methods

Construct development using SCW123 and nptll genes

[0160] Conventional gene cloning techniques using commercial bacterial plasmids, restriction enzyme digestion, and fragment ligation (with ligases) are used to develop the construct for expressing embodiments of the present disclosure.

[0161] The construct of the present disclosure is developed by joining SCW123 with nptll cassettes. T-DNA containing both cassettes is transferred from a cloning plasmid to the base plasmid (binary plasmid vector, which contains in its host spectrum the bacteria E. coli and Agrobacterium tumefaciens') using restriction enzymes, generating a construct.

[0162] After the final cloning step, the construct is inserted into a suitable E. coli strain using heat shock. An isolated colony containing the construct is inoculated into liquid LB medium supplemented with 150 pg/mL spectinomycin and incubated at 37 °C while shaking at 250 rpm for a period of 16 hours. Stocks are then prepared containing bacterial suspension and 10% (v/v) glycerol, which are stored in an ultrafreezer at -80 °C.

[0163] The construct of the present invention is then transferred from E. coli to a suitable Agrobacterium tumefaciens strain by isolation and purification of plasmid DNA and transformation of Agrobacterium by electroporation. As with the E. coli strain, stocks containing the bacterial suspension of Agrobacterium and 10% (v/v) glycerol are stored in an ultrafreezer at -80 °C.

Agrobacterium-mediated plant transformation

[0164] To obtain embryogenic callus tissue, young sugarcane leaf rolls, grown in the field or greenhouse for up to 12 months, are collected for isolation of the initial explants.

[0165] After surface disinfection, transverse sections about 0.05 - 5 mm thick are cut from above the meristem under aseptic conditions. The sections are placed on the surface of the callus induction culture medium [MS - Murashige and Skoog, from Murashige and Skoog (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Plant Physiology, 15, 473-497; sucrose, vitamins B5, amino acids selected from the group comprising proline, casein hydrolyzate, citric acid, mannitol, copper sulfate, glycine, gelling agent, 2,4D]. The cultures are kept in the dark at 26 ± 2 °C and sub-cultured every 15 days for three to five cycles of 7-28 days each. One week before transformation, calli are again selected for characteristics favoring embryogenesis: nodular, compact, opaque, and slightly yellowish.

[0166] Agrobacterium culture, comprising a suitable strain transformed with the construct of the present disclosure, is started from a glycerol stock and kept in the dark at 28 °C for two to three days. The Agrobacterium suspension is prepared by resuspending the culture in MS liquid medium plus acetosyringone, adjusting to a final ODeoo of 0.1-1.0 (MS salts, sucrose, and vitamins B5) for infection of calli.

[0167] The calli with embryogenic characteristics are visually selected and directly transferred to the Agrobacterium suspension, where they remain for 30 minutes in the dark with constant agitation at 50 rpm.

[0168] After this period, calli are separated from the Agrobacterium suspension and excess suspension is removed. Then, calli are cultured for 1-5 days in semi-solid medium (MS salts, sucrose, vitamins B5, citric acid, gelling agent, 2,4D and acetosyringone) at 22 °C in the dark.

[0169] After co-cultivation, callus is transferred to DT rest medium (MS salts; sucrose, B5 vitamins, amino acids selected from the group comprising proline and asparagine, casein hydrolyzate, citric acid, copper sulfate, glycine, gelling agent, 2,4D, timentin) and kept for 5- 14 days at 26 °C in the dark.

[0170] Transformed cells are selected by successive sub-cultures in selection culture medium containing phytoregulators and the selective agent geneticin. The selection medium with geneticin includes: MS salts, sucrose, vitamins B5, amino acids selected from the group comprising proline and asparagine, casein hydrolyzate, copper sulfate, glycine, gelling agent, 2,4D, and timentin. The calli remain in this condition for 21 days at 26 °C in the dark, and then the calli are transferred to the regeneration medium (equivalent to selection medium without 2,4D) and then to elongation medium (including MS salts, sucrose, B5 vitamins, casein hydrolyzate, gelling agent, and timentin). The calli are exposed to a 16-hour photoperiod at 4,000 lux in the presence of the selective agent, and then they are multiplied, rooted, and acclimatized before transfer to the greenhouse. This process is used to generate the clones expressing the protein of interest.

Enzyme-Linked Immunosorbent Assay (ELISA}

[0171] To evaluate SCW123 gene expression via ELISA, different sugarcane tissues are studied at different stages of crop development.

[0172] For the analysis of the SCW123 protein via ELISA, samples of 200mg ± 1 of vegetal tissue are macerated using TissueLyser equipment (QIAGEN, Germantown, Maryland, USA). To the macerated tissue is added 350 pL saline phosphate extraction buffer (PBS) supplemented with Tween™ 20 (0.138 M NaCl; 0.027 mM KC1; 0.05% Thermo Scientific™ Tween™ 20, pH 7.4).

[0173] After buffer addition, vortex homogenization is performed, followed by centrifugation for 20 minutes at maximum speed. The resulting supernatant is collected, and total protein is quantified using the Bradford assay (SCW123).

[0174] The standards used for obtaining the calibration curve are the already-diluted commercial BSA (Bovine Serum Albumin) standards supplied with the kit described above. The 2000, 1000, 500, 250, 125, and 0 pg/mL calibrators (prepared in PBST buffer) are used.

10 pL of each standard calibrator is added in triplicate to plate wells. In total, 6 curves are generated from independent dilutions. For the samples, lOpL of the 3 individual protein extractions are used in each well. Then 200pL of Coomassie Plus Reagent Solution is added to each well containing the calibrators and samples. The plates are covered and incubated for 5 minutes at room temperature. Absorbance is read at 595 nanometers (nm) using SoftmaxPro® 7.0 software (Molecular Device, US).

[0175] Total soluble proteins are obtained in triplicate for each sample studied. After the total protein quantification of each replicate, the sample with the smallest variation of the median quantification value is chosen for ELISA analysis. After quantification of total proteins, samples are diluted 8x.

[0176] Results are obtained by 96-well plate spectrometry reading at two different wavelengths: 450 nm and 630 nm on a SpectraMax® Plate reader (Molecular Devices, USA). SCW123 protein is detected and quantified using a His-tag commercially available kit.

[0177] The analysis is based on the association of the absorbance values of the test samples with the predicted values in an equation estimated by measuring the absorbance of a standard curve. Synthetic proteins are diluted to desired concentrations in PBST buffer. Analysis is performed in experimental duplicate for each sample.

Determination of the number of transgene copies inserted into the host plant germplasm

[0178] The copy number of SCW123 and nptll genes inserted into plants is initially evaluated by quantitative Taqman® PCR (qPCR/Taqman®). The Taqman® real time PCR reactions are realized with QuantStudio 6 and 7 Flex Real-Time PCR (Applied Biosystems™, EUA) in the Fast mode. As endogenous, positive controls of the SCW123 and nptll reactions (to confirm the presence and quality of the used DNA, as well as effectiveness of the reaction), the sugarcane polyubiquitin gene (forward primer: 5 ’ACCATTACCCTGGAGGTTGAGA 3 ' (SEQ ID NO: 4); reverse primer: 5 ’GTCCTGGATCTTCGCCTTCA 3 ' (SEQ ID NO: 5); and probe: VIC -5 ’CTCTGACACCATCGAC 3’-MGB (SEQ ID NO: 6) are used in multiplex mode.

[0179] The qPCR reactions use IX TaqMan® Fast PCR Master Mix II (Applied Biosystems, USA) with 150 - 300 nM from each primer and 100 - 200 nM from the corresponding probes. The thermocycling programming used is: a 50 °C cycle for 2 minutes for uracil N-glycosylase activation, a 95 °C cycle for 20 seconds for DNA polymerase activation, 40 cycles of 95 °C for 3 seconds (denaturation), and 60 °C for 30 seconds (annealing and extension).

[0180] Data analysis is performed by manually entering the threshold at the exponential phase of the amplification curve. For SCW123 and Nptll genes, the copy number is inferred from DeltaCt (dCt) analysis, in which the Ct (cycle at which the fluorescence signal emitted by the amplification product reaches the threshold) of the endogenous gene is subtracted from the Ct of the target gene. In this type of analysis, the number of copies is assumed to double every Ct and the reference number of control copies of the same variety whose value is known is taken as a reference.

In vitro biological tests: Sphenophorus levis

[0181] In vitro biological assays (feeding bioassays) with target pest S. levis are performed with the events generated at Example 3, demonstrating the efficacy on the pest control provided by the in planta expressed insecticidal protein SCW123.

[0182] For biological assay, plant tissue of genetically modified sugarcane plants (successful events) and non-transgenic sugarcane plants (negative control) are collected, lyophilized, mixed with the insect gelled diet, and distributed in bioassay plates. Each well from culture plates is infested with S. levis neonates and incubated at 25 ± 1°C, relative humidity 60 ± 10%, and photoperiod 12:12h (light:dark) for a period of 7 days. At the end of incubation, larval mortality is evaluated.

Results

[0183] As a result of the transformation events conducted for SCW123 and Nptll, the presence of at least one copy of the SCW123 gene and the Nptll gene is found in the event’s genomes (one copy in each of the two, replicated experiments), the assay of the latter of which is based on detection of the gene promoter. Events with the presence of 1 or 2 copies of the SCW123 gene and the Nptll gene are selected to perform the in vitro biological test.

[0184] An average of relative efficacy is determined for the modified plants (SCW123 expressing plants) after 7 days of biological assay.

Claims

CLAIMS What is claimed is:

1. A recombinant polypeptide having at least 40%, at least 60%, at least 90%, or 100% sequence identity to SEQ ID NO: 1, and/or variants or fragments thereof.

2. A recombinant polypeptide comprising at least one amino acid substitution, deletion, and/or insertion compared to SEQ ID NO: 1; and/or at least one addition at the N-terminus or C-terminus compared to SEQ ID NO: 1.

3. The recombinant polypeptide of any one of claims 1-2, wherein the recombinant polypeptide is a fusion protein comprising at least a portion of the polypeptide of any one of claims 1-2, variants, and fragments thereof.

4. The recombinant polypeptide of any one of claims 1-3, wherein the polypeptide has insecticidal activity against at least one agricultural insect pest, wherein the at least one insect pest is a Coleopteran pest, and optionally wherein the Coleopteran pest is Sphenophorus levis.

5. A recombinant polynucleotide encoding the polypeptide of any one of claims 1-4, optionally wherein the recombinant polynucleotide comprises SEQ ID NO: 2.

6. The recombinant polynucleotide of claim 5, wherein the polynucleotide has codons optimized for expression in an agriculturally important crop.

7. The recombinant polynucleotide of claim 5 or 6, wherein the polynucleotide is a non- genomic polynucleotide, optionally wherein the polynucleotide is a synthetic polynucleotide, and/or wherein the polynucleotide is a cDNA.

8. An isolated construct or expression cassette comprising a nucleotide encoding the polypeptide of any one of claims 1-4 or the polynucleotide of any one of claims 5-7, wherein the nucleotide or the polynucleotide is operably linked to a promoter, and optionally operably linked to a heterologous regulatory element.

9. The isolated construct or expression cassette of claim 8, wherein the polypeptide comprises at least SEQ ID NO: 1; and/or wherein the polynucleotide comprises SEQ ID NO:

2.

10. The isolated construct or expression cassette of claim 8 or claim 9, wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a tissue-specific promoter.

11. A transgenic plant, plant part, propagule, seed, tissue, organ, embryo, or plant cell comprising the polypeptide of any one of claims 1-4, the polynucleotide of any one of claims 5-7, or the isolated construct or expression cassette of any one of claims 8-10; optionally wherein the polypeptide is SEQ ID NO: 1; and/or optionally wherein the polynucleotide is SEQ ID NO: 2.

12. A pesticidal composition comprising:

(i) one or more polypeptides of any one of claims 1-4, wherein the one or more polypeptides are present at a concentration sufficient to control at least one agricultural insect pest;

(ii) one or more polynucleotides of any one of claims 5-7, wherein the polynucleotide has codons optimized for expression in an agriculturally important crop; and/or

(iii) one or more isolated constructs or expression cassettes of any one of claims 8-10; optionally wherein the one or more polypeptides are SEQ ID NO: 1; and/or optionally wherein the one or more polynucleotides are SEQ ID NO: 2.

13. The pesticidal composition of claim 12, wherein the at least one agricultural insect pest is a Coleopteran pest, and wherein the one or more polypeptides are present at a concentration sufficient to control at least one agricultural insect pest in or on a plant when the composition is applied to the plant or to a plantation, optionally wherein the plant is a sugarcane plant.

14. The pesticidal composition of claim 12 or claim 13, further comprising one or more inert ingredients and/or acceptable carriers;

(i) wherein the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste, a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier;

(ii) wherein the composition is formulated as an orally acceptable, orally administrable, or orally ingestible diet intended for consumption by the insect pest; (iii) wherein the composition is formulated for direct soil application and/or direct plant pot substrate application; and/or

(iv) wherein the composition is formulated as a controlled release formulation.

15. The pesticidal composition of any one of claims 12-14, wherein control of the Coleopteran pest comprises: a) decreasing pest infestation by 40%, 50%, 60%, 70%, 80%, 90%, or 100%; or b) increasing pest mortality by 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

16. The pesticidal composition of any one of claims 12-15, wherein the Coleopteran pest is Sphenophorus levis.

17. A method for controlling an insect pest population, comprising: a) providing a composition comprising at least one polypeptide of any one of claims 1-4 or providing the composition of any one of claims 12-16; and b) contacting an insect pest population with an effective amount of the composition; optionally wherein the at least one polypeptide is SEQ ID NO: 1.

18. The method of claim 17, wherein

(i) the contacting of step (b) comprises one or more of: providing the pest with the composition formulated as an insect diet; feeding the composition to the pest; applying the composition to the exterior surface of the pest; applying the composition to a plant; applying the composition to a part of a plant where the pest feeds; applying the composition to a soil area where the pest may be present; applying the composition to an area where the pest population may be present; providing the composition formulated as a controlled release formulation to an area where the pest is expected to be; applying the composition to a trap for the insect pest; injecting the composition into a plant; or injecting the composition into the pest; or

(ii) the contacting of step (b) comprises applying the composition to a plant or an area to be planted, optionally wherein the composition is applied to the plant as at least one of a foliar treatment, a seed coating, an injection treatment, a pre-emergence treatment, and/or a postemergence treatment.

19. The method of claim 17 or claim 18, wherein the composition is formulated as a suspension, a solution, an emulsion, a dusting powder, a dispersible granule or pellet, a wettable powder, an emulsifiable concentrate, an aerosol, a spray, an impregnated granule, an adjuvant, a paste, a colloid, a culture medium, an artificial diet, or an encapsulation in an agricultural acceptable carrier; and/or wherein the composition is prepared through desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, cryopreservation, or concentration.

20. The method of any one of claims 17-19, wherein the pest is contacted with an effective amount of the composition by feeding, spraying, dusting, coating, or wetting with the composition, or any combination thereof.

21. The method of any one of claims 17-20, wherein the insect pest population is decreased by 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to an insect pest population not contacted with the composition; optionally wherein the insect pest or insect pest population is resistant to at least one Bt toxin.

22. The method of any one of claims 17-21, further comprising providing a chemical mixture, a pesticidal protein, and/or a biological control agent, and contacting the insect pest population with an effective amount of the chemical mixture, the pesticidal protein, and/or the biological control agent before step (b), in step (b), or after step (b).

23. The method of any one of claims 17-22, wherein the insect pest is a Coleopteran pest, and optionally wherein the Coleopteran pest is Sphenophorus levis.

24. A method of controlling an insect pest population, comprising: a) providing a pesticidal composition comprising one or more of the polypeptides of any one of claims 1-4; b) introducing the pesticidal composition to the insect pest population, wherein introducing is through providing the composition in or on a food source for the insect pest; and wherein the insect pest population is decreased.

25. Use of the polypeptide of any one of claims 1-4 to inhibit growth of an insect, control or kill an insect, and/or control or kill an insect population.