Nematode toxins and methods of use

Abstract

The described technology pertains to the field of agricultural pest control and addresses the technical challenge of insect damage to crops caused by pest resistance to existing control methods. The disclosed approach involves a method for producing insecticidal toxin compositions derived from nematodes. Hie method includes activating nematodes using a waxworm homogenate, inducing the release of excretory / secreted proteins (ESPs), and isolating the insecticidal toxin composition. The composition, which includes proteins such as NIP-1, exhibits oral toxicity to pest insects, including Spodoptera frugiperda, and can be applied to plants as a spray. Primary-- applications include pest control in agricultural systems to reduce crop damage and improve food security. The described technology facilitates large- scale production of ESPs for use as protein insecticides.

Description

[0001]UC-2024-9CR-2-WO / / 3868.092WO1 NEMATODE TOXINS AND METHODS OF USE PRIORITY This application claims the benefit of the filing date of U.S. Application No. 63 / 662,921, filed on June 21, 2024, the disclosure of which is incorporated by reference herein. STATEMENT OF GOVERNMENT RIGHTS This invention was made with government support under grant R35GM137934 awarded by the National Institutes of Health, and grants 2020-67013-31855 and 2024-67014- 42319 awarded by the United States Department of Agriculture. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on June 20, 2025, is named 3868092WO1.xml and is 2,258 bytes in size. BACKGROUND Food security is one of the most important global challenges of our age and insect pests continue to be a problem in agricultural systems, contributing to between 36%-42% losses in some staple crop systems. Reducing crop damage by pest insects is one strategy to increase food production and security. Toxins from the insect pathogen Bacillus thuringiensis (Bt) are widely used in biological control. Bt toxins, which are specifically toxic to insects and not mammals, are applied (sprayed) on crops and produced by transgenic crops to control insect pests in agriculture. However, few of the transgenic Bt technologies currently available for cultivation can prevent infestation by sap-sucking pests, and resistance to these toxins in some populations of insect pests is a problem, making Bt toxins less effective. One way to combat resistance to Bt toxins is to use a variety of toxins with different modes of action, including the development of novel toxins. SUMMARY As disclosed herein, insect-parasitic nematodes, which are considered "beneficial nematodes" since they kill insect pests, produce and release insecticidal proteins or other toxins into their hosts. Those insecticidal toxins have oral toxicity to some insect pests. The present disclosure provides methods to produce those toxins in vitro, meaning without infecting a host, and use of those toxins. In one embodiment, the disclosure provides an in vitro method to produce an insecticidal toxin composition. The method comprises contacting nematodes with a homogenate of waxworm that includes powdered and chunky waxworm tissue, a waxworm- UC-2024-9CR-2-WO / / 3868.092WO1 derived fat layer, and a liquid carrier; incubating the nematodes in the presence of the homogenate for a period sufficient to activate them and induce the release of excretory / secreted proteins; washing the activated nematodes to remove the homogenate; incubating the washed, activated nematodes in a buffer solution for a time sufficient to permit secretion of the excretory / secreted proteins into the buffer; and filtering the buffer solution to remove the nematodes, thereby obtaining an isolated insecticidal toxin composition comprising the excretory / secreted proteins. Additional embodiments include the use of sponge pieces (e.g., small fragments or sections of a sponge material, such as a porous, absorbent material that can retain and distribute the insect homogenate, where the material is often inert and non-toxic to the nematodes) saturated with the homogenate during the contacting step, incubating at about 25°C (such as about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, or about 40°C etc.) for periods ranging from 0.1 to about 18 hours (including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours) during activation, and further incubating with agitation at about 25°C (such as about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, or about 40°C etc.) for about 0.1 to 10 hours (including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 hours) to enhance protein secretion. In some embodiments, the waxworm homogenate is prepared by grinding waxworm tissue in liquid nitrogen and suspending the resulting powder in a liquid carrier so that a lipid layer is retained and equally distributed. The nematodes employed may be entomopathogenic, such as infective juveniles, and may include species like Steinernema and / or Heterorhabditis, for example, S. carpocapsae, S. feltiae, or S. hermaphroditum. In certain embodiments, the insecticidal toxin composition comprises excretory / secreted proteins including NIP-1 (Nematode Insecticidal Protein 1; is a protein identified in the excretory / secreted products (ESPs) of, for example, entomopathogenic nematodes (EPNs), it is one of the insecticidal proteins secreted by nematodes during their infection of insect hosts), and is used against insect pest insects, such as those that chew or suck on plants, including pests like Spodoptera frugiperda, Manduca sexta, or Plutella xylostella. NIP-1 sequence can be found under the accession number L596_023060 in WormBase Parasite (https: / / parasite.wormbase.org / index.html). (L596_023060) MRTLFVLFALIVTVSGIPLQGFAAPHVEQIFSEPALFPLDPEEFFRNLDKVNTTRFVYGGQEARPGQFPQHAFML YKTAEGFFICGASLLSPTHALTAAHCVEGMMAPSQIMAGGLNRRDRRAPNAQWRSIHRATKHAEYKPPSKRNDIA UC-2024-9CR-2-WO / / 3868.092WO1 IVEFHPPMTLNRDVQLTKIVEDDAELLQEKKSYVTGFGTYTFKNKQAVTSDELLWAEIDLFDFSRCQQLWHRRLW QKQICAGAKNLGAGPGDSGGPLQVLHDGTLFQVGLTSYGTSDKFDDEFNQDRFPTVFTRVSSYCDFIAKVTDNAA TCSSLSKKPTAKPDCRF (SEQ ID NO: 1). In another embodiment, the disclosure encompasses a composition comprising the insecticidal toxin composition. This composition may further include additional insecticidal compounds (e.g., neonicotinoids, organophosphates, carbamates, pyrethroids, formamidines, botanical and / or natural insecticides; examples include, but are not limited to, imidacloprid, acetamiprid, clothianidin, thiamethoxam, and / or dinotefuran) and a carrier, which can be a water-soluble liquid and / or formulated to provide sustained release. In some embodiments, the composition is adapted for aerosolization. In yet another embodiment, the disclosure features a method to induce insect mortality, developmental delay, or morphological abnormalities in an insect pest by administering the insecticidal toxin composition. This method can involve spraying the composition on plants or other surfaces frequented by the insect pests so that the pests ingest the lethal compositions, wherein the plants may include a variety of crop plants, such as tomato, cotton, maize, tobacco, eggplant, rice, wheat, potato, sugarcane, soybean, cabbage, cauliflower, bean, mustard, rapeseed, pulse, or sunflower plants. DRAWINGS The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein. FIG. 1 depicts the entomopathogenic nematode (EPN) lifecycle. Entomopathogenic nematodes are a type of insect-parasitic nematode. They kill their hosts quickly and have a partnership with bacteria to facilitate their parasitism. FIG.2 depicts EPNs. FIG.3 depicts homogenate for in vitro activation of toxin production from EPNs. The insect homogenate, which includes pieces of sponge. FIG.4 depicts in vitro activation of productions of toxins. After the nematodes (e.g., S. carpocapsae and S. feltiae) are exposed to the homogenate from FIG 3 for a period of time, usually about 0 to about 18 hours, the nematodes are cleaned and moved into clean buffer where the toxins are collected for about 3 hours. FIG.5 and FIG.6 depict injection of toxic protein, produced as previously described, into insects, so as to cause insect death. Lu et al. (2017; PloS Pathogens; Chang et al., 2019; PloS Pathogens) discussed proteins collected were toxic to insects by injection. Injecting it UC-2024-9CR-2-WO / / 3868.092WO1 mimics what would happen in natural settings. Each individual nematode releases about 0.48 nanograms of protein in a 24-hour period. Infective juvenile (IJ) is the stage that infects insects. FIG.7 demonstrates that NIP-1, a protein isolated from the toxins produced by ENPs, is toxic when orally fed to the insects. FIG. 8 demonstrates that the collected toxin mixture of excreted / secreted proteins (ESPs) is toxic to insects by feeding and shifts the developmental progress of Spodoptera frugiperda. FIG.9 provides a graphical abstract of experimental data. Developmental progression of larvae fed and control (upper panel) and venom protein mixed diet (lower panel). FIGS. 10A-10D demonstrate the effects of S. carpocapsae venom proteins on larval mortality, morphology, and development. (A) Percent mortality of the larvae fed on increasing concentrations of venom protein compared to the control. A significant increased mortality was observed at the 1000 ng dose (*p < 0.05). (B) Representative images showing morphological differences between control and treated larvae. Control larvae appeared healthy and well- developed, whereas ~25% of the treated larvae exhibited signs of melanization, deformation, and arrested development in a dose-dependent manner. (C) Average larval weight across second, fourth, and sixth instar stages under different treatment conditions. later stages showed reduced weight in treated larvae, but the difference was not significant. (D) The larval-to-pupal duration (in days) was found to be significantly prolonged in the treated groups compared to the control, with the most notable delay observed at the 1000 ng concentration (****p < 0.0001). FIGS. 11A-11E demonstrate that oral administration of the venom proteins incurred deleterious impacts on pupal development, adult emergence, and moth lifespan. (A) Representative images of pupae from control and treated groups. Control pupae appeared normal in size and pigmentation. In contrast, treated pupae were smaller and darker with clear darker lines on the ventral surface. (B) Phenotypic defects in pupae and adult moths from treated groups. Upper panels show abnormal pupae, represented by arrested melanized forms. It displays various abnormalities, including dehydrated, smaller, darker, and shrunken pupae. The extent of abnormality increased with increasing venom protein concentration and reached a maximum at the highest tested concentration (1000 ng). The middle and lower two panels depict defective moth emergence and a range of morphological deformities in the moths, respectively. The fly abnormality includes but was not limited to crumpled wings, incomplete eclosion, and deformed bodies in treated groups. (C) Duration (in days) from pupae to moth emergence increases significantly in treated groups compared to control, with the most UC-2024-9CR-2-WO / / 3868.092WO1 pronounced delay observed at 1000 ng (***p < 0.001). (D) Percent emergence of moths was significantly impaired in treated groups. It shows a dose-dependent decline in successful emergence (**p < 0.01, ****p < 0.0001). (E) Moth lifespan was significantly reduced in both male and female moths, which emerged from venom protein-fed larvae. Male moths showed prolonged delay < 0.0001) when compared with the females of the same group (**p < 0.01). FIGS. 12A-12B demonstrate that oral administration of venom proteins significantly reduced the fecundity and enhanced egg abnormality. (A) representing the egg-laying pattern of the moths, developed from the larvae that fed on the control diet (A) and 3 different concentrations of venom protein mixed diet (B-D). The leftmost circular insets show a magnified view of the overall appearance of clustered eggs in various treatment groups. Panel B-D illustrates the morphological alterations in eggs and demonstrates a clear dose-dependent reduction in both the number of eggs laid and the rate of successful emergence. Notably, at higher concentrations (especially 1000 ng / gm of diet), eggs exhibit marked deformities and significantly decreased viability. (B) The bar graph represents the number of healthy (left bars) and deformed eggs per 100 egg counts (right bars) across treatments and control. The number of healthy eggs was significantly reduced in the upper two treatment groups, in a dose- dependent manner. The data is presented as mean ± SEM. Statistical significance was assessed using ANOVA with post-hoc tests: p < 0.01, *p < 0.001, **p < 0.0001. DESCRIPTION Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter. The issue of insect pest damage to crops continues to pose a significant challenge in agricultural systems, leading to considerable losses in food production on a global scale. Current biological control methods, such as the use of Bacillus thuringiensis (Bt) toxins, have been widely implemented due to their ability to target insects without causing harm to mammals. However, these approaches encounter notable limitations. Resistance to Bt toxins has developed in certain insect populations, diminishing their effectiveness. Furthermore, existing Bt technologies are largely inadequate against sap-sucking pests, creating a gap in pest control strategies. The dependence on a limited range of toxins with similar modes of action further intensifies the problem, as it restricts the capacity to address resistance and manage UC-2024-9CR-2-WO / / 3868.092WO1 diverse pest species efficiently. Consequently, there is a demand for innovative insecticidal solutions that provide broader spectrum activity and alternative mechanisms of action. The present disclosure addresses these limitations by leveraging the insecticidal properties of excretory / secreted proteins (ESPs) produced by nematodes. Unlike conventional approaches, the disclosed method enables the in vitro production of these nematode-derived toxins without requiring the nematodes to infect a host. This process involves activating nematodes using a waxworm homogenate, inducing the release of ESPs, and isolating the resulting insecticidal toxin composition. The composition includes proteins such as NIP-1, which exhibit oral toxicity to pest insects. By enabling oral ingestion as a mode of delivery, the disclosed method expands the applicability of these toxins to sap-sucking pests and other insect species that feed externally on plants. The disclosed concept enhances previous approaches by enabling large-scale production of nematode-derived toxins, addressing the environmental and labor-intensive challenges associated with traditional pest control methods. Additionally, the described toxin mixture exhibits broad-spectrum insecticidal properties, including developmental delays, morphological abnormalities, and mortality in pest insects such as Spodoptera frugiperda. This development provides an alternative to Bt toxins and introduces a next-generation protein insecticide that can be applied as a foliar spray. By overcoming the limitations of existing technologies, the disclosed concept offers a transformative approach to advance agricultural pest management and support food security. Further examples of pest insects include, but are not limited to, harmful insects, such as phylloxera, migratory locusts, the boll weevil, Japanese beetle, aphids, mosquitoes, termites, tent caterpillars, blister beetles, bot flies, cockroaches, the Western corn rootworm, and some fly species. Other examples include, but are not limited to, pest insects that cause significant damage to crops, plants, and stored products: Chewing Insect Pests (e.g., Fall Armyworm (Spodoptera frugiperda), Diamondback Moth (Plutella xylostella), Colorado Potato Beetle (Leptinotarsa decemlineata), Cabbage Looper (Trichoplusia ni) and / or European Corn Borer (Ostrinia nubilalis); Sap-Sucking Insect Pests (e.g., Aphids (Aphis spp.), Whiteflies (Bemisia tabaci), Mealybugs (Planococcus spp.), Scale Insects (e.g., Coccus spp.) and / or Leafhoppers (Empoasca spp.)); Stored Product Pests (e.g., Red Flour Beetle (Tribolium castaneum), Rice Weevil (Sitophilus oryzae), Indian Meal Moth (Plodia interpunctella)); Fruit and Vegetable Pests (e.g., Mediterranean Fruit Fly (Ceratitis capitata), Tomato Leafminer (Tuta absoluta), and / or Codling Moth (Cydia pomonella)); and / or Soil-Dwelling Pests (e.g., Wireworms UC-2024-9CR-2-WO / / 3868.092WO1 (Agriotes spp.) and / or Root-Knot Nematodes (Meloidogyne spp.)). Pest insects are responsible for significant economic losses in agriculture and forestry, as well as health risks in some cases. Definitions The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001. For the purposes of clarity and a concise description, features can be described herein as part of the same or separate embodiments; however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations. The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di-substituted. UC-2024-9CR-2-WO / / 3868.092WO1 As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.” The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph. As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle UC-2024-9CR-2-WO / / 3868.092WO1 third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation. As used herein “carrier” refers to a substance or material used to deliver or transport active (a medium or vehicle) ingredients to their intended site of action. Carriers are typically inert or non-reactive and can be chosen to enhance the stability, bioavailability, or effectiveness of the active ingredient. In some embodiment, liquid, solid and / or encapsulation carriers are used herein. Liquid carriers include, but are not limited to, water, saline, phosphate-buffered saline (PBS), oils, or emulsions. These are commonly used in injectable or spray formulations. Solid carriers, include but are not limited to, powders, granules, or microbeads, often used in dry formulations or for controlled release. Encapsulation carriers include, but are not limited to, liposomes, nanoparticles, or polymer matrices, which encapsulate the active ingredient for targeted delivery or sustained release. “Sustained release” refers to a method of delivering an active agent that aims to gradually release the agent over an extended period, typically days or months, following a single administration. This approach allows for a prolonged effect as compared to immediate release. These include diffusion-controlled release, solvent controlled release, and / or degradation-controlled release. Various sustained release systems are available to an art worker. UC-2024-9CR-2-WO / / 3868.092WO1 As used herein “buffer” refers to a solution that resists changes in pH when small amounts of acid or base are added. Buffers are used to maintain a stable pH environment, as used inbiological and chemical processes. Buffers typically consist of a weak acid and its conjugate base or a weak base and its conjugate acid. For example, phosphate-buffered saline (PBS) contains a mixture of sodium phosphate and potassium phosphate to stabilize pH. Buffers are used to maintain the pH of a solution within a narrow range, ensuring optimal conditions for enzymatic activity, protein stability, or other biochemical reactions. Provided herein, the insecticidal toxin composition, the buffer solution is used to resuspend activated nematodes and facilitate the secretion of excretory / secreted proteins (ESPs) into the solution. The buffer ensures that the pH remains stable during the secretion process, preserving the integrity and activity of the proteins. Example of buffers include, but are not limited to, Phosphate-Buffered Saline (PBS), Tris Buffer, HEPES Buffer, MOPS Buffer, Citrate Buffer, Acetate Buffer, Carbonate-Bicarbonate Buffer, Glycine Buffer, MES Buffer or Borate Buffer. Buffers are selected based on their pH range, ionic strength, and compatibility with specific biological or chemical processes. The terms “comprises,” “comprising,” and the like can have the meaning ascribed to them in U.S. Patent Law and can mean “includes,” “including” and the like. As used herein, “including” or “includes” or the like means including, without limitation. Methods to Administer Insect Toxins to Plants In one embodiment, the toxin mixture may be sprayed onto plants or other surfaces frequented by insects or placed in container(s) frequented by insects. Non-limiting effective amounts of the toxin mixture, include but are not limited, to a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of the isolated toxin mixture in a liquid form sprayed on a plant, or from about 10 milligrams per acre to about 100 grams per acre of the isolated toxin mixture applied to a field of plants, or from about 0.001 to about 0.1 microgram per milliliter of the toxin. Where compositions as described herein are topically applied to a plant, the concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants is about 1 nanomole (nmol) of the isolated toxin mixture per plant, for example, from about 0.05 to 1 nmol the isolated toxin mixture per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of the isolated toxin mixture per plant. In certain embodiments, about 40 to about 50 nmol of the isolated toxin UC-2024-9CR-2-WO / / 3868.092WO1 mixture is applied. In certain embodiments, about 0.5 nmol to about 2 nmol of the isolated toxin mixture is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of the isolated toxin mixture is applied. In certain embodiments, a composition of about 0.5 to about 1.5 milligrams per milliliter of the isolated toxin mixture is applied. In certain embodiments, about 1 nmol to about 5 nmol of the isolated toxin mixture is applied to a plant. In certain embodiments, the isolated toxin mixture as topically applied to the plant contains at least one type of isolated protein, peptide or fusion polypeptide at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines can require correspondingly larger amounts of the isolated toxin mixture. Embodiments of such compositions include those where the composition includes a solid, liquid, powder, suspension, emulsion, spray, aerosolized (e.g., particles small and light enough to be carried on the air i.e. into an aerosol), encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment. In many embodiments, the composition is formulated in a form that is ingested by the insect. In embodiments, the composition further includes one or more components selected from a carrier agent, a surfactant, an organosilicone, an herbicidal molecule, a pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, or an insect growth regulator, or any combination thereof. The composition can include or be in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment. In some embodiments, the contacting includes providing the isolated toxin mixture in a composition that further includes one or more components selected from a carrier agent, a surfactant, an organosilicone, an herbicidal molecule, a pesticide, a fungicide, a safener, an insect attractant, or an insect growth regulator, or any combination thereof. In some embodiments, the insecticidal composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment. In some embodiments, the insecticidal composition is provided in a form that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. The insecticidal compositions can further include one or more components selected from a carrier agent, a surfactant, an organosilicone, an herbicidal molecule, a pesticide, a fungicide, a safener, an insect attractant, or an insect growth regulator, or any UC-2024-9CR-2-WO / / 3868.092WO1 combination thereof. The insecticidal compositions can further include at least one pesticidal agent selected from a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and Paenibacillus popilliae. In some embodiments, the combination of the isolated toxin mixture and the pesticidal agent provides a level of insect control that is greater than the sum of the effects of the isolated toxin mixture and the pesticidal agent components if tested separately. The form of a spray treatment (applied prior to, contemporaneously with, or following application of the composition including the isolated protein, peptide or fusion polypeptide) of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, or 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of the isolated protein, peptide or fusion polypeptide into plant cells from a topical application on the surface. Method of Preparing / Isolating Toxin Mixture with Insecticidal Activity from EPNs Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis kill insect hosts quickly. While infecting insects, EPNs spit out excretory / secretory products (ESPs) into the host. An arsenal of proteins is found in EPN ESPs. In particular, EPNs release relatively large quantities (for their size) of a variety of proteins when stimulated by insect tissue. Of the two EPNs that were evaluated, S. carpocapsae and S. feltiae, it was found that they release different complex mixtures of proteins when exposed to the same host tissue, and that collectively, both ESP mixtures are highly toxic to insects (the mixture has insecticidal activity). To obtain a large amount of ESPs, 100 million S. carpocapsae IJs were reared. All the IJs were activated in vitro as described herein (see, for example, the Example below). After activation, IJs are thoroughly washed and excreted / secreted proteins (ESPs) are then collected in PBS for 3 hours at 25 ºC. A total of 7.9 mg ESPs were collected, representing an average of .079 ng / IJ in the 3-hour period. Crude ESPs released by activated IJs of S. carpocapsae were highly toxic to fruit flies, killing them quickly (within hours) with as little as 20 nanograms UC-2024-9CR-2-WO / / 3868.092WO1 (ng) (Figure 3) (Lu et al., 2017). The crude ESPs from S. carpocapsae contain ~500 different proteins and utilizing the published genome sequences and mass spectrometric data, all of the proteins present in the ESPs of S. carpocapsae were identified (Chang et al., 2019a; Dillman et al., 2015; Lu et al., 2017). EPN ESPs themselves were directly toxic to D. melanogaster. The crude ESPs of S. carpocapsae, S. feltiae, and S. hermaphroditum were tested in other insects including Bombyx mori, Manduca sexta, and Galleria mellonella and they were found to be highly toxic to all of these insects, indicating that EPN ESPs has broad spectrum toxicity to insects. EXAMPLES The disclosure can be better understood by reference to the following examples which are offered by way of illustration. The disclosure is not limited to the examples given herein. Example I Introduction Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis kill insect hosts quickly. While infecting insects, EPNs spit out excretory / secretory products (ESPs) into the insect host. An arsenal of proteins is found in EPN ESPs. We have focused on the proteins that are released by EPNs during infection using a modified in vitro technique for stimulating release of excreted / secreted proteins (ESPs) and collecting them (Lu et al., 2017; Chang et al., 2019). Materials and Methods Waxworm homogenate preparation The worm homogenate was prepared as previously published (Lu et al., 2017) with novel modifications. Briefly frozen waxworms (25 g for making 100 ml 25% homogenate) were ground in liquid nitrogen with a mortar and pestle into a fine powder, which was then transferred to a clean beaker. Phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) was added to suspend the powder to reach the desired volume. The beaker was loosely covered with plastic wrap and heated in a microwave oven until the suspension started boiling. The suspension was stirred and microwaved to boil again. The boiling step was repeated 3±4 times in total. The homogenate was cooled to room temperature in a water bath. The contents were transferred to 50 ml tubes and centrifuged at 3000 rcf for 5±10 min at 25ºC. The supernatant, including the top lipid layer, along with part of the insect tissue, was transferred to new tubes and mixed vigorously before being aliquoted and frozen at -20ºC for future use. It is important that the lipid layer be equally distributed among all aliquots. The proportion of aliquot that is lipid has a significant effect on activation UC-2024-9CR-2-WO / / 3868.092WO1 and the release of proteins by the nematodes. The homogenate was supplemented with 1x antibiotics immediately prior to use (penicillin, streptomycin, and neomycin solution; Sigma, P4083-100ML). The modifications include the inclusion of the chunky tissue produced during the process, and not just the supernatant. Another beneficial modification to process includes the equal distribution of the fat layer across the number of aliquots generated. Activation of IJs and venom collection About 2 million IJs were washed at least 3 times with washing solution (autoclaved 0.8% NaCl solution containing 0.01% Triton X-100 which prevents IJs from sticking to plastic and glass surfaces) in a glass vacuum filter holder (Fisher Scientific, Cat# 09-753-1C) with two layers of 11 m nylon net filters (Millipore, NY1104700). The IJ suspension was then transferred to a 15 ml tube and centrifuged at 700 rcf for 0.5±1 min at room temperature. The supernatant was removed leaving about 5 ml of suspended IJs. The IJs were transferred to a 1 L flask containing 8.5 g of autoclaved sponge pieces soaked with 100 ml of 25% waxworm homogenate and 1x antibiotics (Sigma, P4083-100ML). The sponge and IJs were incubated at 25ºC in the dark for a specified amount of time (specified in the experiments) for IJ activation. To recover the nematodes after incubation, the sponge pieces were soaked in a 1 L beaker containing about 500 ml of washing solution for 5±10 min with occasional stirring (squeezing the sponge was avoided since doing so could damage nematodes). The nematode suspension was transferred to another beaker and 500 ml of washing solution was added to the sponge. The soaking step was repeated 5±6 times to recover most of the nematodes. The nematodes were collected in the glass vacuum filter mentioned above. The solution was removed, leaving only a thin layer of solution covering the nematodes. Then 100 ml of washing solution was immediately added and the nematodes were resuspended by pipetting. This washing step was repeated 10 times to remove contamination. The clean nematodes were resuspended in 105 ml of PBS solution and transferred to a sterile 1 L glass flask. The flask was incubated in a shaker set to 25ºC and 200 rpm for about 3 hr for the nematodes to continue to release venom. Then the nematodes were pelleted in 15 ml tubes by centrifugation at 700 rcf for 0.5±1 min at room temperature. The nematodes were saved for quantification of activation rates and microscopy. The supernatant was filtered through a low protein binding 0.22 m syringe filter (Fisher Scientific, Cat# 9719001) to remove residual nematodes and bacteria and collected in two 50 ml tubes (approximately 100 ml). The filtered supernatant containing the nematode venom was stored at -20ºC or immediately concentrated in an Amicon Ultra 15 ml centrifugal 3kDa filter (Millipore, UFC900308). The centrifugal filter was filled with 15 ml of venom and centrifuged UC-2024-9CR-2-WO / / 3868.092WO1 in a swing-bucket rotor at 3000 rcf at 4ºC for 50±60 min to concentrate to less than 1 ml. The flowthrough solution was discarded, and another 15 ml of venom was added to the centrifugal filter. The centrifugation was repeated until the 100 ml of venom was concentrated to the dead volume of the filter (~200 l). The concentrated venom proteins / toxins were transferred to a 1.5 ml low retention tube (Fisher Scientific, Cat# 21402903). Protein concentration was measured using a Bradford assay using the Bio-Rad protein assay dye reagent (Bio-Rad, 500±0006). The remaining concentrated venom / toxic mixture was stored at -80ºC. Three biological replicates of activation and venom collection were performed. Results The general life cycle of these nematodes is depicted in Figure 1. The method to induce production and release of the toxic mixture is illustrated in Figure 2-4. While the nematodes normally will release this toxic mixture into insects when they infect the insects, the induction provided herein is developed for large scale production of the mixture / proteins. This requires culturing the nematodes or purchasing them from a commercial seller. It also requires tens of millions of nematodes to generate milligram amounts of protein. The published method has been modified in several novel ways, as discussed herein. The previously published method shows that collectively, the proteins are toxic if injected into insects, which is the normal course in nature (Lu et al., 2017; Chang et al., 2019). In nature, these proteins / toxins are introduced by the nematodes inside the insect, similar to injection. Whether these toxins were toxic by ingestion was unknown (as these proteins / toxins are not eaten in nature and could therefore be inactive or degraded / inactivated after ingestion and not have any toxic effect). Provided herein, is a method of oral feeding, causing insects to ingest, the toxin mixture produced by the modified in vitro production method. As demonstrated in US Provisional Application Number 63 / 530,831 (filed August 4, 2023), one protein in the complex mixture of about 472 proteins, called NIP-1, is toxic to insects if eaten (Figure 8). Provided herein, it is shown that if eaten, the about toxin mixture, including about 472 proteins, is detrimental to the insects, including Spodoptera frugiperda (Figure 8). Bibliography Chang, D.Z., Serra, L., Lu, D.H., Mortazavi, A., and Dillman, A.R. (2019). A core set of venom proteins is released by entomopathogenic nematodes in the genus Steinernema. Plos Pathogens 15. ARTN e1007626 UC-2024-9CR-2-WO / / 3868.092WO1 Dillman, A.R., Macchietto, M., Porter, C.F., Rogers, A., Williams, B., Antoshechkin, I., Lee, M.M., Goodwin, Z., Lu, X., Lewis, E.E., et al. (2015). Comparative genomics of Steinernema reveals deeply conserved gene regulatory networks. Genome Biol 16, 200. 10.1186 / s13059-015-0746-6. Lu, D., Macchietto, M., Chang, D., Barros, M.M., Baldwin, J., Mortazavi, A., and Dillman, A.R. (2017). Activated entomopathogenic nematode infective juveniles release lethal venom proteins. Plos Pathog 13(4), e1006302.10.1371 / journal.ppat.1006302. Upadhyay, S.K., Chandrashekar, K., Thakur, N., Verma, P.C., Borgio, J.F., Singh, P.K., and Tuli, R. (2011). RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J Biosci 36, 153-161.10.1007 / s12038-011-9009-1. Example II The method of inducing entomopathogenic nematodes to release their venom protein slurry was used to collect and study the excreted / secreted proteins (ESPs) from Steinernema carpocapsae. The use of Steinernema carpocapsae infective juveniles (IJs) as biological control agents is a bio-safe and long-standing practice; however, it is unfortunately highly dependent on environmental conditions and is labor-intensive (FIG. 9). These IJs release a cocktail of venom proteins upon infection, yet their oral activity against insect pests is poorly understood. Three different doses of ES proteins were teseted in an artificial diet feeding assay against Spodoptera frugiperda. Early instars were found to be highly susceptible to ES proteins in a dose-dependent manner. These ES proteins cause a range of developmental abnormalities in the treatment group, including ~22% melatonic early-instar mortality (FIGS. 10A-10D). Surviving larvae required up to 1.8 days longer to pupate, yet gained no extra mass. The carry- over effects were found to be even more profound. Treated pupae were smaller, dorsally dehydrated, and ventrally streaked with brownish-black lines; only 19% of 1000 ng pupae produced viable adults versus 92 % in controls (FIGS.11A-11E). Adults that did emerge lived 30 % fewer days and laid >90 % fewer morphologically normal eggs (FIGS.12A-12B). It was found that the venom proteins from S. carpocapsae, collected using the method of induction, affect insect pests in several ways: they damage the gut and over-activate melanization, flood tissues with ROS that slow growth, and disrupt cuticle tanning. The treatment leads to more larval deaths, delayed development, malformed pupae, brief adult lifespans, and minimal egg production. Together, these results make the venom proteins of S. carpocapsae next-generation protein insecticides that can be used to generate pest-resistant transgenic crops and / or foliar-applied insecticides to combat crop pests in general and UC-2024-9CR-2-WO / / 3868.092WO1 Spodoptera frugiperda in particular. The oral toxicity of S. carpocapse ES proteins to a crop pest has never been shown before. Statements 1. An in vitro method to produce an insecticidal toxin composition comprising a) contacting nematodes with a homogenate of waxworm, wherein the homogenate comprises powdered and chunky waxworm tissue, a waxworm derived fat layer and a liquid carrier; b) incubating the nematodes of a) in contact with the homogenate for a period sufficient to activate the nematodes and induce the release of excretory / secreted proteins; c) washing the activated nematodes of b) to remove the homogenate; d) incubating the washed, activated nematodes in a buffer solution a period sufficient to permit the nematodes to secrete excretory / secreted proteins (ESPs) into the buffer solution; and e) filtering the buffer solution to remove nematodes to obtain an isolated insecticidal toxin composition comprising the excretory / secreted proteins (ESPs) from the nematode. 2. The method of statement 1, wherein a) further comprises sponge pieces saturated with the homogenate. 3. The method of statement 1 or 2, wherein b) incubating the nematodes in contact with the homogenate is carried out at about 25°C for a period ranging from 0.1 to 18 hours. 4. The method of any one of statements 1 to 3, wherein d) incubating is with agitation and performed at about 25°C for about 0.1 to10 hours to permit secretion of the excretory / secreted proteins. 5. The method of any one of statements 1 to 4, wherein the waxworm homogenate is prepared grinding waxworm tissue in liquid nitrogen and suspending the resulting powder in a liquid carrier such that a lipid layer is retained and equally distributed therein. 6. The method of any one of statements 1 to 5, wherein the nematodes are entomopathogenic nematodes (EPNs). 7. The method of any one of statements 1 to 6, wherein the nematodes are infective juveniles (IJs). 8. The method of any one of statements 1 to 5, wherein the nematodes are Steinernema and / or Heterorhabditis nematodes. 9. The method of any one of statements 1 to 8, wherein the nematodes are S. carpocapsae, S. feltiae, S. hermaphroditum or a combination thereof. UC-2024-9CR-2-WO / / 3868.092WO1 10. The method of any one of statements 1 to 9, wherein the insecticidal toxin composition comprises excretory / secreted proteins including NIP-1. 11. The method of any one of statements 1 to 10, wherein the insecticidal toxin composition is insecticidal pest insects. 12. The method of statement 11, wherein the pest insects comprise chewing and sucking insect pests of plants. 13. The method of statement 11 or 12, wherein the insects comprise Spodoptera frugiperda, Manduca sexta, Plutella xylostella or a combination thereof. 14. A composition comprising the insecticidal toxin of any one of statements 1 to 13. 15. The composition of statement 14, further comprising other insecticidal compounds. 16. The composition of statement 14 or 15, further comprising a carrier. 17. The composition of statement 16, wherein the carrier is a water-soluble liquid. 18. The composition of statements 16 or 17, wherein the carrier provides for sustained release. 19. The composition of any one of statements 14 to 18, wherein the composition can be aerosolized. 20. A method to induce of insect mortality, developmental delay, or morphological abnormalities in an insect pest comprising administering orally to the insect the insecticidal toxin composition of any one of statements 14 to 19. 21. The method of statement 20, wherein the composition is sprayed on plants or other surfaces frequented by said insects, wherein ingestion of the composition is lethal to said insects. 22. The method of statement 21, wherein said plants are crop plants. 23. The method of statement 22, wherein the crop plants are tomato, cotton, maize, tobacco, eggplant, rice, wheat, potato, sugarcane, soybean, cabbage, cauliflower, bean, mustard, rape seed, pulse, sunflower plants or a combination thereof. The embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and formulation and method of using changes may be made without departing from the scope of the invention. The detailed description is not to be taken in a limiting sense, and the scope of the invention is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled. UC-2024-9CR-2-WO / / 3868.092WO1 It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. All publications, patents, and patent applications, Genbank sequences, websites and other published materials referred to throughout the disclosure herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application, Genbank sequences, websites and other published materials was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.

Claims

UC-2024-9CR-2-WO / / 3868.092WO1 WHAT IS CLAIMED IS:

1. An in vitro method to produce an insecticidal toxin composition comprising a) contacting nematodes with a homogenate of waxworm, wherein the homogenate comprises powdered and chunky waxworm tissue, a waxworm derived fat layer and a liquid carrier; b) incubating the nematodes of a) in contact with the homogenate for a period sufficient to activate the nematodes and induce the release of excretory / secreted proteins; c) washing the activated nematodes of b) to remove the homogenate; d) incubating the washed, activated nematodes in a buffer solution a period sufficient to permit the nematodes to secrete excretory / secreted proteins (ESPs) into the buffer solution; and e) filtering the buffer solution to remove nematodes to obtain an isolated insecticidal toxin composition comprising the excretory / secreted proteins (ESPs) from the nematode.

2. The method of claim 1, wherein a) further comprises sponge pieces saturated with the homogenate.

3. The method of claim 1, wherein b) incubating the nematodes in contact with the homogenate is carried out at about 25°C for a period ranging from 0.1 to 18 hours.

4. The method of claim 1, wherein d) incubating is with agitation and performed at about 25°C for about 0.1 to10 hours to permit secretion of the excretory / secreted proteins.

5. The method of claim 1, wherein the waxworm homogenate is prepared grinding waxworm tissue in liquid nitrogen and suspending the resulting powder in a liquid carrier such that a lipid layer is retained and equally distributed therein.

6. The method of claim 1, wherein the nematodes are entomopathogenic nematodes (EPNs).

7. The method of claim 1, wherein the nematodes are infective juveniles (IJs).UC-2024-9CR-2-WO / / 3868.092WO1 8. The method of claim 1, wherein the nematodes are Steinernema and / or Heterorhabditis nematodes.

9. The method of claim 1, wherein the nematodes are S. carpocapsae, S. feltiae, S. hermaphroditum or a combination thereof.

10. The method of claim 1, wherein the insecticidal toxin composition comprises excretory / secreted proteins including NIP-1.

11. The method of claim 1, wherein the insecticidal toxin composition is insecticidal pest insects.

12. The method of claim 11, wherein the pest insects comprise chewing and sucking insect pests of plants.

13. The method of claim 11, wherein the insects comprise Spodoptera frugiperda, Manduca sexta, Plutella xylostella or a combination thereof.

14. A composition comprising the insecticidal toxin of claim 1.

15. The composition of claim 14, further comprising other insecticidal compounds.

16. The composition of claim 14, further comprising a carrier.

17. The composition of claim 16, wherein the carrier is a water-soluble liquid.

18. The composition of claim 16, wherein the carrier provides for sustained release.

19. The composition of claim 14, wherein the composition can be aerosolized.

20. A method to induce of insect mortality, developmental delay, or morphological abnormalities in an insect pest comprising administering orally to the insect the insecticidal toxin composition of claim 14.UC-2024-9CR-2-WO / / 3868.092WO1 21. The method of claim 20, wherein the composition is sprayed on plants or other surfaces frequented by said insects, wherein ingestion of the composition is lethal to said insects.

22. The method of claim 21, wherein said plants are crop plants.

23. The method of claim 22, wherein the crop plants are tomato, cotton, maize, tobacco, eggplant, rice, wheat, potato, sugarcane, soybean, cabbage, cauliflower, bean, mustard, rape seed, pulse, sunflower plants or a combination thereof.

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