A lungfish toxin for controlling pests including aedes aegypti

Abstract

The present invention is directed to a natural pore forming toxin (CrTX-A) obtained from African lungfish, Protopterus spp. (Protop-CrTX-A) and its use in the control and / or elimination of Aedes aegypti larva and other pests from areas treated with this pore forming toxin. A number of active Protop-CrTX-A peptides, delivery vehicles and methods of controlling pests are described in detail herein.

Description

[0001] A Lungfish Toxin for Controlling Pests Including Aedes aegypti Field of the Invention

[0002] The present invention is directed to a natural pore forming toxin (CrTXA) obtained from African lungfish, Protopterus spp. (Protop-CrTXA) and its use in the control and / or elimination of Aedes aegypti larva and other pests from areas treated with this toxin. A number of active Proptop-CrTXA peptides, delivery vehicles and methods of controlling pests are described in detail herein.

[0003] Related Applicants and Government Support

[0004] This application claims priority from United States provisional application serial number 63 / 745,035, filed 14 January 2025, the entire contents of which application is incorporated by reference herein.

[0005] This invention was made with government support under grant no. 2212077 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] Background and Overview of the Invention

[0007] Conquering land was an essential step for the success of the vertebrate lineage.

[0008] Water-land transitions required complex morphological and physiological novelties. Thus, studies on transitional fossils and genome sequencing efforts from extant transitional species such as lungfish have painted our current view of vertebrate land adaptation. Specifically, step-wise changes in body plans (neck, appendages), respiratory systems (surfactant molecules) and nervous systems (anxiolytic response) have been attributed to novel locomotion, feeding and respiration strategies needed for life on land. Adventuring onto land, however, drastically impacted vertebrate mucosal surfaces, which faced novel environmental stressors such as dehydration and UV damage. Yet, innovations supporting mucosal barrier transformations for successful water-land transitions in vertebrates are currently unknown.

[0009] African lungfish (Protopterus spp.)., the closest extant relative to all tetrapods, survives extreme environmental changes every year. The dry season that strikes Africa every year forces lungfish to undergo a process known as estivation. During the induction phase of estivation, African lungfish internalize external stimuli, curl up, reduce their metabolism andstart producing large amounts of mucus, which will eventually cover their entire body and form a cocoon that protects them against desiccation. While living dormant underground, lungfish are seemingly vulnerable to predators and pathogens which abound in the surrounding soil. Thus, although currently unknown, cocoons likely contain diverse molecules that protect the dormant lungfish from external attacks during estivation.

[0010] The inventors recently reported that the African lungfish cocoon is not only made of mucus but is also rich in epithelial cells, immune cells (granulocytes) and other cell types acting as an extracorporeal immune organ that shields them against bacterial invasion during this seemingly unguarded state. Cocoon formation is linked to drastic remodeling of the skin and a global state of inflammation that culminates with the shedding of epidermal layers. During this process, the thick columnar epithelium rich in goblet cells characteristic of an aquatic vertebrate turns into a terrestrial-like skin with a thinner epithelium, reduced numbers of goblet cells and flattened epithelial cells. Strikingly, the immune system also comes into play, as large numbers of granulocytes migrate from reservoir tissues, infiltrate the skin of estivating African lungfish and transmigrate into the cocoon where they deploy extracellular traps. Thus, skin remodeling is associated with a form of physiological inflammation in African lungfish water-land transitions.

[0011] Pore-forming toxins (PFTs) are at the forefront of the chemical warfare between hosts and pathogens and prey and predators. PFTs make pores in host membranes, disrupting the integrity of cells. PFTs usually form small (0.5 to 5 nm) or large (20 to 100 nm) pores, leading to several outcomes on target cells, including inflammation, apoptosis and cell death. While PFTs are mostly found in pathogenic bacteria, animals can also produce PFTs; the best-known examples have been found in venomous invertebrates such as cnidarians and spiders. Interestingly, a PFT was recently described in a vertebrate, the frog Bombina maxima. Amphibian Py-CAT, an aerolysin-like PFT, mediates extracellular nutrient scavenging into liver cells of frogs under nutrient deprivation states. Furthermore, Py-CAT is also likely an immune molecule as evidenced by bacterial challenge experiments in frogs and mice. Excitingly, this frog-derived toxin has already been proposed as a therapeutic for skin wound repair as it promotes re-epithelialization in animal models of antibiotic-resistant bacterial infection.In the present application, the inventors identify a PFT (called Protop-CrTX thereafter) encoded in all lungfish genomes that is phylogenetically and structurally related to PFTs from corals and jellyfish but has no other vertebrate counterpart. At the steady state, Protop-CrTX is produced and secreted into the mucus by goblet cells. Upon terrestrialization, granulocytes expressing high amounts of this toxin flood the lungfish skin and cocoon, exocytosing their cargo and inflicting epidermal damage. Producing a PFT that induces skin damage is beneficial for estivating African lungfish, as once in the cocoon, this multifunctional toxin confers likely protection against insects and microbes whose attack would otherwise abort the lungfish's estivating state. These results uncover a novel paradigm by which immunity translates environmental change into adaptive remodeling of mucosal barriers.

[0012] Animals have evolved diverse chemical defense strategies that mediate prey-predator interactions as well as combat pathogens. Toxins are potent, multifunctional chemicals produced by organisms with toxic effects on others. Toxins can be produced by bacteria, fungi, invertebrates and vertebrates, but the vast diversity of toxins across the phylogenetic tree remains largely unexplored. Every dry season, African lungfish (Protopterus spp.) face unfavorable environmental conditions which trigger a process known as estivation. Estivating lungfish secrete copious amounts of mucus and form a cocoon that protects them from evaporative water loss. Estivation is also a dormant, vulnerable state for any animal, unable to escape from the attack of predators or pathogens. The inventors recently reported that the lungfish cocoon is a living antimicrobial structure, rich in granulocytes, antimicrobial molecules and extracellular traps. They also found that estivation is associated with a drastic remodeling of the skin characterized by severe inflammation and epithelial damage. These studies led the inventors to a new observation, the identification of a novel molecule (Protop-CrTXA) that was highly expressed in the skin of estivating lungfish. This toxin has no other vertebrate counterpart except for other lungfish species, including the Australian lungfish (Neoceratodus forsteri) and South America lungfish (Lepidosiren paradoxa). However, Protop-CrTXA gene is significantly different from the other two lungfish counterparts with much higher exon and intron complexity and additional protein domains predicted to have defense functions in African lungfish such as those disclosed here. The inventors theorized that Protrop-CrTXA plays multifunctional roles in skin defense: first it self-inflicts inflammatory damage to form the cocoon, second it provides the cocoon with antimicrobial and insecticidal properties. The inventors further propose that skin goblet cells are the mainproducers of this toxin in free swimming lungfish and that granulocytes secrete this toxin upon activation in the estivating state. It has been discovered by the inventors of this application that Protop-CrTXA is a potent selective toxin and may be used as an antimicrobial (antifungal), insecticide and / or a pest repellent in various agricultural and environmental applications to control or eliminate populations of pests.

[0013] Brief Description of the Invention

[0014] The present invention describes pest control peptides as active components in compositions which are particularly useful as antimicrobial agents, including anti-fungal agents and larvicides / insecticides for controlling pests in various agricultural, food storage and environmental application. These active pest control peptides also find use as pest repellents. In embodiments, the pest control peptides according to the present invention can be formulated in capsules or pellets with other agents for ingestion by a targeted organism to eliminate the targeted organism or repel the targeted organism from a particular area or site. In embodiments, when ingested, these active compositions (as antimicrobial agents or insecticides) are lethal to the targeted species of microbe or insect. In embodiments, these active peptides may also function as deterrents or pest repellents.

[0015] In an embodiment, the present invention is directed to a pest control peptide derived from Protop-CrTXA according to a sequence identified herein below:

[0016] MNGQL I TPKDRVD YDLVT TKGNQS S TMRGKKRMA. VLLC I S 11 FNQMWVKALD I PFFGDVNKLPLV KE AKE G IDD I RN I I AGNADDDDDKQFDVDME AAFDQMSRE GTNE FFDEMH S KKDE E H LHRKRDAP SEQ ID NO: 1

[0017] MYGVYSIRNLLPGSYYHDYLSFDTESPHPWTVEKNDPNDWLLIPRGNRLFTIAS LWGCHKSYEFCRGILSWDDGDTHPTCTIENDDPMYWYLVRKSVVGYKLLNTW KCPDNKWCTAELSYDGRSGTYPIATVQFDDPCVWEVTRLRDYTP SEQ ID NO:2

[0018] Or a peptide sequence comprising at least five contiguous amino acids of peptide SEQ ID NO: 1 or peptide SEQ ID NO:2 set forth above. In embodiments, the active peptide sequences of the present invention comprise from 5-40, 5-35, 5-30, 5-25, 5-20 and 5-15 contiguous amino acids of peptide SEQ ID NO: 1 or peptide SEQ ID NO:2.In an embodiment, the pest control peptide is a 15-mer sequence according to any one of the following peptide sequences of SEQ ID NOs: 3-14:

[0019] MNGQLITPKDRVDYD SEQ ID NO:3;

[0020] LVTTKGNQSSTMRGK SEQ ID NO:4;

[0021] KRMAVLLCISIIFNQ SEQ ID NO: 5;

[0022] MWVKALDIPFFGDVN SEQ IDNO 6;

[0023] KLPLVKEAKEGIDDI SEQ ID NO 7;

[0024] RNIIAGNADDDDDKQ SEQ ID NO:8;

[0025] FDVDMEAAFDQMSRE SEQ ID NO: 9;

[0026] GTNEFFDEMHSKKDE SEQ ID NO: 10;

[0027] EHLHRKRD APATISE SEQ ID NO: 11;

[0028] TSKILQQKLESFKGT SEQ ID NO: 12;

[0029] ASKAKAGINNL VKA Y SEQ ID NO: 13;

[0030] DSASSDTKDNINNVL SEQ ID NO: 14,

[0031] Or a peptide sequence comprising 5-14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 contiguous amino acids within the sequence of SEQ ID NOs:3-14.

[0032] In an embodiment, compositions according to the present invention comprise effective amounts of at least one peptide set forth above in combination with a carrier, additive or excipient to provide microparticles, nanoparticles, powders or sprayable liquids, among other delivery forms which are prepared by traditional techniques including spray drying and / or encapsulating techniques described herein, into a stable form which can be delivered to a site to impact the pest population of a chosen area. These compositions may be formulated to deliver the peptide toxins in an immediate release or controlled release form. These compositions are often combined with a food or nutrient consumable by the pest to be eliminated and / or repelled, such as nectar, sugar, plants / plant components or other foodstuffs consumable by the pest. Such food or nutrient may be combined with the peptides according to the present invention in admixture and / or in a coating / encapsulation in compositions. In embodiments, the composition comprises a bacteria, fungi, plant or food which is palatable tothe targeted pest population and has been engineered to express the toxic peptide to limit the exposure of the active peptides to the environment and / or to enhance the uptake of these peptides by the target pest population.

[0033] In embodiments, formulations for delivery of the active peptides according to the present invention to environmental sites for purposes of impacting (inhibiting, preventing) the growth of pests in the environment comprise the use of active peptides combined with carriers such as sprayable liquids (a concentration of peptide in water, saline or other liquid), powders, microparticles / nanoparticles, emulsions (e.g. water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in water emulsions), solid lipid nanoparticles, protein-based nanoparticles, including emulsion-based delivery systems, nanoparticle-based peptide delivery systems, liposome-based delivery systems, hydrogel-based delivery systems, stimuli-responsive peptide delivery systems including pH responsive systems, thermo-responsive systems, enzyme-responsive systems, light-responsive systems, ultrasound-responsive systems, magnetically responsive systems and multi-stimuli-responsive systems for peptides. These systems comprise active peptide and carrier which is modified to deliver active peptide to a target pest to a particular site or location to maximize stability of the active peptide and exposure of the target pest to active peptide for maximum effect.

[0034] In embodiments, the active peptide is combined with a carrier as otherwise described herein at a weight ratio of about 0.01% to about 75% by weight active peptide and about 25% to about 99.99% by weight carrier, often about 0.1% to about 50% by weight active peptide and about 99.9% to about 50% by weight carrier, often about 0.5% to about 40% by weight active peptide and about 99.5% to about 60% by weight carrier, about 1% to about 25% by weight active peptide and about 99% to about 75% by weight carrier or about 1% to about 10% by weight active peptide and about 90% to about 99% by weight active peptide.

[0035] Brief Description of the Figures

[0036] FIGURE 1 shows survival rates of A. aegypti larva exposed to different concentrations of African lungfish recombinant toxin diluted in vehicle (PBS) for 24h. Negative controls consisted of larvae exposed to PBS only or to a recombinant protein produced in the same expression system (the flu vaccine FluBlok. Statistical analyses (One way ANOVA with Tukey Mean separation) show a significant difference at 2, 3, 4, 5, and 6h between thesurvival rates of mosquito larvae exposed to the recombinant toxin 14 pg / ml compared to the controls (both Flu vaccine and PBS) and the other toxin concentrations tested (1.75, 3.5, and 7pg / ml ) (P<0.05). At 24h, there was a statistically significant difference between the vehicle PBS group and each of all the tested toxin concentrations (P<0.05).

[0037] FIGURE 2 shows that Protop-CrTX-A has insect-repellent and insecticidal activity.

[0038] FIGURE 2A shows a schematic of the two-choice bioassay used to determine attraction preference of imported red ants, Solenopsis invicta (RIF A), little black ants, Monomorium minimum (LBA), and thief ants, Solenopsis sp (TA) over fresh cocoon and mealworm or fresh cocoon and acetone extracted cocoon. FIGURES 2B and 2C show the difference in attraction to fresh cocoon and mealworm or fresh cocoon and acetone extracted cocoon in RIF A, LBA and TA ants. Data were analyzed by unpaired Student’s t test. Data are represented as mean ± SEM. FIGURE 2D shows a schematic of the apparatus used to evaluate the residing preferences of RIF A ants. Two tubes with entrance holes (DI) were filled with either Protop-CrTX-A and Neo-CrTX-2-treated sand or an untreated control sand.

[0039] FIGURES 2E and 2F show quantification of RIF A ants residing in tubes containing either Protop-CrTX-A, Neo-CrTX-2-treated sand or control untreated sand (n=15 ants per assay. Every assay was repeated six times. Data were analyzed by unpaired Student’s t test. Data are represented as mean ± SEM. FIGURE 2G shows a schematic of the apparatus used to evaluate the digging preferences of RIF A ants. Two tubes were filled with either Protop-CrTX-A and Neo-CrTX-2-treated sand (FIGURE 2 G2) or untreated control sand (FIGURE 2 G3). FIGURE 2 G3 shows the control sand being dug out of the tube. FIGURES 2H and 21 show quantification of Protop-CrTX-A, Neo-CrTX-2 -treated sand or control untreated sand removed by RIFA ants from tubes (every assay was repeated six times). Data were analyzed by unpaired Student’s t test. FIGURE J shows Kaplan-Meier survival curves of yellow fever mosquito larvae (Aedes aegypti) over 48 h following immersion in PBS (control), recombinant Protop-CrTX-A (22 pg / ml), or Neo-CrTX-2 (22 pg / ml). Protop-CrTX-A significantly reduced survival compared to both PBS and Neo-CrTX-2 (p = 4.0 x 10" ), while survival did not differ significantly between PBS and Neo-CrTX-2 (p = 0.23). Pairwise comparisons were performed using the log-rank test with Holm’s correction for multiple hypothesis testing. FIGURE K shows Kaplan-Meier survival curves of yellow (Aedes aegypti) larvae over 48 h following immersion in PBS or increasing concentrations of recombinant Protop-CrTX-A (1.75, 3.5, 7, and 14 pg / ml). All concentrations of Protop-CrTX-A significantly reducedlarval survival compared to PBS, with p-values ranging from p = 7.61 x 10'5to p < 2.0 x 10'16(log-rank test, Holm-adjusted for multiple comparisons. See also FIGURES 3 and 4.

[0040] FIGURE 3 shows that heat inactivated Protop-CrTX-A have reduced toxic effects on yellow fever mosquito larvae. This FIGURE shows Kaplan-Meier survival curves of yellow fever mosquito larvae (Aedes aegypti) over 48 h following immersion in PBS (control), Protop-CrTX-A (22 pg / ml), or heat-inactivated Protop-CrTX-A (HI Protop, 22 pg / ml). Both Protop-CrTX-A and HI Protop significantly reduced larval survival compared to PBS (p = 1.3 x IO'09and p = 2.7 x 10', respectively), with the active form showing a more rapid decline in survival.

[0041] FIGURE 4 shows that Protop-CrTX-A N-terminus domain is necessary for functional activity. FIGURE 4A shows AlphaFold 3 models of Protop-CrXT-A and Truncated-CrTX-A. An additional N-terminal helical bundle (shown in yellow in Protop-CrTX-A) was acquired in cocoon-forming African lungfish. The N-Terminal was removed in the Truncated-CrTX-A only leaving the C-Terminal. FIGURE 4B shows a schematic of the ex-vivo exposure experiment and skin remodelling assessment in African lungfish skin explants exposed to DMEM only (control) and recombinant Protop-CrTX-A and Truncated-CrTX-A (14 pg / ml) for 12 h. FIGURE 4C shows PAS stain and SEM micrographs of free-swimming African lungfish skin explants exposed to recombinant Protop-CrTX-A and Truncated-CrTX-A for 12 h. Explants exposed to Truncated-CrTX-A do not manifest estivation features observed in Protop-CrTX-A exposed-explants. FIGURE 4D shows quantification of morphometric changes in P AS-stained skin explants stained with PAS: number of exfoliating cells / area, mean epithelium thickness, number of goblet cells / skin area, goblet cell area, number of actively secreting goblet cells / area and mean mucus thickness. Data are represented as mean ± SEM (n=4 explants / treatment, 10 images / explant). Data are represented as mean ± SEM and were analyzed by unpaired Student’s t test. FIGURE 4E shows survival curves of 3 dpf wildtype zebrafish larvae exposed to recombinant Protop-CrTX-A and Truncated-CrTX-A (14 pg / ml) (n=10 larvae / group). Log-rank test was used to test for statistical significance. FIGURE 4F shows Kaplan-Meier survival curves ofdct / c.s aegypti larvae (n=10 larva / group) monitored over 48 h following immersion in PBS (control), full-length Protop-CrTX-A or truncated Protop-CrTX-A (1 pg / ml and 14 pg / ml). Log-rank test was used to test for statistical significance. Results are representative of two independent experiments.FIGURE 5 shows that Protop-CrTX-A inhibits pathogenic V.anguillarum but enhances the growth of the lungfish skin commensal bacterial er omonas sp. FIGURES 5A, 5B and 5C show quantification of V. anguillarum growth exposed for 24 h to increasing concentrations of Protop-CrTX-A and Neo-CrTX-2 shows that Protop-CrTX-A but not Neo-CrTX-2 inhibits bacterial growth compared to the negative control. FIGURES 5D, 5E and 5F show quantification of E. ictaluri growth exposed for up to 24 h to increasing concentrations of Protop-CrTX-A and Neo-CrTX-2. Negative and positive controls consisted of cells grown in Mueller-Hinton broth only and in broth containing an antibiotic cocktail (ampicillin, kanamycin, and carbenicillin), respectively. FIGURES 5G, 5H and 51 show quantification of Aeromonas sp. growth exposed for 24 h to increasing concentrations of Protop-CrTX-A and Neo-CrTX-2 showed increased Aeromonas sp. growth after 24 h with 1 pg / ml. Data are represented as mean ± SEM. One way ANOVA was used to determine statistical significance with P<0.05.

[0042] FIGURE 6 shows that Protop-CrTX-A reduces the growth of M.circinelloides but not that of C.albicans and S.delica. FIGURES 6A, 6B, 6C and 6D show quantification of Candida albicans and Mucor circinelloides growth exposed for 24 h to increasing concentrations of Protop-CrTX-A and Neo-CrTX-2. Negative and positive controls consisted of fungi grown in glucose-peptone broth only and in glucose-peptone broth containing cycloheximide, respectively. FIGURES 6A and 6B show quantification of C.albicans growth exposed for 24 h to the highest concentration of Protop-CrTX-A and Neo-CrTX-2 (1 pg / ml). Broth only and cycloheximide exposure were respectively implemented to achieve a negative and a positive control. Error bars represent the standard error of triplicated wells. FIGURES 6G and 6H show disk fungal growth assays conducted on glucose-peptone agar plate show's no growth impairment in S.delica cultures exposed to either Protop-CrTX-A and Neo-CrTX-2 (1 pg / ml) for 48 h. A negative and positive control FIGURES 6E and 6F were implemented by adding distilled 'ater or cycloheximide to the disks. Data are represented as mean ± SEM. One way ANOVA was used to determine statistical significance with P<0.05.

[0043] Detailed Description of the Invention

[0044] The present invention provides novel isolated peptides (Protop-CrTXA) from natural pore-forming toxin (CrTXA) obtained from African lungfish, Protopterus spp. and their usein the control and / or elimination of Aedes aegypti and other pests from areas treated with this toxin. A number of active CrTXA peptides, delivery vehicles and methods of controlling pests are described in detail herein. The invention also is directed to compositions which comprise the active CrTXA peptides often in delivery vehicles that are ingestible by and toxic to targeted species, but are non-toxic to humans and non-target species. These formulations, which are cost-effective to manufacture, provide targeted active peptide release that allows for maximum impact of these agents on targeted pest larvae and adult pests and provides for significant flexibility in the concentration and timing of the release of larvicide / adulticide.

[0045] For the purposes of the present disclosure, the target larvae or target species refers to the intended target of compounds and compositions according to the present invention.

[0046] While many of the specific embodiments provided herein refer to mosquito larvae as the intended target, it will be understood that larvae of other insects or other species, including adult insect pests may also be the intended target and that the larvicide / adulticide may be altered, as described herein, to be more particularly suited towards one target or another, including black flies and fungus gnat larvae, among others. Additional targets are described in some detail herein below.

[0047] Furthermore, it will be understood that the novel active peptides and compositions described herein may be designed to be suitable for more than one target and that references to “a” or “the” target species does not necessarily preclude embodiments wherein there is more than one target species.

[0048] In embodiments, the target is a mosquito larvae (e.g., various species of Aedes, Culex and Anopheles'), black fly or fungus gnat larvae. In embodiments, the target species is the larvae or adult of Tarnished plant bug, the common housefly, the com earworm, mealworms and termites, including the Eastern subterranean termite.

[0049] The term “pest” is used to describe any target species for which elimination (eradication) from an area and / or inhibition of its growth by the present invention is a principal goal. Common pests include mosquitoes, flies (especially houseflies, and black flies, among others), hornets, wasps and ants and numerous insects which negatively impact agriculture.In an embodiment, the present invention is directed to a method for eradicating of inhibiting the unfavorable consequences of a targeted insect population, often an insect larvae population, more often a mosquito larval population. In embodiments the targeted insect population is an adult insect population. In embodiments targeted insect (larvae or adults) are the target population.

[0050] In embodiments, the method comprises exposing a target population of insects, fungi and / or microbes, including adult insects and / or insect larva to an effective amount of active peptide or composition according to the present invention. In embodiments, the targets of the present invention include mosquito larvae (e.g., various species of Aedes, Culex and Anopheles), black fly, fungus gnat larvae, black ants, red ants, thief ants. In embodiments, the target species is often the larvae or adult of the tarnished plant bug, the common housefly, the com earworm, mealworms and termites, including the Eastern subterranean termite, among numerous others identified herein below.

[0051] In embodiments, the present invention shows particular effect against target pests which are Aedes aegyptii (mosquito), red imported fire ants (Solenopsis invicta), little black ants (Monomorium minimum) and thief ants (Solenopsis sp), among others.

[0052] Additional exemplary target pests of the present invention include mollusks (slugs, snails), worms (nematodes, flatworms), arthropods (e.g. insects, arachnids, crustaceans), fungi and various microbes, including numerous others.

[0053] In embodiments, the target pests include insects and other invertebrates including ants (carpenter, fire, Argentine, house, Pharoah, Thief), bed bugs, beetles, including woodworms, weevils, fur beetles, spider beetles, mealworm beetles, centipedes, cockroaches, dustmites, earwigs, crickets, firebrats, flies (e.g. house, black, fruit, blue bottle, green bottle), mosquitoes, moths (almond, Indianmeal, clothes, brown house), lice, red spiders, silverfish, termites (dampwood, subterranean) and woodlouse, among others

[0054] In embodiments, the target pests include the Oak Splendour Beetle, the Exotic Wood Borer / Bark Beetle, the Oak Anguina Tritici, the Wheat seed gall nematode, Citrus longhomed beetle, Silver y moth, Beocauluss spp., Colosius spp., Laevicaulis spp., Red ring nematode, Candidatus Phytoplasma australiense 16SrXII, Candidatus Phytoplasma mali16SrX-A, Candidatus Phytoplasma palmae 16Sr-IV, Candidatus Phytoplasma phoenicium 16SrIX-B, Candidatus Phytoplasma prunorum 16SrX-F, Candidatus Phytoplasma solani 16SrXII-A (Bois noir / Stolbur Grape), Candidatus Phytoplasma vitis 16SrV-C, Japanese Wax Scale, Asiatic rice borer, Coconut cadang-cadang, Scots pine blister rust, Christmas berry webworm, Pine tree lappet, Mason pine moth, Siberian silk moth, Curcurbit beetle, Sunn pest, Old world bollworm, Black maize beetle, Ash dieback, Small brown planthopper, Gian African snail, Late wilt of corn, Megaplatypus mutatus (Ambrosia beetle), Chinese slugs, hygromiid snails, Tomato fruit borer, Horse thistle, Cotton seed bug, South American palm borer, Java downy mildew, Philippine downy mildew, Aldar root and collar rot, Beech bleeding canker, Oak ambrosia beetle, rotbrenner, Japanese oak wilt, bacterial wilt, Sarasinula spp., Semperula spp., Cotton cutworm, Potato wart, Guatemalan potato moth, False coddling moth, Pine processionary moth, Oak processionary moth, Cucumber green mottle mosaic, Groundnut bud necrosis, Khapra beetle, Tomato leafminer, Veronicella spp., Bacterial blight and Bacterial leaf Steak, among others.

[0055] The composition of the present invention could be used in conjunction with existing municipal or rural larvicide / insecticide / other pest control programs. Furthermore, because the presently described system can be used in environments where traditional chemical larvicides and insecticides are not used due to safety risks from toxicity, the presently described larvicidal / adulticidal system can be used in high-value breeding sites, specifically in urban areas.

[0056] In embodiments, the target is a mosquito larvae (e.g., various species of Aedes, especially Ae. aegypti, Culex and Anopheles), black fly, fungus gnat larvae, black ants, red ants, thief ants. In embodiments, the target species is the larvae or adult of Tarnished plant bug, the common housefly, the com earworm, mealworms and termites, including the Eastern subterranean termite.

[0057] In embodiments, the present invention targets different species of Aedes, Culex and Anopheles mosquitos (larvae and adult, but often larvae). Particularly important Aedes spp. which may be targeted by the present invention include, but are not limited to Ae. aegypti, Ae. albopictus, Ae. japonicus, which are vectors for the zika virus, yellow fever vims and chikungunya vims. Particularly important Culex spp. which may be targeted by the present invention include, but are not limited to Cx. pipiens, Cx. tarsalis, Cx. Quinquefasciatus,which are vectors for West Nile virus, Japanese encephalitis virus, and Saint Louis encephalitis virus, among others. Important Anopheles spp. which may be targeted by the present invention include, but are not limited to An. atropavrus, An. albimanus, An. arabiensis, An. barberi, An. bellator, An. crucians, An. cruzii, An. culcifacies, An. darlingi, An. Deaneorum An. dims, An. earlei, An. freeborni, An. fenestus, An. gambiae, An. introlatus, An. latens, An. maculipennis, An. moucheti, An. nili, An. punctipennis, An. quadrimaculatus, An. stephensi, An. subpictus, An. sundaicus, An. walkeri. Particularly important Anopheles spp. including An. freeborni and An. quadrimaculatus in the United States, An. gambiae, An. funestus and An. Arabiensis in Africa, An. leucosphyrus and An. dims in Asia and An. darlingi, An. marajoara, and An. deaneorum in Latin America.

[0058] Anopheles spp. mosquitoes are often vectors for Plasmodium spp. malaria, including malaria caused by P. falciparum, P. vivax, P. ovale and P. malariae with P. falciparum malaria being the deadliest form.

[0059] The term “peptide” is used to describe a compound, often an active compound, in which amino acids are linked to form a chain of peptide units. The term “peptide” also includes salts of these amino acid chains. In the present invention, active peptides may also be referred to as “protrop-CtxA peptides” which are more fully described herein above and set forth in peptides according to peptide SEQ ID Nos: 1-14.

[0060] In an embodiment, the present invention is directed to a method for eradicating a targeted insect population often an insect larvae population, more often a mosquito larval population or an ant population. In embodiments the targeted insect population is an adult insect population. In embodiments, the method comprises exposing a target population of insects, including adult insects and / or insect larva to an effective amount of a composition according to the present invention. In preferred embodiments, the present invention targets different species oiAedes, Culex and Anopheles mosquitos (larvae and adult, but often larvae). Particularly important Aedes spp. which may be targeted by the present invention include, but are not limited to Ae. aegypti, Ae. albopictus, Ae. japonicus, which are vectors for the zika virus, yellow fever virus and chikungunya virus. Particularly important Culex spp. which may be targeted by the present invention include, but are not limited to Cx. pipiens, Cx. tarsalis, Cx. Quinquefasciatus, which are vectors for West Nile virus, Japanese encephalitis virus, and Saint Louis encephalitis virus, among others. Important Anopheles spp. which may be targeted by the present invention include, but are not limited to An.atropavrus, An. albimanus, An. arabiensis, An. barberi, An. bellator, An. crucians, An. cruzii, An. culcifacies, An. darlingi, An. Deaneorum An. dims, An. earlei, An. freeborni, An. fenestus, An. gambiae, An. introlatus, An. latens, An. maculipennis, An. moucheti, An. nili, An. punctipennis, An. quadrimaculatus, An. stephensi, An. subpictus, An. sundaicus, An. walkeri. Particularly important Anopheles spp. including An. freeborni and An. quadrimaculatus in the United States, An. gambiae, An. funestus and An. Arabiensis in Africa, An. leucosphyrus and An. dims in Asia and An. darlingi, An. marajoara, and An. deaneorum in Latin America. Anopheles spp. mosquitoes are often vectors for Plasmodium spp. malaria, including malaria caused by P. falciparum, P. vivax, P. ovale and P. malariae with P. falciparum malaria being the deadliest form.

[0061] The term “effective” is used to describe an amount of a peptide polymer, including larvicide / adulticide or other component used to provide compositions according to the present invention which are provided and used to effect an intended result within the context of the use of the component of the invention described herein. The term effective is also used to describe an amount of an active peptide, carrier or a composition comprising an active peptide and a carrier which is used to deliver active peptide to control insect larvae and other pests (larvae and adult) as otherwise described herein.

[0062] The term “about” is used to describe an amount of a compound or component (such as an active peptide or carrier) which is + 5% of an indicated weight range for that compound or component. Thus, by way of example, an amount of a compound or component which is identified as falling within a range of about 1% to about 65% by weight is recognized as being within that range which includes + 5% by weight of that range.

[0063] The term “carrier” is used to describe a component of pesticide / larvicide compositions according to the present invention which comprise effective amounts of pesttoxic peptides for eliminating or inhibiting target pests and target pest growth which is responsible for economic and environmental damage as well as the transfer of pathogens to humans, livestock and other animals. The term carriers as used herein refer to delivery vehicles of active peptides according to the present invention including sprayable liquids (water, saline or other liquid) in which active peptides according to the present invention are formulated for delivery to a site for maximum impact on a target pest population, concentration of peptide in water, saline or other liquid), powders,microparticles / nanoparticles, emulsions (e.g. water-in-oil emulsions, oil-in-water emulsions, water-in-oil -in water emulsions), solid lipid nanoparticles, protein-based nanoparticles, including emulsion-based delivery systems, nanoparticle-based peptide delivery systems, liposome-based delivery systems, hydrogel-based delivery systems, stimuli-responsive peptide delivery systems including pH responsive systems, thermo-responsive systems, enzyme-responsive systems, light-responsive systems, ultrasound-responsive systems, magnetically responsive systems and multi-stimuli-responsive systems for peptides. These systems comprise active peptide and carrier which is modified to deliver active peptide to a target pest at a particular site or location to maximize stability of the active peptide and exposure of the target pest to active peptide for maximum effect.

[0064] In embodiments, the carriers or delivery forms are prepared by traditional techniques including spray drying and / or encapsulating techniques into a stable form which can be delivered to a site to impact the pest population of a chosen area. These compositions may be formulated to deliver the peptide toxins in an immediate release or controlled release form. These compositions are often combined with a food or nutrient consumable by the pest to be eliminated and / or repelled, such as nectar, sugar, plants / plant components or other foodstuffs consumable by the pest. Such food or nutrient may be combined with the peptides according to the present invention in admixture and / or in a coating / encapsulation (wall material) in compositions. In embodiments, the composition comprises a bacteria, fungi, plant or food which is palatable to the targeted pest population. In embodiments, the bacteria, fungi or plant food is bioengineered through transfection of expression vectors and other means to express the toxic peptide to limit the exposure of the active peptides to the environment and / or to enhance the uptake of these peptides by the target population.

[0065] In embodiments, effective amounts / concentrations of active peptides are formulated as powders (e.g. without encapsulation to produce microparticles or nanoparticles), encapsulated into microparticles / nanoparticles to facilitate delivery, stability of active peptide and protection from environmental elements. Powders which may be used to formulate compositions according to the present invention include maltodextrin, cornsyrup solids, gum arabic, modified starch, modified cellulose, gelatin, cyclodextrin, lecithin, whey protein, pectin, hydrogenated fat, chitosan and mixtures thereof. These components may also be used to create microparticles or nanoparticles using spray drying and other techniques well known in the art. In addition, the active peptides according to the present invention may beformulated to provide emulsions (e.g. water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in water emulsions) to facilitate delivery and stability by spraying or coating surfaces with the emulsions containing active peptides. Emulsions for use in the present invention comprise an effective amount of active peptide, an appropriate emulsifier, water and other optional components. For oil-in-water emulsions, surfactants with high hydrophilic-lipophilic balance (HLB) such as lecithin or Tween 80 are used. For water-in-oil emulsions, surfactants with lower HLB such as Span 60 are used. Oils such as medium chain triglycerides (MCTs) or fish oil may favorably form the core of the dispersed phase, providing a protective layer for the peptides, especially against hydrolysis and enzymatic degradation. The oil phase may also be used to influence release characteristics of the peptides, providing immediate or sustained release. The aqueous phase where the peptide is often dissolved in oil-in-water emulsions can be formulated to regulate pH and ionic strength, often by adding ionic salts. Stabilizers, such as polyethylene glycol (PEG) or biopolymers such as albumin can be used to further enhance stability of emulsions by reducing surface tension and / or providing electrostatic or steric stabilization.

[0066] The active peptides described herein may be formulated by encapsulation in microparticles and / or nanoparticles. Microparticles are considerably larger than nanoparticles, which generally fall within the size range about 5-10 nm to about 1000 nm (1 pm). In practice, microparticles and to a lesser extent nanoparticles may be made out of materials as discussed hereinabove, which materials may further include the use of polymers, such as polylactic-co-glycolic (PLGA) or polyethylene glycol (PEG). Polysaccharide polymers such as chitosan and alginate, can also be used to enhance mucoadhesion and release characteristics of the active peptides encapsulated by the particles. Lipid-based microparticles and nanoparticles, including nanostructured lipid carriers (NLCs), solid lipid nanoparticles (SLNs) and lipid-polymer complex nanoparticles are often made from physiological lipids. Protein-polysaccharide binary complexes may also be used to provide microparticles / nanoparticles which encapsulate and deliver active peptides to control target pests. The use of inorganic materials may also be used to provide peptide containing microparticles / nanoparticles. These include silica, metallic / metal oxides (e.g. gold, silver, iron oxide, titanium oxide). Microparticles / nanoparticles according to the present invention may be prepared using a variety of methods that can influence their size, shape and encapsulation efficiency. Among these methods are solvent evaporation, solvent diffusion and salting out are commonly used methods for providing polymericmicroparticles / nanoparticles. Spray drying techniques are often used to provide microparticles / nanoparticles for encapsulating and delivery of active peptides.

[0067] Liposomes may also be used to provide microparticles / nanoparticles for formulation and delivery of active peptides according to the present invention. These nanoparticles may readily accommodate both hydrophilic and / or hydrophobic active peptides according to the present invention. Liposomes may be used to enhance stability, and controlled release of peptides as well as targeting of pests at sites for release. Liposomes are typically formulated using natural lipids such as phospholipids as well as cholesterol to enhance structure integrity of the delivery vehicle. The use of phospholipids of varying chain lengths can be used to modify release characteristics and the inclusion of concentrations of cholesterol up to about 50% by weight of the liposome can be used to influence both release characteristics and structural integrity of the microparticles / nanoparticles. The choice of lipid component as well as the number and thickness of bilayers directly influences the physical properties, encapsulation efficiency and release behavior of the lipid particles. Preparation of liposome particles is well known in the art as is the use of thin-film hydration, anti-solvent (mixing of lipids with an aqueous phase), freeze-thaw extrusion and supercritical fluid technology, among others to provide these particles.

[0068] Hydrogels are another material for formulating and delivering active peptides according to the present invention. These are three dimensional soft material networks comprised of cross-linked polymer networks. Typically, hydrogels are formed from synthetic polymers such as PEG, polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) as well as natural polymers such as gelatin, casein, whey proteins, soy proteins, fish proteins and polysaccharides such as alginates, carrageenan, pectin and various starches, which are preferred for their environmental compatibility. Various hydrogel crosslinkers may be used in weight ratios ranging from about 0.01% to about 25%, often 0.1% to about 10% by weight of the polymers to provide structural integrity to the hydrogel particles. Typical crosslinkers include glutaraldehyde, carbodiimide, boric acid, sodium trimetaphosphate, N, N’ -methylene bisacrylamide, polycarboxylic acid, transglutaminase, tyrosinase, sortase, laccase, genipen, vanillin, tannic acid and phytic acid, among others.

[0069] Stimuli-responsive delivery systems may also be used to deliver active peptides pursuant to the present invention. These systems include pH-responsive polymeric microparticles / nanoparticles (containing polyanions and / or polycations, their mixtures orcrosslinked polymers), pH-responsive liposomes and pH-responsive hydrogels, thermos-responsive polymers (microparticles / nanoparticles comprising polymers capable of undergoing a phase transition), liposomes and hydrogels, enzyme-responsive microparticles / nanoparticles, liposomes and hydrogels responsive to phospholipases, proteases, oxidoreductases, glycosidases and lipases, light-responsive microparticles / nanoparticles, liposomes and hydrogels comprising photosensitive polymers and light responsive phospholipids / lipids responsive to photoisomerization, photochemistry and photothermolysis, ultrasound responsive microparticles / nanoparticles, liposomes and hydrogels responsive to thermal and mechanical effects of ultrasound, magnetically responsive microparticles / nanoparticles, liposomes and hydrogels that can be activated and / or directed by external magnetic fields, using a magnetic core surrounded by a protective core comprising iron oxide and materials such as silica or a polymeric material, liposomes comprising magnetoliposomes and hydrogels comprising magnetic hydrogels. In addition, combinations of these approaches can be mixed or combined into a single delivery vehicle responsive to multiple stimuli for effective delivery of active peptides according to the present invention.

[0070] The following examples further support the present invention and establish the bioactivity of the active peptides described herein. These examples should not be taken to limit the present invention in any way.

[0071] Examples

[0072] Protop-CrTx-A Exhibits Potent Activity Against Aedes Aegypti as an Insecticide

[0073] A. aegypti was tested against several concentrations of Protop-CrTxA in PBS to determine the impact and activity of Protop-CrTx-A against this mosquito pest. Survival rates of A. aegypti larva exposed to different concentrations of African lungfish recombinant toxin diluted in vehicle (PBS) for 24h were determined. Negative controls consisted of larvae exposed to PBS only or to a recombinant protein produced in the same expression system (the flu vaccine FluBlok). Statistical analyses (One way ANOVA with Tukey Mean separation) show a significant difference at 2, 3, 4, 5, and 6h between the survival rates of mosquito larvae exposed to the recombinant toxin at a concentration of 14 pg / ml compared to the controls (both Flu vaccine and PBS) and the other toxin concentrations tested in a doseresponse effect (1.75, 3.5, and 7pg / ml) (P<0.05). FIGURE 1 shows the effects of toxin at the various concentrations tested. At 24h, there was a statistically significant difference between the vehicle PBS group and each of the tested toxin concentrations (P<0.05), evidencing a substantial toxic effect of toxin against aegypti mosquitoes. The experiments evidenced that Protrop-CtxA exhibited inhibition of A. aegypti larva at all concentrations tested (1.75pg / ml, 3.5pg / ml, 7pg / ml and 14 pg / ml) when compared to controls.

[0074] Protop-CrTX-A exhibits both insect-repellent and insecticidal activities

[0075] Ants inhabit nearly every terrestrial habitat and have thrived for millions of years (Holldobler and Wilson 1990). Some species, such as fire ants, are highly aggressive and can overwhelm prey much larger than themselves thanks to their numerical abundance. Ants are also able to dig into soil and exploit buried resources, reaching those organisms that rely on subterranean refuges. Estivating African lungfish are therefore likely susceptible to ant attack. The inventors hypothesized that Protop-CrTX-A may confer protection to African lungfish against ants by exerting toxic, repellent, or feeding-deterrent effects. In addition, although mosquitoes exert no feeding pressure on lungfish, we tested the potential larvicidal effects of Protop-CrTX-A on mosquito larvae, because of the considerable public health importance they hold.

[0076] The inventors first examined whether three ant species were attracted to African lungfish cocoons (FIGURES 2A and 2B). Neither red imported fire ants (Solenopsis invicla). little black ants (Monomorium minimum), nor thief ants (Solenopsis sp.) showed attraction to the cocoon compared to mealworm, a known ant food source (Figure 9B). However, when the cocoon was acetone-extracted, it attracted significantly more ants than the untreated one, suggesting that acetone either removed or deactivated bioactive substances in the cocoon (FIGURE 2C).

[0077] Next, the inventors evaluated whether Protop-CrTX-A or Neo-CrTX-2 altered red imported fire ant digging behavior (FIGURES 2D and 2G). In nature, fire ants dig whenever an adequate substrate, such as sand, is available, and then reside in the space they create.This behavioral response has been widely used in bioassays to assess fire ant attractants and repellents (Chen and Zhang 2013).

[0078] The bioassay apparatus for the current experiment used a two-choice design (FIGURES 2D, 2E, 2F, and 2G) (see details in the Materials and Methods section below). Protop-CrTX-A did not significantly affect the amount of sand removed compared to the control (FIGURE 2H), but it significantly reduced the number of ants residing in the treated tube after digging (FIGURE 2E). In contrast, Neo-CrTX-2 had no significant effect on either digging or residing behavior (FIGURES 9F, 9G, 9H, and 91).

[0079] The inventors additionally used an immersion bioassay to test whether

[0080] Protop-CrTX-A and Neo-CrTX-2 have insecticidal effects on Aedes aegypti larvae. Results show that, at high doses (22 pg / ml), Protop-CrTX-A has significantly more potent insecticidal activity compared to Neo-CrTX-2 (FIGURE 2J). Dose-response experiments with serial dilutions of Protop-CrTX-A showed 100% larval mortality within 24 h compared to PBS controls (FIGURE 9K). Interestingly, heat-inactivated recombinant proteins retained larvicidal activity, though with reduced potency compared to the non-denatured counterpart (FIGURE 3).

[0081] Together, the results presented in these experiments indicate that African lungfish CrTX toxins, but not Australian lungfish CrTX toxins, possess insect-repellent and larvicidal activities, which may help African lungfish survive underground during estivation.

[0082] Protop-CrTX-A N-terminus domain is important for enhancing functional activity

[0083] Since Protopterus CrTX-A molecules are considerably larger compared to invertebrate and Australian lungfish CrTX proteins, the inventors next evaluated the functional properties of a truncated Protop-CrTX-A recombinant protein lacking this additional N-terminal region (FIGURE 4A). Exposure of skin explants from free-swimming lungfish to either full-length recombinant Protop-CrTX-A or the truncated version of the recombinant protein (FIGURE 4B) revealed that the N-terminus domain is necessary for some of the skin remodeling functions of Protop-CrTX-A. Specifically, truncated Protop-CrTX-A failed to induce any significant epithelial cell desquamation compared to the full-length toxin (FIGURES 4C and 4D). Additionally, the inventors observed that truncated Protop-CrTX-A-treated explants had significantly lower numbers of actively secreting goblet cells compared to controls and full-length toxin-treated explants (FIGURES 4C and 4D). Interestingly, removing the N-terminus from Protop-CrTX-A resulted in complete loss of toxicity in zebrafish larva assays (FIGURE 4E), indicating that the N-terminus domain is an important feature for the pore-forming toxic effects of the protein. Finally, we observed that truncated Protop-CrTX-A retained some insecticidal functions against yellow fever mosquito larvae, with the high-dose treatment significantly reducing A. aegypti mosquito larvae survival compared to the PBS control. However, its potency was diminished compared to the full-length toxin (FIGURE 4F). Combined, these results indicate that the evolution of CrTX genes in African lungfish leading to the acquisition of exons encoding for an extended N-terminal domain fine-tunes toxicity, tissue remodeling and insecticidal functions of this PFT.

[0084] Protop-CrTX-A Exhibits Anti-Microbial and Antifungal Activity

[0085] The inventors next evaluated the bacterial and fungal killing activities of Protop-CrTX-A and compared them to Neo-CrTX-2, the Australian lungfish single exon molecule. We tested two gram-negative pathogenic bacteria, Vibrio anguillarum and Edwardsiella iclahiri. as well as the gram-negative commensal Aeromonas sp. isolated from the skin of a healthy Protopterus sp. individual. Interestingly, while V. anguillarum growth was significantly reduced by Protop-CrTX-A exposure (20% inhibition) after 24 h, Neo-CrTX-2 exposure led to a 40% increase in growth of this bacterium (FIGURES 5A, 5B and 5C). The growth of E. ictaluri was not significantly impacted by Protop-CrTX-A, while it was inhibited by Neo-CrTX-2 by 10% by 24 h (FIGURES 5D, 5E and 5F). Interestingly, the growth of the commensal Aeromonas sp. was significantly higher when grown in the presence of Protop-CrTX-A but not Neo-CrTX-A (FIGURES 5G, 5H and 51). These results suggest that pore-forming toxins in lungfish can inhibit pathogenic bacteria and promote commensal bacteria growth.

[0086] The inventors next tested the effects of both toxins on Candida albicans, Mucor circinelloides and Saprolegnia delica growth. C. albicans growth was only significantly reduced by Neo-CrTX-2 exposure (16% inhibition) (FIGURES 6 A, 6B, 6C and 6D), whereas we observed a significant 25% inhibition of AT. circinelloides growth when cultures were exposed to Protop-CrTX-A but not Neo-CrTX-2 for 24 h (FIGURE 6F). No inhibition of S.delica was observed on plate assays (FIGURES 6G and 6H). Applicant notes that C. albicans mAMucor circinelloides represent phyla of true fungi (Ascomycota and Mucoromycota, respectively), whereas S. delica is a member of the non-fungal phylum Oomycota. Also notable, incubation of PMA-stimulated lungfish spleen granulocytes with Mucor circinelloides revealed the deposition of Protop-CrTX-A on hyphae (FIGURE 6G). Combined, the results of these experiments indicate that granulocyte-derived Protop-CrTX-A contributes to the host ET antimicrobial response in the skin and cocoon during estivation and shows potential as an anti-microbial and antifungal agent.

[0087] Materials and Methods

[0088] Recombinant protein production

[0089] Recombinant proteins were commercially generated by GenScript using a baculovirus expression system. Briefly, gene sequences were synthesized and cloned into the pFastBacl vector using 5’-EcoRI and 3’-HindIII restriction sites. Subsequently, bacmids were generated, followed by Pl and P2 virus generation. The resulting viruses were then used for Sf9 cell infection and protein expression. Purifications were performed using a combination of affinity (hexahistidine or FLAG) chromatography followed by either ion-exchange or sizeexclusion chromatography. Expression and purity were confirmed using Coomassie blue staining and Western blot analyses.

[0090] For the African lungfish Protop-CrTX-A recombinant protein, the full length 594 aa long protein (SEQUENCE ID NO: 15:

[0091] MNGQLITPKDRVDYDLVTTKGNQSSTMRGKKRMAVLLCISIIFNQMWVKALDIPFFG DVNKLPLVKEAKEGIDDIRNIIAGNADDDDDKQFDVDMEAAFDQMSREGTNEFFDE MHSKKDEEHLHRKRDAPATISETSKILQQKLESFKGTASKAKAGINNLVKAYDSASS DTKDNINNVLSTT VS SLDKF VNAESNPIGAVQ ATLDIVS SMAAFVEP AGAVISMGLSF ISGILGLFGQAPEAEKPIGDVVREQIEKALAKFYDQTLKNEAEGCIEAFQHSKAFLDG VASAVDGQVPDNAVSSLSAHVPVYHGLTFMGKLASSIRDVIRKDSPKEGEKCMRYIE LYAKLATLKNMILLQLAALIPDSQSAIRAGVLGVRNSLSSTSRTMLRFLYSHKLKYV NTVSYFDPYRYPITDGYMMKILKLEAPDRSMYGVYSIRNLLPGSYYHDYLSFDTESP HPVVTVEKNDPNDWLLIPRGNRLFTIASLWGCHKSYEFCRGILSWDDGDTHPTCTIE NDDPMYWYLVRKSVVGYKLLNTWKCPDNKWCTAELSYDGRSGTYPIATVQFDDPC VWEVTRLRDYTPENLYFQGHHHHHH).The Australian lungfish toxin Neo-CrTX-3 consisted of a 362 aa long protein (Sequence ID NO: 16:

[0092] MNNMVSTTVTAIGKFANAEQDPIGAVQATLDIVSSISAFAGPVGGLISMGLSFISGILG LFGQGLEAQKPIGEVVREVIEKALDKFYDRTLKDQAHGCIEAFQHSKAFLDGVASAV DGQISDNSVSSLSAHVPVYSGLEFMGTLASVIRDLITKNSVEDGNKIMSYIELYSELAT LKDMILLQLAALIPDSQNGIRAGVWGVHDSVQSTSRAMLKFLYSSDLRYYRIVSYFD PYRYPITDGYLQKILNLAAPDRSMYGVYSIRNLNPGQWYLDYLSFDRESPRSVVTVE KNDPVDWLLIPRGNNYFTIESLWGCDNGNELCRAMLSWDFVRGHPYCTIEHNDPVY WQLKNNGNGYRHHHHHH).

[0093] To generate a truncated Protop-CrTX-A recombinant protein, the inventors expressed the Protop-CrTX-A protein without the N-terminal domain, creating a version of the protein lacking the extended N-terminal helical bundle (Sequence ID NO: 17:

[0094] MATISETSKILQQKLESFKGTASKAKAGINNLVKAYDSASSDTKDNINNVLSTTVSSL DKFVNAESNPIGAVQATLDIVSSMAAFVEPAGAVISMGLSFISGILGLFGQAPEAEKPI GDVVREQIEKALAKFYDQTLKNEAEGCIEAFQHSKAFLDGVASAVDGQVPDNAVSS LSAHVPVYHGLTFMGKLASSIRDVIRKDSPKEGEKCMRYIELYAKLATLKNMILLQL AALIPDSQSAIRAGVLGVRNSLSSTSRTMLRFLYSHKLKYVNTVSYFDPYRYPITDGY MMKILKLEAPDRSMYGVYSIRNLLPGSYYHDYLSFDTESPHPVVTVEKNDPNDWLLI PRGNRLFTIASLWGCHKSYEFCRGILSWDDGDTHPTCTIENDDPMYWYLVRKSVVG YKLLNTWKCPDNKWCTAELSYDGRSGTYPIATVQFDDPCVWEVTRLRDYTPENLYF QGDYKDDDDK).

[0095] Antibody generation

[0096] Three distinct polyclonal rabbit antisera generated against three different peptide antigens of Protop-CrTX-A were generated by GenScript by immunizing New Zealand rabbits, verified by MS and HPLC, and conjugated to KLH (Keyhole Limpet Hemocyanin) using the MBS method (maleimide-thiol chemistry). Reactivity of rabbit antisera was evaluated by indirect ELISA using the corresponding peptide as coating antigen. To further validate the specificity of the antibody labeling, lungfish skin cryosections were incubated with the rabbit anti-Protop-CrTX-A antibody as the primary antibody of anti -Protop-CrTX-A antibody incubated under constant slow rotation with a 10-fold excess of the peptide used to generate the antibody for 1 h.Antibacterial Assays

[0097] The antibacterial activity of both Protop-CrTX-A and Neo-CrTX-2 was measured in vitro against three different gram-negative bacterial species. V. anguillarum, a pathogen of teleost fish, was obtained from E. ictaluri, originally isolated from sick channel catfish and previously used by us to infect Protopterus sp. was also tested. In order to test effects on commensal strains, we swabbed the skin of a healthy free-swimming African lungfish in the laboratory. The swab was streaked onto a TSA plate under sterile conditions and incubated at room temperature for 2 days. A single pale colony was streaked into a fresh plate to ensure a pure culture prior to picking one colony and growing it in 4 ml of Tryptic Soy Broth (TSB) overnight. Bacteria were pelleted, DNA extracted using the QlAprep®spin DNA miniprep kit, and identity obtained by PCR using 16S P46 forward primer and P943 reverse primers (P46 forward, 5'-GCYTAAYACATGCAAGTCG-3', and P943 reverse, 5'-ACCGCTTGTGCGGGYCC-3') as previously described. See, Kelly, et al., Sci. Rep. 7, 41753 (2017).

[0098] V. anguillarum, Aeromonas sp. and E. ictaluri were grown in Remel Tryptic Soy Broth (TSB) with 1% NaCl at 22°C. The minimum inhibitory concentration (MIC) of recombinant Protop-CrTX-A and Neo-CrTX-2 at increasing concentrations (0.0001, 0.01 and 1 pg / ml) was assessed by the broth microdilution method. See, Kowalska-Krochmal, Pathogens, 10, 165, 2021. Briefly, on the day of the experiment, a lOOpl suspension of either V. anguillarum, Aeromonas sp. or E. ictaluri at a density of was prepared in Mueller-Hinton broth and placed into a 96-well plate. Wells containing a pool of standard antibiotics (ampicillin, kanamycin, and carbenicillin at a concentration of 495ng / pl) were used as positive killing controls, while bacteria suspended in Mueller-Hinton broth only as a negative control. The assay was performed in replicates, at 22°C, and ODeoo was recorded 1, 3, 6, 12, 16 and 24 h after exposure using a Bio-Rad iMark™ microplate absorbance reader.

[0099] Background-corrected absorbance values were plotted to generate growth curves.

[0100] Antifungal Assays

[0101] M. circinelloides, C. albicans and X delica were grown on sterile glucose-peptone agar (10 g / 1 glucose, 2 g / 1 yeast extract, and 15 g / 1 agar). On the day of the experiment, C. albicanscolonies were harvested using a sterile loop, while M. circinelloides and 5. delica were scraped from the plates using a sterile scalpel. The colonies were resuspended in 1 ml of sterile glucose-peptone broth, and the mycelia were gently fragmented by brief vortexing or filtration through a 100 pm nylon mesh. C. albicans and Al circinelloides suspensions were placed in 96-well plates at a final concentration of l x 105cells / ml in a total volume of 200 pl of glucose-peptone broth per well. Fungal cultures were then supplemented with Protop- CrTX-A and Neo-CrTX-2 at increasing concentrations (0.0001, 0.01 and 1 pg / ml). Wells with cycloheximide (10 mg / 1) were used as a positive killing control, while fungal cells exposed to medium only were used as negative controls. Plates were incubated at 25°C, and OD«x) was recorded 1, 3, 6, 12, 16 and 24 h after exposure using a Bio-Rad iMark™ microplate absorbance reader. Background-corrected absorbance values were plotted to generate growth curves

[0102] To evaluate antifungal effects on X delica, mycelia were resuspended in broth and then seeded on glucose -peptone agar plates. Sterile disks were then placed at the center of the agar plate and inoculated with 15 pl of either sterile H2O, cycloheximide (10 mg / 1), recombinant Protop-CrTX-A (22.0 pg / ml in PBS) or recombinant. Neo-CrTX-2 (22.0 pg / ml in PBS). Plates were incubated at 25°C. Halos without fungal growth around each disk were observed to assess growth inhibition every 12 h for 48 h.

[0103] Two-choice ant attraction bioassay

[0104] Pairwise comparisons of attraction preference were conducted between African lungfish cocoon and mealworm pupae, and between acetone-extracted and fresh cocoon. Bioassays were performed in the USDA-ARS Southeast Area in Stoneville, MS using three ant species: red imported fire ants (Solenopsis invicta, RIF A), little black ants (Monomorium minimum, LB A), and thief ants (Solenopsis sp., TA). For each species, soybean oil bait was used to identify foraging spots. Thereafter, 60 mg of fresh cocoon, mealworm, or acetone-extracted cocoon were placed in individual 0.5-ml sterile Eppendorf tubes. Pairs of tubes were then simultaneously presented to RIF A, LB A, or TA colonies by placing them at the predetermined foraging spots. After 30 min, the tubes were collected, sealed, and placed in a Fluon-coated container. The container was stored at -80 °C for at least 10 min, after which the number of ants present in each tube was counted. Percentages of ants in each tube were used to evaluate attraction preferences. Each assay was replicated six times for each antspecies and each comparison. Replicates were conducted at different foraging spots, with spots at least 10 m apart. Data for each species were analyzed using paired / -tests.

[0105] Ant digging and residing assay

[0106] The insect repellent effects of recombinant Protop-CrTX-A and Neo-CrTX-2 were evaluated by conducting a two-choice digging and residing bioassay on imported fire ants. This bioassay is based on the premise that ants will either dig more extensively in tubes containing a preferred substrate or show increased residence in such tubes following digging (FIGURES 2D, 2E, 2F, 2G, 2H, 21). The apparatus described in Figures 2D-2I and the assays were originally developed by Chen (2005) and frequently used to develop fire ant repellants and attractants (Chen 2009; George et al. 2025; Paudel et al. 2023). Briefly, four 2 ml Eppendorf tubes were mounted under an 8.7 cm x 2.3 cm Petri dish and filled with 3 g of moist sand (12% water). Entrance holes were drilled in two of the tubes, and the sand within was treated with 20pl of either PBS (control), Protop-CrTX-A (22 pg / ml in PBS) or Neo-CrTX-2 (22 pg / ml in PBS). Fifteen fire ants were then introduced into the petri dish and kept at room temperature for 24 h. To determine digging preference, the moist sand removed from each tube was collected, dried at 121 °C for 4 h and weighed. At the same time, to assess the residing preference, the number of ants in each tube was recorded. A paired t-test was used to analyze both digging and residing data for each recombinant protein.

[0107] Mosquito larva immersion toxicity assays

[0108] Ten Aedes aegypti (yellow fever mosquito) larvae collected from the wild in Stoneville, Mississippi, were assigned to each treatment and submerged in 2-ml tubes containing 100 pl of either Protop-CrTX-A (22, 14, 7, 3.5, or 1.75 pg / ml in PBS), Neo-CrTX-2 (22 pg / ml in PBS), Truncated-CrTX-A (14 pg / ml in PBS), or PBS (control). Larval mortality was recorded every 6 h for up to 48 h post-exposure. Larvae that remained motionless after gentle tapping of the tube were considered dead. Each assay was replicated three times.

[0109] Quantification and statistical analysis

[0110] Data were expressed as means ± SENT Statistical analysis was performed by unpaired Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s multiplecomparison tests Differences were considered statistically significant when P < 0.05. Statistical analyses were performed in R v4.5.0 and Prism GraphPad version 10. For R. T- qPCR assays, relative changes in gene expression were determined using the Pfaffl method.

[0111] Survival analysis was performed using Kaplan-Meier (KMJ estimates and Firth’s penalized Cox proportional hazards regression. KM curves were generated using the survfit() function from the survival R package (Themeau, 2023), visualized with ggsurvplot() from the survminer package (Kassambara et ah, 2023), and further customized using ggplot2 (Wickham, 2.016). Pairwise group comparisons were performed using the log-rank test via the pairwise survdiff() function, with p-values adjusted for multiple comparisons using Holm’s method.

[0112] 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.

Claims

Claims:

1. A pest control peptide according to any one of SEQ ID Nos: 1-14, or a peptide comprising at least five contiguous amino acids which comprise the peptide residues of SEQ ID NO: 1 or which comprise the peptide residues of SEQ ID NO: 2.

2. The peptide according to claim 1, corresponding to SEQ ID NO: 1.

3. The peptide according to claim 1, corresponding to SEQ ID NO:

24. The peptide according to claim 1 comprising at least five contiguous amino acids of peptide SEQ ID NO: 1 or peptide SEQ ID NO:2.

5. The peptide according to claim 1 which comprises from 5-40, 5-35, 5-30, 5-25, contiguous amino acids of peptide SEQ ID NO: 1 or peptide SEQ ID NO:2.

6. The peptide according claim 1 wherein the peptide is a 15-mer sequence according to any one of SEQ ID Nos: 3-14.

7. A pest control composition comprising an effective amount at least one peptide according to any one of claims 1-6.

8. The composition according to claim 7 further comprising a carrier, additive or excipient in order to facilitate delivery of said peptide to a chosen area.

9. The composition according to claim 7 or 8 wherein said composition is formulated as a sprayable liquid.

10. The composition according to any one of claims 7-9 wherein the composition comprises a foodstuff to facilitate ingestion of said peptide by a pest.

11. The composition according to any one of claims 7-10 wherein said composition is formulated as a powder, microparticle / nanoparticle, emulsion, solid lipid nanoparticle, protein-based nanoparticle, liposome, hydrogel or a stimuli-responsive system.

12. The composition according to claim 11 wherein said stimuli-responsive system is a pH responsive system, a thermo-responsive system, an enzyme-responsive system, a light-responsive system, an ultrasound-responsive systems, magnetically responsive systems and multi-stimuli-responsive systems for peptides.

13. The composition according to claim 11 wherein said emulsion is a water-in-oil emulsion, an oil-in-water emulsion or a water-in-oil-in water emulsion.

14. A method of inhibiting or eradicating a target population of pests comprising exposing said population to an effective amount of a compound according to any one of claims 1-6.

15. A method of inhibiting or eradicating a target population of pests or a composition according to any one of claims 7-13.

16. The method according to either of claims 14 or 15 wherein said target population is insect larvae.

17. The method of claim 16 wherein said insect larvae are mosquito larvae (e.g., various species of Aedes, Culex and Anopheles), black fly or fungus gnat larvae.

18. The method according to claim 14 or 15 wherein said target population is the larvae or adult population of Tarnished plant bug, the common housefly, the com earworm, mealworms or termites, including the Eastern subterranean termite.

19. The method according to claim 14 or 15 wherein said target population of pests is selected from the group consisting of Aedes aegyptii (mosquito), red imported fire ants (Solenopsis Invicta), little black ants (Monomorium minimum) or thief ants (Solenopsis sp).

20. The method according to claim 14 or 15 wherein said target population of pests is selected from the group consisting of ants (carpenter, fire, Argentine, house, Pharoah, Thief), bed bugs, beetles, including woodworms, weevils, fur beetles, spider beetles, mealworm beetles, centipedes, cockroaches, dustmites, earwigs, crickets, firebrats, flies (e.g. house, black, fruit, blue bottle, green bottle), mosquitoes, moths (almond, Indianmeal, clothes, brown house), lice, red spiders, silverfish, termites (dampwood, subterranean) or woodlouse.

21. The method according to claim 14 or 15 wherein said target population of pests is the Oak Splendour Beetle, the Exotic Wood Borer / Bark Beetle, the Oak Anguina Tritici, the Wheat seed gall nematode, Citrus longhorned beetle, Silver y moth, Beocauluss spp., Colosius spp., Laevicaulis spp., Red ring nematode, Candidatus Phytoplasma australiense 16SrXII, Candidatus Phytoplasma mali 16SrX-A, Candidatus Phytoplasma palmae 16Sr-IV, Candidatus Phytoplasma phoenicium 16SrIX-B, Candidatus Phytoplasma prunorum 16SrX-F, Candidatus Phytoplasma solani 16SrXII-A (Bois noir / Stolbur Grape), Candidatus Phytoplasma vitis 16SrV-C, Japanese Wax Scale, Asiatic rice borer, Coconut cadang-cadang, Scots pine blister rust, Christmas berry webworm, Pine tree lappet, Mason pine moth, Siberian silk moth, Curcurbit beetle, Sunn pest, Old world bollworm, Black maize beetle, Ash dieback, Small brown planthopper, Gian African snail, Late wilt of corn, Megaplatypus mutatus (Ambrosia beetle), Chinese slugs, hygromiid snails, Tomato fruit borer, Horse thistle, Cotton seed bug, South American palm borer, Java downy mildew, Philippine downy mildew, Aldar root and collar rot, Beech bleeding canker, Oak ambrosia beetle, rotbrenner, Japanese oak wilt, bacterial wilt, Sarasinula spp., Semperula spp., Cotton cutworm, Potato wart, Guatemalan potato moth, False coddling moth, Pine processionary moth, Oakprocessionary moth, Cucumber green mottle mosaic, Groundnut bud necrosis, Khapra beetle, Tomato leafminer, Veronicella spp., Bacterial blight and Bacterial leaf Steak.

22. A method for inhibiting or eradicating the unfavorable consequences of a targeted insect population comprising exposing said targeted population to an effective amount of a compound or composition according to any one of claims 1-13.

23. The method according to claim 22 wherein said target population is insect larvae.

24. The method of claim 22 wherein said insect larvae are mosquito larvae (e.g., various species of Aedes, Culex and Anopheles), black fly or fungus gnat larvae.

25. The method according to claim 22 wherein said target population is the larvae or adult population of Tarnished plant bug, the common housefly, the corn earworm, mealworms or termites, including the Eastern subterranean termite.

26. The method according to claim 22 wherein said target population of pests is selected from the group consisting of Aedes aegyptii (mosquito), red imported fire ants (Solenopsis Invicta), little black ants (Monomorium minimum) or thief ants (Solenopsis sp).

27. The method according to claim 22 wherein said target population of pests is selected from the group consisting of ants (carpenter, fire, Argentine, house, Pharoah, Thief), bed bugs, beetles, including woodworms, weevils, fur beetles, spider beetles, mealworm beetles, centipedes, cockroaches, dustmites, earwigs, crickets, firebrats, flies (e.g. house, black, fruit, blue bottle, green bottle), mosquitoes, moths (almond, Indianmeal, clothes, brown house), lice, red spiders, silverfish, termites (dampwood, subterranean) or woodlouse.

28. The method according to claim 22 wherein said target population of pests is the Oak Splendour Beetle, the Exotic Wood Borer / Bark Beetle, the Oak Anguina Tritici, the Wheat seed gall nematode, Citrus longhorned beetle, Silver y moth, Beocauluss spp., Colosius spp., Laevicaulis spp., Red ring nematode, Candidatus Phytoplasma australiense 16SrXII, Candidatus Phytoplasma mali 16SrX-A, Candidatus Phytoplasma palmae 16Sr-IV, Candidatus Phytoplasma phoenicium 16SrIX-B, Candidatus Phytoplasma prunorum 16SrX-F, Candidatus Phytoplasma solani 16SrXII-A (Bois noir / Stolbur Grape), Candidatus Phytoplasma vitis 16SrV-C, Japanese Wax Scale, Asiatic rice borer, Coconut cadang-cadang, Scots pine blister rust, Christmas berry webworm, Pine tree lappet, Mason pine moth, Siberian silk moth, Curcurbit beetle, Sunn pest, Old world bollworm, Black maize beetle, Ash dieback, Small brown planthopper, Gian African snail, Late wilt of corn, Megaplatypus mutatus (Ambrosia beetle), Chinese slugs, hygromiid snails, Tomato fruit borer, Horse thistle, Cotton seed bug, South American palm borer, Java downy mildew, Philippine downy mildew, Aldar root and collar rot, Beech bleeding canker, Oak ambrosia beetle, rotbrenner,Japanese oak wilt, bacterial wilt, Sarasinula spp., Semperula spp., Cotton cutworm, Potato wart, Guatemalan potato moth, False coddling moth, Pine processionary moth, Oak processionary moth, Cucumber green mottle mosaic, Groundnut bud necrosis, Khapra beetle, Tomato leafminer, Veronicella spp., Bacterial blight and Bacterial leaf Steak.

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