Venom pesticide

Abstract

Pesticidal compositions comprising isolated arthropod venom proteins, typically of arachnids and scorpions, and zinc at least partly inhibit or prevent arthropod pest infestation of plants and animals. Zinc is typically in the form of zinc chloride. Methods of administering pesticidal compositions to treat or prevent arthropod pest infestation of animals and plants are also provided.

Description

[0001] TITLE

[0002] VENOM PESTICIDE TECHNICAL FIELD

[0003] THIS INVENTION relates to pesticidal compositions and their use for preventing or treating pest infestations of plants. More particularly, this invention relates to pesticidal compositions comprising arthropod venom proteins and methods of use for preventing or treating pest infestations of animals and plants.

[0004] BACKGOUND

[0005] Arthropods, such as arachnids and scorpions, comprise some of the oldest and most complex venoms that evolved as powerful tools for prey capture and defence (Herzig, 2021). Their venoms are dominated by peptide toxins that can be exquisitely selective and potent for certain molecular structures in their target organisms (Bende et al., 2014). However, peptide synthesis is energetically expensive and therefore arachnids developed sophisticated strategies to minimise venom wastage (Evans et al., 2019, Morgenstern and King, 2013). For example, spiders can tightly control and adapt the volume of venom delivery to the specific requirements of each prey, which forms the basis of the "venom optimisation hypothesis" (Wigger et al., 2002). Synergism between venom components further potentiates the activity of the venom. Several examples of chemically diverse spider venom components have been reported to act synergistically with venom peptides, including acylpolyamines (Adams, 2004), histamine (Wullschleger et al., 2005), peptides (Wullschleger et al., 2005) proteins (Kuhn-Nentwig et al., 2019), and even potassium (Wullschleger et al., 2005).

[0006] SUMMARY

[0007] Surprisingly, the present inventors have identified zinc as a functional component of arthropod venoms that synergizes with venom peptides in facilitating venom toxicity.

[0008] Accordingly, the present invention is broadly directed to use of arthropod venom protein(s) in combination with zinc as a pesticide. An aspect of the invention provides a composition comprising, consisting essentially of, or consisting of one or more isolated arthropod venom proteins and zinc.

[0009] Suitably, the composition elicits more venom activity than the one or more arthropod venom proteins in the absence of zinc.

[0010] In an embodiment, zinc is in the form of a zinc salt, such as zinc chloride (ZnCI2).

[0011] In a particular embodiment, the composition is a pesticidal composition.

[0012] Another aspect of the invention provides a method of producing a composition by combining one or more isolated arthropod venom proteins and zinc.

[0013] In an embodiment, zinc is in the form of a zinc salt, such as zinc chloride (ZnCI2).

[0014] This aspect also provides a composition produced by the method.

[0015] Suitably, the one or more isolated arthropod venom proteins and zinc elicit more venom activity than the one or more arthropod venom proteins in the absence of zinc.

[0016] In some embodiments, the amount or concentration or amount of zinc may be, or may be equivalent to, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 7080, 90, 100, 120, 150, 200 or more times the concentration or amount of zinc normally present in arthropod venom.

[0017] In some embodiments, the mass ratio of zinc:venom protein may be from about 0.05:1 to about 100:1, inclusive of about 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.15:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or any range between these stated values.

[0018] In some embodiments, the amount or concentration of zinc is that which produces a desired pesticidal effect wherein the amount of venom protein required to produce said desired pesticidal effect is reduced by a factor of at least about 2, 5, 10, 15, 20, 50, 80, 100, 150, 200 or more compared to that in the absence of zinc.

[0019] In an embodiment, the isolated arthropod venom protein is obtained, or obtainable from, an arachnid or scorpion. In particular embodiments of these aspects, the isolated arthropod venom protein comprises an amino add sequence set forth in any one of SEQ ID NOS:l-397, or a variant, fragment or derivative thereof.

[0020] In a particular embodiment, the composition is a pesticidal composition.

[0021] Yet another aspect of the invention provides a method of at least partly increasing or potentiating the pesticidal activity of one or more isolated arthropod venom proteins including the step of combining the one or more isolated arthropod venom proteins with zinc to thereby at least partly increase or potentiate the pesticidal activity of the one or more isolated arthropod venom proteins.

[0022] This aspect of the invention also provides a composition comprising one or more isolated arthropod venom proteins produced according to this method.

[0023] In an embodiment, zinc is in the form of a zinc salt, such as zinc chloride (ZnCI2).

[0024] Another further aspect of the invention provides a method of at least partly preventing or inhibiting pest infestation of an organism, said method including the step of administering one or more isolated arthropod venom proteins and zinc to the organism, to thereby at least partly prevent or inhibit pest infestation of the organism.

[0025] In particular embodiments, the isolated arthropod venom protein comprises an amino acid sequence set forth in any one of SEQ ID NOS:l-397, or a variant, fragment or derivative thereof.

[0026] A further aspect of the invention provides a method of at least partly preventing or inhibiting pest activity including the step of administering one or more isolated arthropod venom proteins and zinc to a pest or to an environment containing, or potentially containing the pest, to thereby at least partly prevent or inhibit pest activity.

[0027] A still further aspect of the invention provides a method of increasing or potentiating at least partial inhibition or blocking of a pest ion channel by one or more arthropod venom proteins, said method including the step of administering zinc and the one or more arthropod venom proteins to the pest, or to an environment containing the pest, to thereby increase or potentiate at least partial inhibition or blocking of the pest ion channel. In an embodiment, the isolated arthropod venom protein is obtained, or obtainable from, an arachnid or scorpion.

[0028] In particular embodiments, the isolated arthropod venom protein comprises an amino acid sequence set forth in any one of SEQ. ID NOS:l-397, or a variant, fragment or derivative thereof.

[0029] The one or more isolated arthropod venom proteins and zinc may be administered sequentially or in combination, such as in the aforementioned composition.

[0030] The one or more isolated arthropod venom proteins and zinc may be administered sequentially ( / .e in any order) or in combination, such as in the aforementioned composition.

[0031] In an embodiment, the pest is an Arthropod inclusive of insects and arachnids.

[0032] In one embodiment, the organism is an animal.

[0033] In another embodiment, the organism is a plant, such as a crop plant.

[0034] In yet another embodiment, the organism is a fungus.

[0035] In some embodiments of the aforementioned methods, the amount or concentration or amount of zinc may be, or may be equivalent to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70 80, 90, 100, 120, 150, 200 or more times the concentration or amount of zinc normally present in arthropod venom.

[0036] In some embodiments, the mass ratio of zinc:venom protein may be from about 0.05:1 to about 100:1, inclusive of about 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.15:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, , or any range between these stated values.

[0037] In some embodiments of the aforementioned methods, the amount or concentration of zinc is that which produces a desired pesticidal effect wherein the amount of venom protein required to produce said desired pesticidal effect is reduced by a factor of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70 80, 90, 100, 120, 150, 200 or more compared to that in the absence of zinc.

[0038] Throughout this specification, unless the context suggests otherwise, "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

[0039] In the context of the present invention, by "consists essentially of" is meant that the composition may consist of the one or more isolated arthropod venom proteins and zinc together with one, two or three additional non-stated components.

[0040] It will also be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" venom protein includes one venom protein, one or more venom proteins and a plurality of venom proteins.

[0041] The term "about" is used herein to refer to a tolerance or variation in a stated amount. The tolerance or variation may be no more than ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1% of a stated amount.

[0042] BRIEF DESCRIPTION OF THE FIGURES

[0043] Figure 1. Synergistic insecticidal effects of zinc and spider venom peptides in sheep blowflies. A: ZnCI2was found to be 1-2 orders of magnitude more potent than CaCI2or NaCI, respectively, in causing insecticidal toxicity in sheep blowflies (Lucilia cuprina). B-F: Comparison of the expected (assuming additive effects, black) and observed effects (red) of co-administration of ZnCI2(5 μg / fly) with the insecticidal spider toxins ω-HxTX-Hv1a (co-Hv1a, B), k -HxTX-Hv1c (K-HV1C, C), Ui-AGTX-Tala (, D), μ-DGTX-Dcla (Dcla, E), and ω / k -HXTX-Hv1a (ω / k- Hv1a, F). Statistical comparison using CalcuSyn for Windows (Biosoft, Cambridge, UK) indicates synergism (+++, Cl = 0.3-0.7), moderate synergism (++, Cl = 0.7-0.85), nearly additive (±, Cl = 0.9-1.1) slight antagonism (-, Cl = 1.1-1.2) and antagonism ( — , Cl = 1.45-3.3). * denotes that no Cl value could be calculated for zinc and toxin combinations that resulted in 0% or 100% lethality. Fly lethality was always determined 24h post-injection

[0044] Figure 2: Zinc and spider venom fractions inhibit Cav3.2 channels. A: Zinc and three reversed-phase HPLC fractions of A. theraphosoides spider venom containing, or potentially containing, different zinc quantities were found to inhibit Cav3.2 channels (percentage values indicate Cav3.2 inhibition ± SEM). Increasing concentrations of EDTA blocked the Cav3.2 inhibition caused by the venom fractions or by zinc. B, C: Representative calcium current traces for human Cav3.2 expressed in HEK-293 cells before (black) and after (red) treatment with zinc or A. theraphosoides venom fraction 3 (left panels). Addition of EDTA to zinc or venom fraction 3, respectively, abolished this inhibition (right panels).

[0045] Figure 3: Zinc supplement feeding trial. Juvenile A. geniculata spiders were fed with crickets dusted with zinc-containing, or potentially containing, supplement (treatment group) or without (control group). A: Average zinc content over different milkings for the control and treatment groups. B: Average zinc content for individual A. geniculata spiders over all milkings. Error bars indicate the respective STDEV.

[0046] Figure 4: Oral toxicity test. Results of oral toxicity tests of spider venom peptide Dcla (SEQ. ID NO:258) with (+) and without (-) Zn (= 2 ug / fly of ZnCI2). All treatments were dissolved in 5% sucrose and delivered orally to adult sheep blowflies ( / .. cuprina). Mortality was recorded after 24 hours.

[0047] DETAILED DESCRIPTION

[0048] The present invention is at least partly predicated on the identification of zinc as a functional component of arthropod venom proteins that synergizes with venom proteins in facilitating venom toxicity towards pest arthropods. It is therefore proposed that pesticidal compositions comprising isolated arthropod venom proteins and zinc may be useful in at least partly inhibiting or preventing arthropod pest infestation of plants and other animals. A particular advantage of such compositions is that zinc is an essential trace element that assists plant growth and also has benefits for animal skin health. Accordingly, the present invention may facilitate both pest inhibition and beneficial zinc treatment of plants and animals.

[0049] An aspect of the invention provides a composition comprising, consisting essentially of, or consisting of one or more isolated arthropod venom proteins and zinc.

[0050] In a particular embodiment, the composition is a pesticidal composition.

[0051] Another aspect of the invention provides a method of producing a composition by combining one or more isolated arthropod venom proteins and zinc. Yet another aspect of the invention provides a method of at least partly increasing or potentiating the pesticidal activity of one or more isolated arthropod venom proteins including the step of combining the one or more isolated arthropod venom proteins with zinc to thereby at least partly increase or potentiate the pesticidal activity of the isolated arthropod venom protein.

[0052] The invention also provides a pesticidal composition comprising the one or more isolated arthropod venom proteins of this aspect.

[0053] The composition may comprise one or a plurality of different arthropod venom proteins.

[0054] Suitably., the composition disclosed herein elicits more pesticidal activity than the one or more arthropod venom proteins in the absence of zinc.

[0055] As will be evident from the experimental data shown in FIG. 1, in particular, zinc and isolated arthropod venom proteins in combination show greater than additive pesticidal activity and typically demonstrating moderate to strong synergism. In some embodiments, the zinc and isolated arthropod venom proteins in combination elicit substantially more pesticidal activity than the one or more isolated arthropod venom proteins in the absence of zinc. By way of example, the combination of ω / k -HXTX-Hv1a venom protein and zinc is about 134-fold more potent than the venom protein alone. Effectively, the amount of venom protein required to produce a desired pesticidal effect may be reduced by a factor of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70 80, 90, 100, 120, 150, 200 or more in the presence of zinc, or any range between these stated values.

[0056] For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be partly, substantially or essentially free from, or depleted of, components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form. In some embodiments, isolated material may be in enriched, partially purified or purified form.

[0057] Zinc may be in atomic form of a metal, in ionic form (typically Zn2+or more rarely [Znz]2+) such as a salt, oxide, hydride and / or peroxide, an isotopic form such as64Zn, as a zinc co-ordination complex or chelate and / or in the form of a Zinc (I) compound and / or Zinc (li) compound, although without limitation thereto. Nonlimiting examples of zinc salts include halides, sulfates, nitrates and molybdates. Preferably, zinc is in the form of a zinc halide, more preferably zinc chloride (ZnCI2).

[0058] The concentration of zinc in combination with the one or more isolated arthropod venom proteins is suitably that which enhances or potentiates a desired level of pesticidal activity of the one or more isolated arthropod venom proteins. In the context of pesticidal use, it will be appreciated that the amount of zinc and one or more arthropod venom proteins in the composition will at least partly depend upon the type of pest(s), the particular organism to which the composition is administered, the level of pest infestation and the isolated arthropod venom proteins(s) that are administered and also the zinc requirements and tolerance of the organism to which the composition is administered.

[0059] Tables 2 and 3 provide zinc content as a percentage of dried venom protein mass in a plurality of different spider and scorpion venoms. It will be appreciated that the concentration of zinc in combination with the one or more isolated arthropod venom proteins may be the same or different to that which is normally found in arthropod venom ( / .e higher or lower). In some embodiments, the concentration or amount of zinc may be, or may be equivalent to, at least about 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200 or more times the concentration or amount of zinc normally present in arthropod venom. Given that the majority of dried venom mass is venom protein, the abovementioned zinc concentrations disclosed herein may be approximated to venom protein mass.

[0060] In some embodiments, the mass ratio of zinc:venom protein may be from about 0.05:1 to about 100:1, inclusive of about 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.15:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or any range between these stated values.

[0061] As generally used herein the term "protein" refers to an amino acid polymer. Amino acids may include natural or non-natural amino acids, D- and L-amino acids. The term "peptide" typically refers to a protein comprising at least, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 but no more than 100 contiguous amino acids. The term "polypeptide" typically refers to a protein comprising more than 100 contiguous amino acids. The term "protein" may also include and encompass aptamers, protein-nucleic acid hybrids and modified proteins such as glycoproteins and lipoproteins, although without limitation thereto.

[0062] The isolated proteins disclosed herein may be referred to as "venom proteins". Suitably, the isolated arthropod venom proteins are obtained, or are obtainable from, invertebrates of the phylum Arthropoda and have pesticidal activity which is enhanced or potentiated by zinc. By "pesticidal activity" is meant that the venom proteins may at least partly inhibit, interfere with, suppress or otherwise adversely affect pest activity. In this context, "pest activity" may include or at least be partly dependent on one or more of pest viability, structural integrity, predator defences, feeding, reproduction, fecundity, movement, flight, energy metabolism, lipid metabolism, hormonal system, haemolymph, sensory perception, respiration, excretion, secretion, ion transport and / or the nervous system, although without limitation thereto. This pesticidal activity may be effective against some or all developmental forms and stages of the pest, inclusive of adults, juveniles, eggs, pupae and larvae. It will also be understood that this pesticidal activity does not necessarily kill, eliminate or eradicate pest arthropods.

[0063] As generally used herein a "pest" is an Arthropod which has an undesired, negative effect on other organisms such as animals and / or plants, particularly animals and / or plants of agricultural, commercial and / or economic importance. Pest Arthropods may typically be insects (class Insecta) although some pests may be arachnids (class Arachnida).

[0064] Non-limiting examples of pests include Diptera (e.g flies such as sheep blowflies, mosquitoes, gnats; Lepidoptera (e.g. butterflies and moths such as cotton bollworm); Orthoptera (e.g. locusts and crickets); Parasitiformes (e.g. ticks and mites); Coleoptera (e.g beetles such as small hive beetle); Formicidae (e.g ants); Phthiraptera (e.g. lice); Siphonaptera (e.g. fleas); Hemiptera (e.g. aphids, bedbugs and whiteflies); Acariformes (e.g mites such as spider mites), Varroidae (e.g varroa mites), Blattaria (e.g cockroaches); and Thysanoptera (e.g thrips), although without limitation thereto.

[0065] In particular embodiments, the isolated arthropod venom proteins are obtained, or are obtainable from one or more arachnids or scorpions. Non-limiting examples of isolated arthropod venom proteins are set forth in Table 1 (SEQ. ID NOS: 1-397), which includes the particular arachnid or scorpion species from which each of the venom proteins originate.

[0066] It will also be appreciated that each venom protein may display or possess an activity that is deleterious to one or more particular arthropod pests. By way of example only: Dcla may have suitable pesticidal activity against Lepidoptera (e.g butterflies, moths) and / or Diptera (e.g flies); Omega-Hv1a may have suitable pesticidal activity against Orthoptera (e.g locusts, crickets), Diptera (e.g. flies), Lepidoptera (e.g butterflies, moths) and / or Parasitiformes (e.g ticks); Kappa-Hv1c may have suitable pesticidal activity against Lepidoptera (e.g butterflies, moths), Diptera (e.g flies) and / or Orthoptera (e.g locusts, crickets); Tala may have suitable pesticidal activity against Lepidoptera (e.g butterflies, moths); Diptera (e.g flies) and / or Coleoptera (e.g beetles); and ω / k -HXTX-Hv1a toxin contained in Spear® (Vestaron Corp; also see King 2019, DOI 10.1002 / ps.5452) may have suitable pesticidal activity against Lepidoptera (e.g butterflies, moths), Diptera (e.g flies), Orthoptera (e.g locusts, crickets), Hemiptera (e.g aphids, whiteflies), Acariformes (e.g spider mites) and / or Thysanoptera (e.g thrips).

[0067] It will also be appreciated that also contemplated are variants, fragments and derivatives of the isolated proteins disclosed herein. In some embodiments, protein variants may be homologs, orthologs, mutagenized and / or chemically-modified forms of the isolated proteins disclosed herein. It will be appreciated that the isolated proteins disclosed herein may be mutagenized by one or more amino acid deletions, additions and / or substitutions. Conservative amino acid substitutions preferably retain substantial protein function, such as by conserving amino acid hydrophobicity, hydrophilicity, charge and / or side-chain bulk, although without limitation thereto.

[0068] By way of example, conservative amino acid substitutions may occur within groups such as: small aliphatic, non-polar or slightly polar amino acids Als, Ser, Thr, Pro, Gly; polar negatively charged amino acids Asp, Asn, Glu, Gin; polar positively charged amino acids His, Arg, Lys; large, aliphatic non-polar amino acids Met, Leu, He, Vai, Cys; and large aromatic amino acids Phe, Trp, Tyr.

[0069] Accordingly, in one embodiment a variant amino acid sequence may have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ. ID NOS: 1-397.

[0070] The term "sequence identity" is used herein in its broadest sense to include the number of exact amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a "comparison window". Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window', determining the number of positions at which the identical amino acid occurs in both sequences to thereby yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison {i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0071] With reference to the foregoing, a "comparison window" refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions {i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as: the BLAST family of algorithms including BLAST-P and psi-Blast; FASTA; and ClustalW and Clustal Omega (EMBL-EBI), although without limitation thereto.

[0072] A more detailed discussion of amino acid sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons inc NY, 1995-2015) and Unit 2 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al. (John Wiley & Sons Inc NY, 1995-2016).

[0073] Fragments of the isolated arthropod venom proteins may comprise at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 or more amino acid portions of a "full length" isolated arthropod venom protein. These portions may be contiguous or non-contiguous. Suitably, the fragment has pesticidal activity.

[0074] It will also be appreciated that derivatives are contemplated, such as chemically modified venom proteins disclosed herein. By way of example, chemical modification of proteins may be by way of biotinyiation, avidin conjugation, fluorophore conjugation, acylation, amidation of carboxyl groups, phosphorylation, reductive alkylation, succinylation, performic acid modification of cysteines and / or carboxymethylation, although without limitation thereto. A more detailed discussion of chemical modification of proteins may be found in Unit 2 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al. (John Wiley & Sons Inc NY, 1995-2016).

[0075] Isolated proteins, inclusive of variants and derivatives, may be produced by purification or partial purification of native proteins, chemical synthesis or by recombinant DNA technology. Chemical synthesis of peptides is well known in the art, such as described in Unit 9 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al. (John Wiley & Sons Inc NY, 1995-2016).

[0076] In an embodiment, the isolated protein is produced as a recombinant protein. Suitably, recombinant protein expression is by way of a nucleic acid encoding the recombinant protein.

[0077] The term "nucleic acid' as used herein designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, thiouridine, thiothymidine, methylcytosine, methylinosine and methyladenosine, although without limitation thereto.

[0078] Recombinant protein production is well known in the art, such as described in Unit 5 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-2015) and Chapter 19 of Molecular Cloning: A Laboratory Manual. 4thEd. Sambrook & Russell (CSHL Press, NY, 2012). Recombinant proteins may be modified to comprise one or more fusion partners or tags that facilitate isolation and / or detection of recombinant proteins. Non-limiting examples include fusion partners for recombinant protein isolation such as maltose-binding protein (MBP), glutathione-S-transferase (GST), polyhistidine (e.g hexahistidine) and FLAG, c- myc and hemagglutinin (HA) epitope tags.

[0079] Typically, a nucleic acid encoding the protein is present as a genetic construct, such as an expression vector, that further comprises one or more regulatory nucleotide sequences that facilitate, initiate and / or control transcription of the nucleic acid, expression and / or purification of the recombinant protein.

[0080] Regulatory nucleotide sequences may comprise one or more of: a promoter (which may be inducible, repressible or constitutively active); enhancer; RNA splice site(s); polyadenylation signal; termination sequence fusion partner nucleotide sequence; host cell origin of replication; and / or nucleotide sequences encoding antibiotic resistance; although without limitation thereto. Suitably, regulatory nucleotide sequences are utilized according to suitability with the host organism or cell type used for expression of the recombinant protein. Host cell types may be of bacterial (e.g E. coH), mammalian (e.g. HEK-293, CHO and derivatives such as CHO- Kl, CHO pro-3, DUKX-X11 and DG44), plant (e.g tobacco, rice and microalgae), insect (e.g Sf9), yeast (e.g Pichia pastoris) and / or amphibian (e.g Xenopus oocytes) origin, although without limitation thereto.

[0081] Genetic constructs may be introduced into a host cell by transfection techniques such as electroporation, DEAE-dextrose, calcium phosphate, liposomes, microinjection, viral transduction, microparticle bombardment and / or Agrobacterium-mediated transfection, although without limitation thereto. It will be appreciated that the mode of host cell transfection will depend on the host cell type and genetic construct, as is well understood in the art.

[0082] It will also be appreciated that a genetic construct may be transiently expressed by a host cell, such as for up to about 48- 96 hrs or may be stably expressed thereby enabling recombinant protein expression for the duration of the life of the host cell and its progeny. Accordingly, the genetic construct, host cell type and mode of transfection can be chosen according to whether transient or stable recombinant protein expression is required.

[0083] Suitably, the composition is formed by combining purified or partially purified native venom protein(s), chemically synthesized or recombinant venom protein(s) with zinc to achieve a zinc concentration as hereinbefore described. Preferably, the venom protein is combined with a zinc halide such as zinc. The composition may further comprise one or more carriers, diluents and / or excipients suitable for delivery to plants or animals. These may include fillers, stabilizers, surfactants, solid supports, solvents inclusive of water and organic solvents such as alcohols, emulsifiers, buffers or other pH modifiers, oils, fats and / or waxes, sugars, sugar alcohols, salts in addition to zinc salts, vitamins, minerals and / or nutrients, although without limitation thereto. In some embodiments, the composition may be in solution form (e.g an aqueous or other solvent solution), as a suspension or in solid form, such as in a dried, lyophilized, desiccated or powdered form, as an emulsion, a gel, in colloidal form, encapsulated such as in liposomes and / or may comprise the isolated arthropod venom proteins coupled to a solid support such as microspheres or latex beads, although without limitation thereto.

[0084] As hereinbefore described, a particular advantage of such compositions is that zinc is an essential trace element that assists plant growth and also has benefits for animal skin health.

[0085] In some embodiments, the composition may be combined with, or present in, another composition suitable for administration to a plant or animal. Said another composition may be another pesticide composition, a herbicide, a fertilizer, a soil conditioner, a topical composition for treating animals such as a dermatitis, tick or flea treatment, shampoo or livestock dip, although without limitation thereto.

[0086] In some embodiments, the composition alone or combined with, or present in, another composition, may be in the form of a concentrate suitable for subsequent dilution prior to use.

[0087] A further aspect of the invention provides a method of at least partly preventing or inhibiting pest infestation of an organism, said method including the step of administering one or more isolated arthropod venom proteins and zinc to the organism, to thereby at least partly prevent or inhibit pest infestation of the organism.

[0088] In this context "infestation" includes pest colonization and reproduction and the effects of infestation such as disease, damage and destruction of the organism.

[0089] It will be appreciated that pest infestation of the organism may be at least partly prevented or inhibited by the pesticidal activity of the one or more venom proteins and zinc, as hereinbefore described.

[0090] Suitably, the one or more isolated arthropod venom proteins and zinc is administered at a pesticidal-effective concentration or amount which is sufficient to at least partly prevent or inhibit pest infestation. It will be appreciated that the pesticidal-effective concentration or amount will at least partly depend upon the type of pest(s), the particular organism, the level of pest infestation and the isolated arthropod venom proteins(s) that are administered. Further to this, the amount of zinc and arthropod venom protein will at least partly depend upon similar factors and also the zinc requirements and tolerance of the organism.

[0091] In some embodiments, the concentration or amount of zinc may be, or may be equivalent to, at least about 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200 or more times the concentration or amount of zinc normally present in arthropod venom.

[0092] In some embodiments, the mass ratio of zinc:venom protein may be from about 0.05:1 to about 100:1, inclusive of about 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.15:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, , or any range between these stated values.

[0093] In particular embodiments, the isolated arthropod venom protein comprises an amino acid sequence set forth in any one of SEQ. ID NOS:l-397, or a variant, fragment or derivative thereof.

[0094] Suitably, the one or more isolated arthropod venom proteins and zinc is administered at a pesticidal-effective concentration or amount which is sufficient to at least partly prevent or inhibit pest infestation of the organism.

[0095] The one or more isolated arthropod venom proteins and zinc may be administered sequentially or in combination, such as in the composition disclosed herein.

[0096] In one embodiment, the organism is an animal.

[0097] Preferably, the animal is a mammal or an avian. Mammals may include human and non-human mammals.

[0098] The animal may be livestock, domestic or companion animals, poultry, performance animals and zoo or other display animals, although without limitation thereto.

[0099] Non-limiting examples of non-human mammals include sheep, cattle, goats, pigs, horses, alpacas, camels, donkeys, dogs and cats, although without limitation thereto. In another embodiment, the organism is a plant.

[0100] Generally, the term "plant" as used herein refers to any member of the plant kingdom. This term includes any plants which grow naturally and / or which are grown as a result of human intervention. In particular, plants may be obtained by conventional breeding, selection and optimization methods or by biotechnological and genetic engineering methods or combinations of these methods.

[0101] In particular embodiments, the plants are of agronomic value or importance, such as crop plants.

[0102] In particular embodiments, plants inclusive of crop plants, may be timber plants, grasses, fruit-bearing plants, cereals, vegetables, oil-producing plants, vines, fibre plants, nuts, legumes, herbs and / or ornamental plants, although without limitation thereto.

[0103] In some embodiments, plants, inclusive of crop plants, may include: timber plants such as conifers (e.g pine, cypress, fir and spruce), cedar, eucalypts, oak, mahogany, willow, beech and ash; grasses such as sugarcane, bamboo, hay and turf grasses; cereals such as wheat, rye, barley, rice, maize and sorghum; fruits such as stone fruit {e.g. peaches, plums and cherries), bananas, mangoes, papaya, melons, pineapples, pomegranate, apples, pears, avocadoes, citrus fruit such as oranges, limes, lemons, grapefruit, mandarins and tangerines, berries such as blueberries, strawberries, raspberries and blackberries; nuts such as macadamia, almond, walnut, hazelnut and Brazil nut; legumes such as beans, lentils, peas and soya beans; oil- producing crops such as canola, mustard, poppies, olives, sunflowers, coconuts, castor-oil plants, cacao and peanuts; fibre plants such as cotton, flax, hemp and jute; vegetables such as spinach, lettuce, pumpkin, cucumber, asparagus, broccoli, cabbage, carrots, onions, tomatoes, potatoes, beet and capsicum; herbs such as marjoram, oregano, basil, coriander, sage and thyme; tobacco; vines such as grapes, passionfruit, kiwifruit and hops; and ornamental flowers such as daisies, tulips, roses, geraniums, dahlias, carnations, azaleas, lilies, rhododendrons and violets, although without limitation thereto.

[0104] In another embodiment, the organism may be a fungus such as a mushroom.

[0105] The composition disclosed herein may be administered to plants and / or fungi in broadacre crops, farmland, grassland, gardens such as home gardens and public gardens, nurseries, arboretums, in viticulture and timber forests and plantations, although without limitation thereto.

[0106] Administration of the composition may be to plants, fungi or to plant or fungi parts. Parts of plants or fungi may be above-ground or below-ground parts, such as shoots, stems, leaves, flowers, bark, trunks, branches roots, fruits, fruiting bodies, seeds, tubers and rhizomes. Parts of plants and fungi also include harvested material and vegetative and generative propagation material, for example seedlings, tubers, rhizomes, spores, cuttings and seeds.

[0107] Another further aspect of the invention provides a method of at least partly preventing or inhibiting pest activity including the step of administering one or more isolated arthropod venom proteins and zinc to a pest or to an environment containing, or potentially containing the pest, to thereby at least partly prevent or inhibit pest activity.

[0108] It will be appreciated that this pest activity may be at least partly prevented or inhibited by the pesticidal activity of the one or more venom proteins and zinc, as hereinbefore described.

[0109] The one or more isolated arthropod venom proteins and zinc may be administered sequentially or in combination, such as in the composition disclosed herein.

[0110] Suitably, the one or more isolated arthropod venom proteins and zinc is administered at a pesticidal-effective concentration or amount which is sufficient to at least partly prevent or inhibit pest infestation. It will be appreciated that the pesticidal-effective concentration or amount will at least partly depend upon the type of pest(s), the particular environment, the level or potential level of pest infestation and the isolated arthropod venom proteins(s) that are administered.

[0111] In some embodiments, the concentration or amount of zinc may be, or may be equivalent to, at least about 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200 or more times the concentration or amount of zinc normally present in arthropod venom.

[0112] In some embodiments, the mass ratio of zinc:venom protein may be from about 0.05:1 to about 100:1, inclusive of about 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.15:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, , or any range between these stated values.

[0113] In particular embodiments, the isolated arthropod venom protein comprises an amino acid sequence set forth in any one of SEQ. ID NOS:l-397, or a variant, fragment or derivative thereof.

[0114] According to this aspect, the one or more isolated arthropod venom proteins and zinc may be administered to an environment where pests may be present, or where pests may become present. By way of example, administration may be to plant soil, surrounding air, equipment that may come into contact with plants, animal bedding, housing, cages and / or clothing, although without limitation thereto. By administration to the plant or animal environment, the method may at least partly prevent or inhibit pest activity in that environment, thereby at least partly minimizing the possibility that pests may infest plants or animals that enter or otherwise become present in that environment.

[0115] Plants and animals are as hereinbefore described.

[0116] Administration of the one or more isolated arthropod venom proteins and zinc according to the methods disclosed herein may be by spraying, painting, immersion, impregnation or any other suitable delivery method known in the art. Spraying may be achieved using a spray bottle, pressurized aerosol, pump or other hand-held device for house-hold, glasshouse or garden administration of the composition, or by larger scale agricultural spraying equipment inclusive of aerial spraying or vehicle-based spraying suitable for broadacre crops.

[0117] For administration to animals, the one or more isolated arthropod venom proteins and zinc may be administered topically as a spray (e.g aerosol or pump), bodywash, shampoo, orally, as a dipping solution (e.g for livestock), paint, ointment, cream or other mode of topical administration known in the art.

[0118] As previously described, in some embodiments, the one or more isolated arthropod venom proteins and zinc may be combined with, or present in, another composition for administration to a plant or animal. Said another composition may be another pesticide composition, a herbicide, a fertilizer, a soil conditioner, a topical composition for treating animals such as a dermatitis, tick or flea treatment, shampoo or livestock dip, although without limitation thereto. In some embodiments, the one or more isolated arthropod venom proteins and zinc alone or combined with, or present in another composition may be in the form of a concentrate suitable for subsequent dilution prior to use according to the aforementioned methods.

[0119] Although not wishing to be bound by theory, it is proposed that zinc, such as zinc chloride, acts to increase, improve and / or potentiate blocking of one or more pest cell ion channels, to thereby exert pesticidal activity such as hereinbefore described.

[0120] Accordingly, in a still further aspect the invention provides a method of increasing or potentiating at least partial inhibition or blocking of a pest ion channel by one or more arthropod venom proteins, said method including the step of administering zinc and the one or more arthropod venom proteins to the pest, or to an environment containing the pest, to thereby increase or potentiate at least partial inhibition or blocking of the pest ion channel.

[0121] Ion channels may include voltage-gated and ligand-gated ion channels inclusive of calcium, sodium and potassium ion channels and nicotinic acetylcholine receptors (nAChR) ion channels, although without limitation thereto.

[0122] So that the invention may be readily understood and put into practical effect, reference is made to the following non-limiting examples.

[0123] EXAMPLES

[0124] INTRODUCTION

[0125] Zinc is the 23rdmost abundant element in the earth's crust and the second most common trace metal (after iron) in the human body (National Minerals Information Center, 2021). Zinc is an essential trace element enabling the proper function of a variety of enzymes and proteins (Plum et al., 2010). In arthropods, zinc is for example used to cross-link histidine-rich proteins to confer hardness to the cuticula (Politi et al., 2017). Interestingly, a few recent studies detected zinc in the venoms of scorpions, honeybees and snakes, but did not further examine its ecological role (Al-Asmari et al., 2016, Choinska et al., 2021, Lemon et al., 2020). Zinc is an allosteric modulator of a wide range of voltage-gated ion channels and ligand-gated ionotropic receptors (Peralta and Huidobro-Toro, 2016). One study even concluded that "almost all ion receptor channels are modulated by zinc, an indication that probably trace metal modulation is a primitive, ancient, and versatile mechanism to modulate the biophysical properties of agonist-gated channels" (Peralta and Huidobro-Toro, 2016). The present inventors reasoned that zinc's remarkably broad-spectrum of molecular targets renders it an ideal candidate as a chemical weapon for use in venom, even more so if it synergises with venom peptides, thereby enhancing the overall venom activity. To test the hypothesis of venoms containing, or potentially containing, weaponised zinc, the present inventors examined the content of zinc in comparison to seven other metals in 85 arthropod venoms. In addition, the present inventors examined the insecticidal effects of zinc by itself and in combination with a range of insecticidal spider venom peptides to determine potential synergistic activities. Intriguingly, the venom contents of zinc were considerably higher than for any of the other metals and in some venoms the zinc amount was sufficient to cause insecticidal activity by itself, while zinc was also found to synergise when co-applied with all arachnid venom peptides tested.

[0126] MATERIALS AND METHODS

[0127] Venoms

[0128] All arthropod venoms were collected by electrical stimulation (spiders, centipedes, assassin bugs), aggravation assisted by electrical stimulation (scorpions) or by parafilm-assisted manual collection (caterpillars) as previously described (Guo et al., 2018, Herzig and Hodgson, 2009, Walker et al., 2018, Walker et al., 2021).

[0129] Elemental metal quantification by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

[0130] Elemental metal analysis was performed by the Environmental Chemistry Laboratory of The University of Queensland (Australia). A diverse panel venoms from 85 arthropod species (each at 20 pg of dried venom, see Table 2) from 66 spider, 16 scorpion and one species each of centipede, assassin bug, and lepidopteran were analyzed for their content of the following heavy metals:52Cr,55Mn,56Fe,59Co,60Ni,63Cu,66Zn, and111Cd, using an Inductively-Coupled Plasma Mass Spectrometer (ICP-MS 7900, Agilent, Mulgrave, Victoria, Australia). The dried venom was dissolved in 5 ml of 2% ultra-clean nitric acid (HNO3). The sample solutions were thoroughly shaken to obtain a complete homogenization. A set of six multi-element standards with known concentrations ranging from 0.1 ppb to 50 ppb was prepared and used as calibration standards. During analysis, the sample, standard and monitor solutions, and blanks were all systematically mixed with a constant amount of internal standard solution (6Li,61Ni,103Rh,115ln). These internal standards help to correct for any mass bias during analysis and enable the quantification of the metal amounts according to their natural isotopic distribution. A monitor solution was prepared and repeatedly measured at an interval of 5 samples throughout the run. The data of this monitor solution is used to correct for any instrumental drift. A set of two standards with known concentrations were measured at the beginning and at the end of each run and the data is used to assess the data quality. These data showed that the precision of the heavy metals' measurement is better than 2% while the accuracy is better than 6%.

[0131] Mock pooling experiment

[0132] A mock pooling experiment was performed using three different types of tubes from two manufacturers (1.5 ml tubes (lot 1806244) from Sarstedt (Mawson Lakes, SA, Australia); 1.5 ml DNA LoBind tubes (lot H179467N) and 2.0 ml Safe-Lock tubes (lot 11824611) from Eppendorf (Macquarie Park, NSW, Australia). For each tube type, both a single and 30 tubes were filled with 50 pl of mill iQ. water and then kept at - 20°C for three weeks. Thereafter, the tubes were thawed and the liquid from the single or the 30 tubes were pooled separately for each tube type, dried by vacuum centrifugation, and then subjected to the analysis of the elemental66Zn content by ICP-MS. A dried sample weight of 20 pg was assumed for calculating the percental content of metals in the mock pooling experiment to enable comparison with the ICP-MS results from the venoms. The data from the mock pooling experiments can therefore be considered as procedural blanks. The zinc enrichment factor was defined as "zinc content in 30 pooled tubes / zinc content in 1 pooled tube". The range of the zinc enrichment factor is therefore expected to be between 1 (i.e. no zinc enrichment) and 30 (i.e. max. zinc enrichment from all 30 tubes).

[0133] Determination of unbound zinc ratio

[0134] Two of the venoms that exhibited > 1% zinc content based on our initial analysis (Table 3) were selected, including a spider ( / .. megatheloides) and a scorpion (P. hoffmanni) representative. For each venom, 3 technical repeats of 20 pg / venom were separately processed. Each venom sample was dissolved in 200 pl of milliQ water and then concentrated (always using 6000 g) through a 3 kDa cut-off centrifugal filter (Amicon Ultra, Merck, Darmstadt, Germany) to about 100 pl. Then 400 pl of milliQ. water was added and the sample concentrated again to about 100 pL, before adding another 200 pl of milliQ water and concentrating it to the final volume of about 100 pl. For each repeat of both venoms, all supernatants (SN) and flowthroughs (FT) were separately collected, dried by vacuum centrifugation, and then subjected to the analysis of the elemental66Zn content by ICP-MS.

[0135] Cav3.2 patch-clamp electrophysiology

[0136] Patch pipettes were made from borosilicate glass capillary tubes (Warner Instruments, Holliston, MA, USA) using a P-97 Flaming-Brown type micropipette puller (Sutter Instrument, Novato, CA, USA) with a tip resistance of 3~4 MQ. Ionic currents and membrane potentials were recorded using an Axon multiclamp 700B amplifier (Molecular Devices, San Jose, USA). Voltage and current commands and digitization of membrane voltages and currents were controlled using a Digidata 1440A interfaced with Clampex 10.5 (Molecular Devices, San Jose, USA). Obtained data were analyzed with pCLAMP10.5 software (Molecular Devices, San Jose, USA). To record calcium current, cells were perfused with a Na+-free, K+-free solution (in mmol / L): 145 TEA-CI, 5 CsCI, 5 CaCI2, 10 HEPES, 1 MgCI2, and 5 glucose, adjusted to pH 7.4 with TEA-OH. The pipette solution (in mmol / L): 130 CsCI, 5 MgCI2, 20 HEPES, 10 EGTA, 3 Mg-ATP, and 0.3 Tris-GTP, adjusted to pH 7.3 with CsOH. All experiments were performed at room temperature (22~24 °C). To test the inhibitory effect of venom fractions on Cav3.2 currents, calcium currents were recorded from Cav3.2 expressing HEK 293 cells. Calcium currents were elicited from -90 mV holding potential to -30 mV testing potential. Each tested venom fraction was dissolved in 100 pl bath solution. The venom fraction was puffed via a Picospritzer III (Parker Hannifin, Hollis, NH, USA) onto Cav3.2-expressing HEK 293 cells to measure the calcium currents before and after toxin treatment.

[0137] Insect toxicity assays

[0138] Direct insecticidal activity of Na+, Ca2+and Zn2+(each tested as the respective chloride salt) was determined by injection into the ventro-lateral thoracic region of adult sheep blowflies (Lucilia cuprina) with average masses between 21.1 and 32.5 mg according to previously published methods (Guo et al., 2018). Lethal effects were determined 24 hours after injection. For all L. cuprina experiments, n=10 flies were tested for each dose and 3 repeated experiments were performed for each dose and the resulting LD50 values were calculated using Prism 9.2.0 for Mac (GraphPad Software, San Diego, CA, USA).

[0139] The same blowfly injection toxicity assay in adult L. cuprina (Guo et al., 2018) was used to study possible interactions of zinc with a selection of well-known and potent insecticidal spider venom peptides, i.e. co-HxTX-Hv1a (co-Hv1a), / c-HxTX-Hv1c (K- Hv1c), U1-AGTX-Tala (Tala), p-DGTX-Dc1a (Dcla), and ω / k -HXTX-Hv1a (u) / «-Hv1a). All spider venom peptides were either chemically synthesized or recombinantly produced according to previously published methods (de Araujo et al., 2013, Klint et al., 2013). Firstly, the lethal potency of ZnCI2was determined at 10 different doses (0, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, and 50 pg of ZnCI2 / fly). Each spider venom peptide was then tested by using a standardized treatment schedule, comprised of testing 6 different doses of peptide (0, 0.008, 0.004, 0.02, 0.1, and 0.5 pg per fly) either alone or in combination with 5 pg of ZnCI2per fly (average fly masses between 21.7 and 33.3 mg for all zinc and zinc-peptide interaction experiments). The dose of 5 ug / fly of ZnCI2was chosen for co-administration with the spider venom peptides, because the results from the zinc dose-response curve indicated that this zinc dose only caused low levels of paralysis (40%) and lethality (13.3%) in the blowflies.

[0140] The interaction of zinc with the various toxins was examined with isobologram analysis, based on the median effect principle described by Chou and Talay (Chou and Talalay, 1984), using CalcuSyn for Windows (Biosoft, Cambridge, UK). Based on the resulting Combination Index (Cl) values, the interactions at each separate concentration of toxin were classified as <0.1= very strong synergism (+++++), 0.1- 0.3= strong synergism (++++), 0.3-0.7= synergism (+++), 0.7-0.85= moderate synergism (++), 0.85-0.9= slight synergism (+), 0.9-1.1= nearly additive (±), 1.1-1.2= slight antagonism (-), 1.2-1.45= moderate antagonism (--), 1.45-3.3= antagonism ( — ), 3.3-10= strong antagonism (- — ), >10= very strong antagonism ( - ). The interactions of zinc with the toxins were also visualised by comparing the observed dose responses for zinc and toxin combinations with the dose responses expected according to the Bliss Independence model (Bliss, 1939).

[0141] Zn supplement feeding trial

[0142] For the supplement feeding trial, 10 juvenile Acanthoscurria geniculata (body size 3 cm) were split into two equal-sized groups (treatment and control, each n=5) and kept in BraPlast enclosures (15x15x15cm, Bergheim, Germany) filled with coconut fibre as substrate and a cork bark as hide. Both groups were fed with house crickets (Acheta domestica, 1cm body-size) at 7-day intervals. The treatment group received crickets dusted in zinc-containing supplement powder, whereas the control group received non-supplemented crickets. Zinc supplementation was performed by placing crickets in a small plastic box filled with 800 mg Pro Mineral + D3 powder (Lucky Reptile, Waldkirch, Germany), the food supplement with highest zinc content (1 g / kg) available in Germany. Shaking for 30 seconds ensured that the entire cricket was covered in supplement powder. The average amount of supplement powder per cricket was determined by weighing 42 A. domestica before and after dusting with the supplement powder and the corresponding zinc content per cricket was calculated as 0.1% of supplement mass per cricket. Before the feeding trial commenced, the venoms of all spiders were collected to serve as a base value for their respective zinc contents. After commencing the feeding trial, venom was extracted monthly for three consecutive months. All venoms were collected individually for each spider and milking and then freeze-dried and stored at -20°C until being used for analysis.

[0143] Oral bioactivity of spider venom peptide Dcla in Lucilia cuprina blowflies Oral testing against sheep blowflies ( / .. cuprina) was performed according to the previously described method (Guo et al., 2018). In short, flies were deprived of food or water for 12 h and then placed inside of a 2 mL tube. The spider venom peptide Dcla was dissolved in 5% sucrose solution (either with or without adding zinc) and 3 pL of toxin solution was applied to the inside of a 2 mL tube containing a single blowfly. Thereafter, flies were kept in these tubes overnight at room temperature without additional food or water, before being transferred into 250 mL plastic boxes containing vermiculite as bedding material (to absorb any spilled water), a sugar cube, and a piece of wet cotton wool placed into the lid of a 50 mL Falcon tube. Two different doses of Dcla were tested (0.4 or 2 ug / fly) either with or without ZnCI2(2 ug / fly). Three groups of n=5 flies were used for each treatment and n=6 flies were used as control group, receiving only 2 ug / fly of ZnCI2but no Dcla. Another control group of n=6 flies received 5% sucrose solution. Mortality of flies was observed after 24 hours.

[0144] RESULTS AND DISCUSSION

[0145] Molecular Zinc is abundant in arachnid venoms

[0146] Given the current scarcity of information on the metal content of most animal venoms, we performed a systematic large-scale screen to quantify a range of eight metal isotopes (52Cr,55Mn,56Fe,59Co,60Ni,63Cu,66Zn, and111Cd) in venoms from 85 diverse venomous arthropod species, including, assassin bugs, centipedes, lepidopterans, scorpions and spiders. To our surprise, several arthropod venoms contained considerable amounts of zinc. On average, the content of66Zn in the tested venoms was higher than for any of the other metal isotopes we examined, i.e. 135- fold higher compared to52Cr, 22 x55Mn, 24 x56Fe, 10,799 x59Co, 10 x60N i, 18 x63Cu, and 2,376 x111Cd (Table 2). Arachnid venoms exhibited particularly high levels, up to 6.3% (based on the dried venom mass) for scorpions and 1.7% for spiders (Table 3). This is interesting and points to some kind of accumulation process by the arachnids, as based on the relative abundance of metals in the earth's crust, Fe and Mn are 714- and 14-times more abundant than Zn, respectively, and Cr, Ni, Cu, and Co are roughly similar in abundance to Zn (Pandey, 2012). On the other hand, our data supports previous findings of biomagnification of zinc in arthropods through the food chain (Kim and Kim, 2016).

[0147] Due to the abundance of zinc in the environment, we first wanted to exclude the possibility of accumulation artefacts from the observed high zinc contents in the venoms, e.g. from the plastic samples tubes in which the venoms were stored, the pipette tips used for pooling the venom samples or the water used for dissolving the venoms. With the largest of our venom pools being compiled of 21 individual venoms, we performed an experiment to recapitulate the pooling process used for the venoms. For the mock pooling, we either pooled from a single or from 30 tubes containing 50 pl milliQ. water each (using 3 different types of tubes from 2 manufacturers). The average "enrichment factors" (= zinc content in 30 pooled tubes / zinc content in 1 pooled tube) ranged from 0.98 to 1.24 for all three types of tubes tested (Sarstedt tubes: 1.11; Eppendorf DNA LoBind: 0.98; Eppendorf Safe-Lock: 1.24, Table 4), arguing against any inherent environmental accumulation of zinc caused by the pooling procedure used for the venoms. This is further supported by the lack of correlation between the content of zinc in each respective venom pool and the number of individual tubes that were combined when creating each venom pool (Spearman correlation, P = 0.4529, for details see Table 5). To further assess whether zinc in the venom is bound to larger proteins or available in solution, we selected one spider and scorpion venom each with > 1% zinc content and filtered it through a 3 kDa centrifugal filter. A larger percentage of zinc in both venoms was found in the flow-through (56% for Linothele megatheloides and 60% for Paravaejovis hoffmanni, Table 6), implying that most of the zinc in these arachnid venoms is not bound to larger proteins, but freely available for interaction with various ion channels and receptors. Our results therefore imply that several arachnid venoms contain molecular zinc in quantities exceeding 1% of the dried venom mass, which cannot be attributed to sampling or environmental contamination artefacts.

[0148] Zinc in arachnid venom synergizes with venom toxins to facilitate insect prey capture Arachnids venoms serve two ecological purposes: hunting and anti-predator defense. We therefore investigated the potential function of such elevated zinc contents in both scenarios via different in vitro and in vivo functional studies. The first ecological function considered was hunting, hence we focussed on effects on potential insect prey. Zinc can damage the insect midgut epithelium (Bednarska et al., 2016) and has been reported to be toxic to some aquatic insects (Clements et al., 2013, Liess et al., 2017). Furthermore, zinc modulating presynaptic ion channels (Peralta and Huidobro-Toro, 2016) could result in paralysis of the prey, thereby aiding in the prey capture. To assess the possibility of zinc in the venom being used to aid in prey capture, we first compared the acute toxicity of ZnCI2with CaCh and NaCI by intrathoracic injection into sheep blowflies, which we regularly use as a sensitive model for detecting insecticidal effects of venom toxins (Guo et al., 2018, Nixon et al., 2021). We found ZnCI2to be 32-times more potent than CaCh and 163-times more potent than NaCI in causing toxic effects in blowflies (Figure 1A). Despite being significantly more toxic than Na or Ca, the LD50 of zinc was still only in the micromolar range (2.21 pmol ZnCI2 / g — 144.5 pg Zn / g), compared to low pmol / g LD50 values for some potent insecticidal venom peptides (Guo et al., 2018). We therefore then examined whether sufficient zinc doses for insecticidal activity could be delivered by the zinc quantities present in some of the venoms. The diplurid spider L. megatheloides with a venom zinc content of 1.6% yielded an average of 1 mg dried venom per milking (average yields for A. theraphosoides: 15.8 mg; G. swammerdami: 4.7 mg). Based on their average venom yields, the content of zinc in the venom and an average blowfly weight of 25 mg / fly, each L. megatheloides could therefore deliver 16 pg of zinc, which is equivalent to about 4-times the LD50 observed in blowflies (74-times for A. theraphosoides or 82-times for G. swammerdami). In conclusion, some arachnids contain sufficient zinc quantities in their venoms to directly cause acute toxic effects in their insect prey.

[0149] Given that zinc modulates the activity of a wide variety of ion channels and arachnid venom peptides primarily target ion channels in their insect prey (King and Hardy, 2013, Saez and Herzig, 2019), we next examined whether zinc might influence the potency of insecticidal peptide toxins. We selected a panel of known potent insecticidal spider venom peptides with a diverse range of molecular targets in insects: co-HxTX-Hv1a (co-Hv1a) targeting medium to low voltage activated calcium (M-LVA Cav) channels (Wang et al., 1999), k -HxTX-Hv1c (K-HV1C) targeting calcium activated potassium (Kca) channels and nicotinic acetylcholine receptors (nAChR) (Chambers et al., 2019, Gunning et al., 2008), Ui-AGTX-Tala (Tala) with unknown molecular target (Undheim et al., 2015), p-DGTX-Dcla (Dcla) targeting voltage gated sodium (Nav) channels, and ω / k -HXTX-Hv1a (ω / k -Hv1a) targeting nAChR (Chambers et al., 2019). Intriguingly, zinc co-application provided synergistic interactions with all tested insecticidal spider venom peptides (Figure 1B-F). While synergistic mechanisms are often proposed or suggested for various venom compounds, there is surprisingly little experimental evidence confirming synergism. Well-known examples of synergistic activity comprise the toxin cabals reported from cone snail venoms (Jimenez et al., 2003, Safavi-Hemami et al., 2015) or the potentiating effect of phospholipase A2 on venom cytotoxins in spitting cobras (Kazandjian et al., 2021). One example of synergism in spider venoms has been reported between the peptidic p-Agatoxins and the a-Agatoxins (which are acylpolyamines) in the venom of the American funnel web spider Agelenopsis aperta, which aid in insect prey capture (Adams, 2004). Synergistic interactions between several peptide toxins, but also between potassium and some peptide toxins have further been reported from wandering spider Cupiennius salei venom for enhancing the overall insecticidal activity (Wullschleger et al., 2005), with a more detailed discussion of these interactions provided in a recent review (Luddecke et al., 2021).

[0150] Zinc in arachnid venoms is functionally restricted to hunting

[0151] Both spiders and scorpions employ venom defensively, however, the human body is estimated to contain about 3 g of zinc per kg of body weight and it is therefore unsurprising, that zinc is only toxic at very high doses (Plum et al., 2010). It therefore seems exceedingly unlikely that small arthropod venoms contain sufficient quantities of zinc to exert any toxic effects in larger organisms such as humans. On the other hand, arachnid predators are usually much smaller than humans and induction of toxic symptoms or high zinc concentrations across the entire body are not essential for a defensive compound to be effective. Pain-inducing toxins for example cause their effects on sensory pathways through high local tissue concentrations at the site of envenomation, which activate sensory neurons that mediate pain transmission (Bohlen and Julius, 2012, Herzig et al., 2020, Osteen et al., 2016, Robinson et al., 2022). Thus, the question remains if the quantities of zinc in these venoms might be sufficient to induce pain in small vertebrate predators. The results of a recent study would argue against such a defensive role, as intraplantar injection of 20 pg of zinc (which is similar to the zinc quantity contained in the average venom yield of L. megatheloides) in mice was found to even have analgesic activity (Luo et al., 2018). Analgesic effects of zinc are further supported by other studies (Nozaki et al., 2011, Tamba et al., 2013) and consistent with our observations of zinc-containing, or potentially containing, spider venom fractions inhibiting voltage-gated calcium channels of the subtype 3.2 (Cav3.2), which play a pivotal role in processing of pain signals (Cai et al., 2021, Sekiguchi and Kawabata, 2013). Our results show that the first three reversed-phase HPLC fractions of A. theraphosoides venom (containing, or potentially containing, between 0.35 to 0.72% of zinc) caused 66-87% inhibition of Cav3.2 currents, which was abolished by addition of the chelating agent EDTA (Figure 2). Given the apparent analgesic effects of zinc at the quantities present in these venoms, a defensive ecological role of zinc seems implausible.

[0152] Implementation of zinc into arachnid venoms is limited

[0153] It is intriguing though that the ability to utilize zinc in the venom for assisting with prey capture does not follow any clear phylogenetic pattern. For example, two buthid scorpion venoms contained between 0.5% and 0.7% of zinc, whereas four other buthid species exhibited much lower zinc contents (< 0.25%). Of 54 species of theraphosid spiders examined, only four exhibited venom zinc contents above 0.3%, belonging to three different subfamilies broadly spread across the phylogenetic tree of the family Theraphosidae (Foley et al., 2019). The most apparent example for this phylogenetic disparity is Gigantometrus swammerdami (Loria and Prendini, 2021), which exhibited the highest zinc content (6.3%) among all 85 examined venoms, while three species of Heterometrus and Pandinus imperator from the same scorpion family Scorpionidae only had zinc contents below 0.22%. One possibility could be that some of the highest zinc contents were caused by individual specimen with extreme zinc contents that contributed over proportionally to the zinc content of each pooled venom. This implies that a much broader range of spiders and scorpions could have the theoretical ability to incorporate zinc into their venoms, provided there is an abundant and easy to utilize zinc source available.

[0154] The primary route of uptake for metal elements in terrestrial arthropods is diet, and so we hypothesised that the observed zinc in venom is accumulated from prey and that a diet with increased zinc content will accordingly result in higher zinc uptake into venom. We therefore performed a 3-month feeding trial in which juvenile A. geniculata spiders (n=5 per group) were fed with crickets dusted with a zinc- containing, or potentially containing, supplement. On average each feeding with zinc- supplemented insect prey contained 18 pg (± 8 pg) of zinc and there were 4 feeding cycles in between each milking. Accordingly, a zinc dose corresponding to 4.6-fold the average zinc content present in L. megatheloides venom was fed to each spider between milkings. We therefore anticipated that the spiders fed with the zinc- supplemented crickets would continuously increase their venom zinc content over subsequent milkings beyond the content of the control group which was fed on nonsupplemented crickets. However, our data did not support this hypothesis, with no significant difference in zinc content between both groups for any of the monthly milkings (Table 7A,C and Figure 3). This could indicate that these spiders already accumulate sufficient zinc amounts through their normal non-enriched diet without the need for any additional zinc supplementation. Given that zinc can affect arthropod reproductivity (Shu et al., 2009, Xie et al., 2014) or even be toxic at higher doses (Fosmire, 1990, Hussain et al., 2022), there may be regulatory processes involved that prevent the accumulation of zinc beyond a certain maximum capacity for each arachnid. The fact that the average zinc content we observed for the juvenile A. geniculata in our feeding trial was 0.3% (ranging from 0.2-0.7% in the individual spiders, see Table 7B) compared to the 1.5% detected in the venom from adult specimen (Table 3) could further indicate that the zinc accumulating capacity in A. geniculata increases during the spiders' growth. Another possible explanation for our findings is that the diet is not the main source for zinc, which could also be accumulated from the environment through the water or the soil. More research is therefore required to determine the source of the zinc and the mechanisms for how it gets accumulated in the venom of arachnids.

[0155] Fig.4 shows the results of oral toxicity tests of spider venom peptide Dcla (SEQ. ID NO:258) with (+) and without (-) Zn (= 2 ug / fly of ZnCI2). Combining Zn with the spider venom peptide Dcla increases its mortality, resulting in a trend (P = 0.069) for the highest Dcla dose being more potent in the presence of Zn.

[0156] Conclusion

[0157] Overall, our results provide evidence that some arachnids are capable of accumulating elemental zinc in their venoms to enhance their insecticidal potential and thereby facilitating insect prey capture. We demonstrated synergism between zinc and arachnid venom peptides, suggesting that arachnids capable of sequestering zinc into their venoms (even if only used infrequently when an opportunity arises) are endowed with an ecological and evolutionary advantage by reducing their metabolic cost of peptide toxin production. Saving on the venom expenditure is well in line with the venom optimisation hypothesis (Morgenstern and King, 2013, Wigger et al., 2002) initially postulated for spider venoms which are metabolically expensive and therefore frugally used. It is also in line with other mechanisms evolved in various venomous lineages for separately storing and deploying different venoms optimised for designated tasks such as predation or defence (Walker et al., 2018, Inceoglu et al., 2003, Dutertre et al., 2014). The postulated zinc plus peptide co-application strategy might therefore be considered for agricultural applications. For example, the required amounts of arachnid venom derived bioinsecticides (Saez and Herzig, 2019) could be reduced when co-applied with zinc, provided the employed amounts of zinc are limited to minimize environmental accumulation of zinc and thereby any negative impacts on non-target species (Baran et al., 2018, Bednarska et al., 2016, Clements et al., 2013). In addition, foliar application with zinc as a plant micronutrient has been discussed for improving plant growth and health (Hamzah Saleem et al., 2022). Thus, zinc co-application could increase plant productivity and protect against pest insects at the same time. Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

[0158] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

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[0217] TABLES

[0218] Table 1. Venom protein amino acid sequences designated SEQ ID NOS:l-397 in sequential order from top to bottom of table.

[0219]

[0220]

[0221]

[0222] Table 2 Table 3; Zinc (Zn) content in invertebrate venoms. The zinc contents of a diverse panel of venoms from 85 arthropod species were analysed using an Inductively-Coupled Plasma Mass Spectrometer (ICP-MS). N indicates the number of pooled individual venom samples. The top 10 venoms with the highest zinc content as percent of dry venom mass (after subtraction of the average zinc content in the blank) are presented below. For the complete list of all venoms and the other metal contents see Table 2.

[0223] Table 4

[0224] *Zn percentage for mock pooling experiment was calculated by assuming a dried weight of 20 pg to enable comparison with the ICP-MS results from the venoms.

[0225] Table 5 Table 6

[0226] Table 7

[0227] Table 7A A. geniculata - variability within milkings

[0228] Treatment Milking# av. Zn (%) STDEV Control 1 0.4 0.2 Control 2 0.5 0.3 Control 3 0.4 0.2 Control 4 0.3 0.2 av (control) 0.4 0.2

[0229] Treatment Milking# av. Zn (%) STDEV

[0230] Supplement 1 0.2 0.1

[0231] Supplement 2 0.3 0.1

[0232] Supplement 3 0.3 0.1

[0233] Supplement 4 0.3 0.1 av (supplement) 0.3 0.1 av (all) 0.3 0.2

[0234] Table 7B A. geniculata - variability within spiders over milkings

[0235] Treatment Spider ID av. Zn (%) STDEV

[0236] Control 2 0.2 0.1

[0237] Control 4 0.2 0.1

[0238] Control 6 0.7 0.3

[0239] Control 8 0.4 0.2

[0240] Control 10 0.4 0.1 av (control) 0.4 0.1

[0241] Treatment Spider ID av. Zn (%) STDEV

[0242] Supplement 1 0.3 0.2

[0243] Supplement 3 0.3 0.1

[0244] Supplement 5 0.3 0.1

[0245] Supplement 7 0.3 0.1

[0246] Supplement 9 0.2 0.1 av (supplement) 0.3 0.1 av (all) 0.3 0.1 Table 7C Statistical comparison within milking periods

[0247] Table Analyzed milkingl Table Analyzed milking2

[0248] Column B vs. Column A supplement vs. control Column B vs. Column A supplement vs. control

[0249] Mann Whitney test Mann Whitney test

[0250] P value 0.2222 Rvalue 0.4206

[0251] Exact or approximate P value? Exact Exact or approximate P value? Exact

[0252] P value summary ns P value summary ns

[0253] Significantly different (P < 0.05)? No Significantly different (P < 0.05)? No One- or two-tailed P value? Two-tailed One- or two-tailed Pvalue? Two-tailed

[0254] Sum of ranks in column A,B 34 , 21 Sum of ranks in column A,B 32 , 23 Mann-Whitney U 6 Mann-Whitney U 8

[0255] Difference between medians Difference between medians Median of column A 0.2504, n=5 Median of column A 0.3521, n=5 Median of column B 0.1749, n=5 Median of column B 0.2721, n=5 Difference: Actual -0.0755 Difference: Actual -0.08

[0256] Difference: Hodges-Lehmann -0.0818 Difference: Hodges-Lehmann -0.1069 96.83% Cl of difference -0.5096 to 0.1812 96.83% Cl of difference -0.6054 to 0.1744 Exact or approximate Cl? Exact Exact or approximate Cl? Exact

[0257] Table Analyzed milklng3 Table Analyzed mllklng4

[0258] Column B vs. Column A supplement vs. control Column B vs. Column A supplement vs. control

[0259] Mann Whitney test Mann Whitney test

[0260] Rvalue 0.6905 Pvalue 0.8413

[0261] Exact or approximate P value? Exact Exact or approximate Pvalue? Exact

[0262] Rvalue summary ns Pvalue summary ns

[0263] Significantly different (P < 0.05)? No Significantly different (P < 0.05)? No One- or two-tailed Rvalue? Two-tailed One- or two-tailed Pvalue? Two-tailed Sum of ranks in column A,B 30 , 25 Sum of ranks in column A,B 26 , 29 Mann-Whitney U 10 Mann-Whitney U 11

[0264] Difference between medians Difference between medians Median of column A 0.3644, n=5 Median of column A 0.2764, n=5

[0265] Median of column B 0.3396, n=5 Median of column B 0.2366, n=5

[0266] Difference: Actual -0.0248 Difference: Actual -0.0398

[0267] Difference: Hodges-Lehmann -0.0675 Difference: Hodges-Lehmann 0.0534 96.83% Cl of difference -0.4293 to 0.1550 96.83% Cl of difference -0.2980 to 0.2642 Exact or approximate Cl? Exact Exact or approximate Cl? Exact

[0268] Table 8

[0269] No. Mass Supplement (mg) Mass zinc (mg)

[0270] 1 18.3 0.0183

[0271] 2 18.8 0.0188

[0272] 3 15.6 0.0156

[0273] 4 34.9 0.0349

[0274] 5 39.0 0.039

[0275] 6 20.5 0.0205

[0276] 7 18.5 0.0185

[0277] 8 12.5 0.0125

[0278] 9 20.5 0.0205

[0279] 10 25.9 0.0259

[0280] 11 21.8 0.0218

[0281] 12 22.5 0.0225

[0282] 13 18.8 0.0188

[0283] 14 17.4 0.0174

[0284] 15 17.2 0.0172

[0285] 16 45.4 0.0454

[0286] 17 21.2 0.0212

[0287] 18 40.1 0.0401

[0288] 19 17.8 0.0178

[0289] 20 19.5 0.0195

[0290] 21 14.2 0.0142

[0291] 22 12.4 0.0124

[0292] 23 19.2 0.0192

[0293] 24 12.2 0.0122

[0294] 25 12.4 0.0124

[0295] 26 32.1 0.0321

[0296] 27 11.3 0.0113

[0297] 28 20.1 0.0201

[0298] 29 7.0 0.007

[0299] 30 17.6 0.0176

[0300] 31 11.8 0.0118

[0301] 32 12.8 0.0128

[0302] 33 9.0 0.009

[0303] 34 10.0 0.01

[0304] 35 9.2 0.0092

[0305] 36 16.1 0.0161

[0306] 37 13.6 0.0136

[0307] 38 9.2 0.0092

[0308] 39 12.3 0.0123

[0309] 40 19.2 0.0192

[0310] 41 13.5 0.0135

[0311] 42 11.1 0.0111

[0312] Average of fed supplement = 18.3929 mg. SD = 8.5797.

[0313] Average of fed zinc = 0.0184 mg. SD = 0.0086

Claims

CLAIMS1. A composition comprising, consisting essentially of, or consisting of one or more isolated arthropod venom proteins and zinc.

2. A method of producing a composition by combining one or more isolated arthropod venom proteins and zinc.

3. A method of at least partly increasing or potentiating the pesticidal activity of one or more isolated arthropod venom proteins including the step of combining the one or more isolated arthropod venom proteins with zinc to thereby at least partly increase or potentiate the pesticidal activity of the one or more isolated arthropod venom proteins.

4. A composition produced by the method of Claim 2 or comprising one or more isolated arthropod venom proteins produced according to the method of Claim 3.

5. The composition of Claim 1 or Claim 4 or the method of Claim 2 or Claim 3, wherein the one or more isolated arthropod venom proteins elicit more pesticidal activity than the one or more arthropod venom proteins in the absence of zinc.

6. The composition according to Claims 1, 4 or 5 or the method of any one of Claims 2-5 wherein zinc is a zinc salt.

7. The composition or method of Claim 6, wherein the zinc salt is zinc chloride (ZnCI2).

8. The composition of any one of Claims 1 or 4-7, or the method of any one of Claims 2-7, wherein the amount or concentration of zinc is that which produces a desired pesticidal effect wherein the amount of venom protein required to produce said desired pesticidal effect is reduced by a factor of at least about 2, 5, 10, 15, 20, 50, 80, 100, 150, 200 or more compared to that in the absence of zinc.

9. The composition of any one or Claims 1 or 4-8 or the method of any one of Claims 2-8, wherein the concentration or amount of zinc is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70 80, 90, 100, 120, 150, 200or more times the concentration or amount of zinc normally present in arthropod venom.

10. The composition of any one of Claims 1 or 4-9 or the method of any one of Claims 2-9, wherein the mass ratio of zinc:venom protein is from about 0.05:1 to about 100:1.

11. The composition of any one of Claims 1 or 4-10 or the method of any one of Claims 2-10, wherein the isolated arthropod venom protein is obtained, or obtainable, from an arachnid or scorpion.

12. The composition of any one of Claims 1 or 4-11 or the method of any one of Claims 2-11, wherein the isolated arthropod venom protein comprises an amino acid sequence set forth in any one of SEQ. ID NOS:1-397, or a variant, fragment or derivative thereof.

13. A method of at least partly preventing or inhibiting pest activity including the step of administering one or more isolated arthropod venom proteins and zinc to a pest or to an environment containing, or potentially containing the pest, to thereby at least partly prevent or inhibit pest activity.

14. A method of at least partly preventing or inhibiting pest infestation of an organism, said method including the step of administering one or more isolated arthropod venom proteins and zinc to the organism, to thereby at least partly prevent or inhibit pest infestation of the organism.

15. A method of increasing or potentiating at least partial inhibition or blocking of a pest ion channel by one or more arthropod venom proteins, said method including the step of administering zinc and the one or more arthropod venom proteins to the pest, or to an environment containing the pest, to thereby increase or potentiate at least partial inhibition or blocking of the pest ion channel.

16. The method of Claim 15, wherein the ion channel is a calcium channel, a sodium channel, a potassium channel and / or a nicotinic acetylcholine receptor.

17. The method of any one of Claims 13-16, wherein the isolated arthropod venom protein comprises an amino acid sequence set forth in any one of SEQ. ID NOS:l-397, or a variant, fragment or derivative thereof.

18. The method of any one of Claims 13-17 wherein the one or more isolated arthropod venom proteins and zinc are administered sequentially.

19. The method of any one of Claims 13-17, wherein the one or more isolated arthropod venom proteins and zinc are administered in combination.

20. The method of any one of Claims 13-19, wherein the concentration or amount of administered zinc is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 7080, 90, 100, 120, 150, 200 or more times the concentration or amount of zinc normally present in arthropod venom.

21. The method of any one of Claims 13-20, wherein the mass ratio of zinavenom protein is from about 0.05:1 to about 100:1.

22. The method of any one of Claims 13-21, wherein the amount or concentration of zinc is that which produces a desired pesticidal effect wherein the amount of venom protein required to produce said desired pesticidal effect is reduced by a factor of at least about 2, 5, 10, 15, 20, 50, 80, 100, 150, 200 or more compared to that in the absence of zinc.

23. The method of any one of Claims 13, 14 or 17-22, wherein the organism is an animal.

24. The method of any one of Claims 13, 14, or 17-22, wherein the organism is a plant or fungus.

25. The method of Claim 24, wherein the plant is a crop plant.

26. The method of any one of Claims 13-25, wherein the pest is an arthropod.

27. The method of Claim 26, wherein the arthropod is an insect.

28. The method of any one of Claims 13-27, wherein the one or more isolated arthropod venom proteins and zinc are administered as the composition of any one of Claims 1 or 4-12.

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