MX2008003704A - Acaricidal compositions and methods of use thereof - Google Patents

Abstract

A method of controlling acarine pests comprises applying to the locus of the acarine pests, an isolated polypeptide toxin, wherein the polypeptide toxin has acaricidal activity. In one embodiment, the polypeptide toxin comprises three intrachain disulfide bonds and/or is a component of a venom of an Australian funnel web spider of the genus Atrax or Hadronyche.The polypeptide toxins may be applied to the acarine pests themselves, to the environment of the acarine pests, to the hosts of the acarine pests, or to an animal vector of the acarine pests, for example.

Description

ACARICIDAL COMPOSITIONS AND METHODS OF USING THE SAME BACKGROUND OF THE INVENTION Ticks and mites are both members of the Acari taxonomic order, within the Arachnida Class, and collectively referred to as acarines. They are not related to insects. Numerous species of acarines are key pests of wildlife, farm and companion animals, humans and crops. Ticks are essential ectoparasites that infest mammals, birds, reptiles and amphibians. It has been estimated that approximately 80% of the world's cattle are infected with ticks, causing economic losses of $ 7,500 dollars. Many species of ticks are considered a problem mainly because of their ability to transmit numerous pathogens of significant public health importance in humans and veterinary. Selected examples of many tick species of importance for pathogen transmission include, but are not limited to, the dog tick Rhipicephalus sanguineus, the solitary star tick Amblyomma americanum, the Amblyomma hebraeum hair tick, the Amblyomma tropical hair tick variegatum, the winter tick Dermacentor albipictus, the tropical horse tick Dermacentor ni tens, the tick Ref.: 191138 American dog Dermacentor variabilis, the wood tick of Rocky Mountains Dermacentor andersoni, the cattle tick Boophilus microplus and Boophilus annulatus, Ixodes ricinos, and the deer tick Ixodes scapularis. Bacterial pathogens (including ric ettsiales), protozoa and arbovirals transmitted by ticks are responsible for a wide variety of human and animal diseases, including, for example, Lyme disease, tularemia, livestock hemoglobinuria (hydropericardium disease), dermatophilosis , anaplasmosis, theileriosis, encephalitis, babesiosis, and several rickettsial diseases of the cerebrospinal meningitis group, including cerebrospinal meningitis of the Rocky Mountains. For some ticks, however, the main issue is not the transmission of pathogens, but rather the secretion of paralytic neurotoxins that can sometimes be fatal to animals and humans; ticks in this category include, but are not limited to, the Australian paralysis tick Ixodes holocyclus and the African parasite tick Ixodes rubicundis. Ticks also cause significant losses in the livestock industry due to bite injuries and the occurrence of secondary infections. There are approximately 7,000 species of plant-eating mites (phytophagous), many of which are pests of wood, fruits, vegetables, forest crops, ornamental plants, and stored grains. The majority of these mites correspond to the Eriofioidea superfamilies (gall mites, erinosa, outbreak and rust) and the agronomically important Tetranichoidea (red spiders and flat mites). In addition, some mites are endo- or ecto-parasitic pests of livestock and companion animals, causing diseases such as mange and scab, while dust mites produce allergens associated with asthma and other allergic conditions in humans. Mites often acquire rapid resistance to acaricidal agents due to their extremely fast life cycle (for example, 1-4 weeks) and the ability to deposit large numbers of eggs. Some mites are resistant to virtually all existing pesticide agents. The acarids and ticks are acarine and are not closely related to insects. As a result, most insecticides are not effective against acarines. The chemicals that are effective against acarines are called acaricides. There are at least two major problems with the few acaricides available that are effective against mites and ticks. First, many species of ticks and mites have developed resistance to various kinds of these chemicals. There is already widespread resistance to coumaphos and pyrethroids, and increasing reports of amitraz resistance. The macrocyclic lactone endectocides are effective against Boophilus but not against multiple host ticks. Second, many acaricides are under intense regulatory scrutiny by the United States Environmental Protection Agency and some have already been deregistered (eg, chlorpyrifos and diazinon). The loss of major classes of acaricides due to the development of resistance or deregistration, combined with more demanding registration requirements for new acaricides, will probably decrease the use of effective chemical acaricides in the near future. In this way, there is an urgent need to isolate new and safe acaricidal compounds. Several researchers have recognized spider poisons as a possible source of insect toxins. A class of peptide toxins known as ega-atracotoxins, described in U.S. Patent No. 5,763,568, were isolated from Australian funnel web spiders by selecting the venom for "anti-cotton caterpillar" activity. One of these compounds, designated or ega-ACTX-Hvla, has been shown to selectively inhibit the voltage-gated, calcium channel currents of the insect, as opposed to mammalian. A second unrelated family of blockers Calcium channel peptides, specific to insects, are described as being isolated from the same family of spiders in U.S. Patent No. 6,583,264. However, there is no suggestion in any of these references that these peptides are useful in the annihilation of pests other than insects.

Brief Description of the Invention The present inventors have discovered that polypeptide toxins such as the omega-ACTX-1 and omega-ACTX-2 families have acaricidal activity. The biological activity of a mature toxin representative of each family of toxins has been characterized. These two prototypic toxins cause irreversible toxicity when injected into the solitary star tick Amblyomma americanum. The omega-ACTX-1 toxins are also highly lethal when distributed orally to the tick. In one embodiment, a method for controlling acarine pests, comprising applying to the site of acarine pests, an isolated polypeptide toxin, wherein the polypeptide toxin has acaricidal activity. In another embodiment, a method "for inhibiting the infestation of acarine pests in a farm animal, a companion animal, or an animal vector comprises applying to site of acarine pests, an isolated polypeptide toxin, wherein the polypeptide toxin has acaricidal activity. Another aspect of the invention relates to an isolated polypeptide toxin comprising any of SEQ ID NOS: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54 Brief Description of the Figures Figure 1 shows an alignment of omega-ACTX-Hvla (SEQ ID NO: 1) with the complete prepropolypeptide sequences of 16 omega-ACTX-Hvla orthologs (SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49 or 52). The highly conserved amino acid residues are shaded gray. Vertical arrows indicate the signal peptide and propeptide cleavage sites. Figure 2 is a dose-response curve resulting from the injection of recombinant omega-ACTX-Hvla into the solitary star tick Amblyomma americanum. Figure 3 is a dose-response curve resulting from feeding recombinant omega-ACTX-Hvla to the solitary star tick Amblyomma americanum.

Detailed Description of the Invention Methods for controlling acarine pests, comprising applying to the site of the acarus pests an isolated polypeptide toxin, wherein the polypeptide toxin has acaricidal activity. In one embodiment, the polypeptide toxin is a component of a spider venom, such as the venom of the spiderweb Australian spider of the genera Atrax and Hadronyche. Specific polypeptide toxins include the omega-ACTX polypeptide toxins such as the families of omega-ACTX-1 and omega-ACTX-2 polypeptides, as well as the polynucleotides encoding these polypeptides. These polypeptides and the polynucleotides encoding them can be used as acaricides, either alone or in combination with other acaricidal polypeptides, or genes thereof, or other acaricidal agents. An acaricide or an acaricidal composition is one that is toxic to one or more acarine species (mites and ticks). Acaricidal activity refers to the ability of polypeptides to annihilate or paralyze acarines or to inhibit their growth or development. The LD50 is the dose of an omega-ACTX polypeptide that results in the death of 50% of the acarines tested. In one embodiment, the LD50 for a polypeptide toxin such as an omega-ACTX polypeptide dosed to an acarin is less than about 5000 pmol / g, less than about 2500 pmol / g, less than about 1000 pmol / g, less than about 750 pmol / g, less, about 500 pmol / g, or less than about 250 pmol / g. Toxins suitable for use in the described methods are collectively referred to as omega-ACTX polypeptides and include the omega-ACTX-Hvla polypeptides (SEQ ID NO: 1) and omega-ACTX-Hv2a (SEQ ID NO: 2) ), as well as their counterparts. SEQ ID NO: l (omega-ACTX-Hvla): SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD SEQ ID NO: 2 (omega-ACTX-Hv2a): LLACLFGNGRCSSNRDCCELTPVCKRGSCVSSGPGLVGGILGGIL As used herein, an omega-ACTX-Hvla polypeptide is a polypeptide having a molecular weight of about 4000 Da and a length of about 36 to about 37 amino acids, and that is capable of forming three disulfide bridges intra-chain. An omega-ACTX-Hv2a polypeptide is a polypeptide having a molecular weight of about 4500 Da and a length of about 41 to about 45 amino acids, and which is capable of forming three intrachain disulfide bridges. In one embodiment, an omega-ACTX-Hvla peptide has greater than or equal to 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 1 and has insecticidal activity and / or acaricide. In other embodiment, an omega-ACTX-Hv2a polypeptide has greater than or equal to 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 2 and has insecticidal activity and / or acaricide. The omega-ACTX polypeptide can be in the form of a mature polypeptide, a prepropolypeptide or a propolypeptide. Without being bound by theory, it is believed that the biologically active form of the omega-ACTX polypeptide is produced by post-translational proteolytic processing (eg, cleavage) of the prepropolypeptide precursor to produce the mature polypeptide. The cleavage can be endoproteolytic cleavage of the prepropolypeptide by a protease that recognizes a particular amino acid sequence motif in the prepropolypeptide. The "pre" portion of the prepropolypeptide refers to the signal peptide portion of the prepropolypeptide. Without being bound by theory, it is believed that the signal sequence is responsible for the direction of prepropolypeptide a, as well as its translocation through the endoplasmic reticulum membrane, into cells that produce omega-ACTX. In one embodiment, the signal peptide sequence comprises SEQ ID NO: 3 MNTATGX? IALLVLATVIGCIX2A, where Xi is V O F and X2 is S or E. Other signal sequences that function in a similar manner may also be employed. In another embodiment, the "pro" part of the prepropolypeptide refers to the sequence SEQ ID NO: 4 EDTRADLQGGEAAEKVFRR; SEQ ID NO: 5 DFX3GXFEX5X6X7X8EDAERIFRR, where X3 is Q or E, X is G or S, X5 is P or S, X is Y or S, X7 is E or is absent, and Xs is G, E or V; SEQ ID NO: 6: GESHVREDAMGRARR, or other sequences covalently linked in the 5 'direction of a mature omega-ACTX polypeptide. Without being bound by theory, possible rules for the pro sequence include facilitating the export of toxin from the endoplasmic reticulum, assisting in the enzyme-catalyzed oxidative fold of the mature toxin sequence, and signaling enzymes comprised in proteolytic processing. and post-translational modification. The RR motif in the pro sequence is believed to be the endoprotease cleavage site. A purified polypeptide comprising an omega-ACTX polypeptide in this manner may further comprise a signal peptide sequence (the "pre" sequence, a pro sequence, or a combination thereof). The signal sequence of approximately 22 residues (the "pre" sequence and the propeptide sequence of approximately 15 residues (the "pro" sequence) are well conserved through the omega-ACTX orthologs. mature toxin sequences that in the signal sequence and the excision motive.

Five omega-ACTX-Hvla orthologous preprotein (SEQ ID N0: 7, 10, 13, 16 and 19) complete proteins that were isolated by analysis of a cDNA library derived from the venom gland of a spider web Australanian spider funnel, Atrax robus tus: In one embodiment, the omega-ACTX-Hvla orthologue comprises the prepropeptide sequence of SEQ ID NO: 7: SEQ ID NO: 7: MNTATGVIALLVLATVIGClEAEDTRADLQGGEAAEKVFRRSPTCIPSGQPCPYNENCCS QSCTFKENENGNTVKRCD A polynucleotide that codes for SEQ ID NO: 7 is SEQ ID NO: 8: SEQ ID NO: 8 ATGAATACCG CTACAGGTGT CATCGCTCTT TTGGTTCTGG CGACAGTCAT CGGATGCATT GAAGCAGAAG ATACCAGAGC AGATCTTCAA GGAGGAGAAG CCGCCGAGAA AGTATTTCGC CGCTCCCCGA CTTGCATTCC ATCTGGTCAA CCATGTCCCT ACAACGAAAA TTGCTGCAGC CAATCGTGTA CATTTAAGGA AAATGAAAAC GGCAACACTG TTAAAAGATG CGAC The mature polypeptide toxin corresponding to SEQ ID NO: 7 is SEQ ID NO: 9: SEQ ID NO: 9: SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ. ID NO: 10: SEQ ID NO: 10: MNTATGVIALLVLVTVIGCIEAEDTRADLQGGEAAEKVFRRSPTCIPSGQPCPYNENCCS QSCTFKENENGNTVKRCD A polynucleotide coding for SEQ ID NO: 10 is SEQ ID NO: 11: SEQ ID NO: 11: ATGAATACCG CTACAGGTGT CATCGCTCTT TTGGTTCTGG TGACAGTCAT CGGATGCATT GAAGCAGAAG ATACCAGAGC AGATCTTCAA GGAGGAGAAG CCGCCGAGAA AGTATTTCGC CGCTCCCCGA CTTGCATTCC ATCTGGTCAA CCATGTCCCT ACAACGAAAA TTGCTGCAGC CAATCGTGTA CATTTAAGGA AAATGAAAAC GGCAACACTG TTAAAAGATG CGAC The mature polypeptide toxin corresponding to SEQ ID NO: 10 is SEQ ID NO: 12: SEQ ID NO: 12: SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 13: SEQ ID NO: 13: MNTATGVI LLVLATVIGCIEAEDTRADLQGGEAAEKVFRRSPTCIPSGQPCPYNENCCS QSCTFKENETGNTVKRCD A polynucleotide coding for SEQ ID NO: 13 is SEQ ID NO: 14: SEQ ID NO: 14: ATGAATACCG CTACAGGTGT CATCGCTCTT TTGGTTCTGG CGACAGTCAT CGGATGCATT GAAGCAGAAG ATACCAGAGC AGATCTTCAA GGAGGAGAAG CCGCCGAGAA AGTATTTCGC CGCTCCCCGA CTTGCATTCC ATCTGGTCAA CCATGTCCCT ACAACGAAAA TTGCTGCAGC CAATCGTGTA CATTTAAGGA AAATGAAACC GGCAACACTG TTAAAAGATG CGAC toxin mature polypeptide corresponding to SEQ ID NO: 13 is SEQ ID NO: 15: SEQ ID NO: 15: SPTCIPSGQPCPYNENCCSQSCTFKENETGNTVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 16: SEQ ID NO: 16 MNTATGVIALLVLATVIGCIEAEDTRADLQGGEAAEKVFRRSPTCIPSGQPCPYNENCCS QSCTFKENENANTVKRCD A polynucleotide coding for SEQ ID NO: 16 is SEQ ID NO: 17: SEQ ID NO: 17: ATGAATACCG CTACAGGTGT CATCGCTCTT TTGGTTCTGG CGACAGTCAT CGGATGCATT GAAGCAGAAG ATACCAGAGC AGATCTTCAA GGAGGAGAAG CCGCCGAGAA AGTATTTCGC CGCTCCCCGA CTTGCATTCC ATCTGGTCAA CCATGTCCCT ACAACGAAAA TTGCTGCAGC CAATCGTGTA CATTTAAGGA AAATGAAAA.C GCCAACACTG TTAAAAGATG CGAC toxin mature polypeptide corresponding to SEQ ID NO: 16 is SEQ ID NO: 18: SEQ ID NO: 18: SPTCIPSGQPCPYNENCCSQSCTFKENENANTVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 19: SEQ ID NO: 19: MNTATGVIALLVLATVIGCIEAEDTRADLQGGEAAEKVFRRSPTCIPSGQPCPYNENCCS KSCTYKENENGNTVQRCD A polynucleotide coding for SEQ ID NO: 19 is SEQ ID NO: 20: SEQ ID NO: 20: ATGAATACCG CTACAGGTGT CATCGCTCTT TTGGTTCTGG CGACAGTCAT CGGATGCATT GAAGCAGAAG ATACCAGAGC AGATCTTCAA GGAGGAGAAG CCGCCGAGAA AGTATTTCGC CGCTCCCCGA CTTGCATTCC ATCTGGTCAA CCATGTCCCT ACAACGAAAA TTGCTGCAGC AAATCGTGTA CATATAAGGA AAATGAAAAT GGCAACACTG TTCAAAGATG CGAC The mature polypeptide toxin corresponding to SEQ ID NO: 19 is SEQ ID NO: 21: SEQ ID NO: 21: SPTCIPSGQPCPYNENCCSKSCTYKENENGNTVQRCD Also included is the complete preprotein sequence of an omega-ACTX-Hvla ortholog (SEQ ID NO: 22) which was isolated by analysis of a cDNA library derived from the venom gland of an Australian spider funnel web spider Atrax robustus: SEQ ID NO: 22 MNTATGFIVLLVLATVLGCIEAGESHVREDAMGRARRGACTPTGQPCPYNESCCSGSCQE QLNENGHTVKRCV A polynucleotide encoding SEQ ID NO: 22 is SEQ ID NO: 23: SEQ ID NO: 23: ATGAATACCG CAACAGGTTT CATTGTCCTT TTGGTTTTGG CGACAGTTCT TGGATGCATT GAAGCAGGAG AATCTCATGT GAGAGAAGAC GCCATGGGAA GAGCTCGCCG GGGGGCTTGC ACTCCAACTG GTCAACCGTG CCCGTATAAC GAAAGTTGTT GCAGCGGTTC CTGCCAAGAA CAGCTAAATG AAAACGGACA CACTGTTAAA AGATGCGTT toxin polypeptide mature which corresponds to SEQ ID NO: 22 is SEQ ID NO: 24: SEQ ID NO: 24: GACTPTGQPCPYNESCCSGSCQEQLNENGHTVKRCV Also included are the complete sequences of ten omega-ACTX-Hvla orthologous preproteins (SEQ ID NOs: 25, 28, 31, 34, 37, 40, 43, 45, 49 and 52) that were isolated by analysis of a cDNA library derived from the venom gland of an Australian spider of Hadronyche funnel spider web infensa: SEQ ID NO : 25 MNTATGFIVLLVLATVIGCISADFQGGFEPYEGEDAERIFRRSPTCIPTGQPCPYNENCC SQSCTYKANENGNQVKGCD A polynucleotide that codes for SEQ ID NO: 25 is SEQ ID NO: 26: SEQ ID NO: 26: ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCAGATT TTCAAGGAGG TTTCGAACCT TATGAAGGAG AAGACGCCGA AAGAATATTT CGCCGCTCCC CAACTTGCAT TCCAACTGGT CAACCGTGTC CCTACAACGA AAATTGCTGC AGCCAATCCT GTACATATAA GGCAAATGAA AACGGCAACC AAGTTAAAGG ATGCGAC The mature polypeptide toxin corresponding to SEQ ID NO: 25 is SEQ ID NO: 27: SEQ ID NO: 27: 10 SPTCIPTGQPCPYNENCCSQSCTYKANENGNQVKGCD In one embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 28. SEQ ID NO: 28 MNTATGFIVLLVLATVIGCISADFQGGFEPYEEEDAERIFRRSPTCIPTGQPCPYNENCC NQSCTYKANENGNQVKRCD -j_5 A polynucleotide coding for SEQ ID NO: 28 is SEQ ID NO: 29: SEQ ID NO: 29: ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCAGATT TTCAAGGAGG TTTCGAACCT TATGAAGAAG 0 AAGACGCCGA AAGAATATTT CGCCGCTCCC CAACTTGCAT TCCAACTGGT CAACCGTGTC CCTACAACGA AAATTGCTGC AACCAATCCT GTACATATAA GGCAAATGAA AACGGCAACC AAGTTAAAAG ATGCGAC The mature polypeptide toxin corresponding to SEQ ID NO: 28 is SEQ ID NO: 30: 5 SEQ ID NO: 30: SPTCIPTGQPCPYNENCCNQSCTYKANENGNQVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 31: SEQ ID NO: 31: MNTATGFIVLLVLATVIGCISADFQGGFEPYEEEDAERIFRRSPTCIPTGQPCPYNENCC SQSCTYKANENGNQVKRCD A polynucleotide coding for SEQ ID NO: 31 is SEQ ID NO: 32: SEQ ID NO: 32: ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCAGATT TTCAAGGAGG TTTCGAACCT TATGAAGAAG AAGACGCCGA AAGAATATTT CGCCGCTCCC CAACTTGCAT TCCAACTGGT CAACCGTGTC CCTACAACGA AAATTGCTGC AGCCAATCCT GTACATATAA GGCAAATGAA AACGGCAACC AAGTTAAAAG ATGCGAC The mature polypeptide toxin corresponding to SEQ ID NO: 31 is SEQ ID NO: 33: SEQ ID NO: 33: SPTCIPTGQPCPYNENCCSQSCTYKANENGNQVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 34 SEQ ID NO: 34 MNTATGFIVLLVLATVIGCISVDFQGGFESYEEEDAERIFRRSPTCIPTGQPCPYNENCC SQSCTYKANENGNQVKRCD A polynucleotide coding for SEQ ID NO: 34 is SEQ ID NO: 35: SEQ ID NO: 35: ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGTATT TCTGTAGATT TTCAAGGAGG TTTCGAATCT TATGAAGAAG AAGACGCCGA AAGAATATTT CGCCGCTCCC CAACTTGCAT TCCAACTGGT CAACCGTGTC CCTACAACGA AAATTGCTGC AGCCAATCCT GTÁCATATAA GGCAAATGAA AACGGCAACC AAGTTAAAAG ATGCGAC The mature polypeptide toxin corresponding to SEQ ID NO: 34 is SEQ ID NO: 36: SEQ ID NO: 36: SPTCIPTGQPCPYNENCCSQSCTYKANENGNQVKRCD In another embodiment, the ortholog of. omega-ACTX-Hvla comprises the prepropeptide sequence of SEQ ID NO: 37: SEQ ID NO: 37 MNTATGFIVLLVLATVIGCISADFQGGFESSVEDAERLFRRSSTCIRTDQPCPYNESCCS GSCTYKANENGNQVKRCD A polynucleotide coding for SEQ ID NO: 37 is SEQ ID NO: 38: SEQ ID NO: 38: ATGAATACCG CTACAGGTTT CATCGTTCTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCAGATT TTCAAGGAGG TTTCGAATCT TCTGTAGAAG ACGCCGAAAG ATTATTTCGC CGCTCCTCAA CTTGCATTCG AACTGATCAA CCGTGCCCCT ACAACGAAAG TTGCTGCAGC GGTTCCTGTA CATATAAGGC AAATGAAAAC GGAAACCAAG TTAAAAGATG CGAC The mature polypeptide toxin corresponding to SEQ ID NO: 37 is SEQ ID NO: 39: SEQ ID NO: 39: SSTCIRTDQPCPYNESCCSGSCTYKANENGNQVKRCD In another embodiment, the ortholog of omega-ACTX-HVLA comprising the sequence of prepro of SEQ ID NO: 40: SEQ ID NO: 40: MNTATGFIVLLVLATVIGCISADFQGGFEPYEEEDAERIFRRSTCTPTDQPCPYHESCCS GSCTYKANENGNQVKRCD A polynucleotide encoding SEQ ID NO: 40 is SEQ ID NO: 41: SEQ ID NO: 41: ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCAGATT TTCAAGGAGG TTTCGAACCT TATGAAGAAG AAGACGCCGA AAGAATATTT CGCCGCTCAA CTTGCACTCC AACTGATCAA CCGTGCCCCT ACCACGAAAG TTGCTGCAGC GGTTCCTGTA CATATAAGGC AAATGAAAAC GGCAACCAAG TTAAAAGATG CGAC The mature polypeptide toxin corresponding to SEQ ID NO: 40 is SEQ ID NO: 42: SEQ ID NO: 42: STCTPTDQPCPYHESCCSGSCTYKANENGNQVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 43: SEQ ID NO: 43: MNTATGFIVLLVLATVIGCISADFEGSFEPYEEEDAERIFRRSTCTPTDQPCPYDESCCS GSCTYKANENGNQVKRCD A polynucleotide that codes for SEQ ID NO: 43 SEQ ID NO: 44: SEQ ID NO: 44: ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCTGATT TTGAAGGAAG TTTCGAACCT TATGAAGAAG AAGACGCCGA AAGAATATTT CGCCGCTCAA CTTGCACTCC AACTGATCAA CCGTGCCCCT ACGACGAAAG TTGCTGCAGC GGTTCCTGTA CATATAAGGC AAATGAAAAC GGCAACCAAG TTAAAAGATG CGAC The mature polypeptide toxin corresponding to SEQ ID NO: 43 is SEQ ID NO: 45: SEQ ID NO: 45: STCTPTDQPCPYDESCCSGSCTYKANENGNQVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 46: SEQ ID NO: 46: MNTATGFIVLLVLATVIGCISADFQGSFEPYEEEDAERIFRRSTCTPTDQPCPYDESCCS GSCTYKANENGNQVKRCD A polynucleotide coding for SEQ ID NO: 46 is SEQ ID NO: 47: SEQ ID NO: 47: ATGAATACCG CTACAGGTTT CATCGTTCTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCAGATT TTCAAGGAAG TTTCGAACCT TATGAAGAAG AAGACGCCGA AAGAATATTT CGCCGCTCAA CTTGCACTCC AACTGATCM CCGTGCCCCT ACGACGAAAG TTGCTGCAGC GGTTCCTGTA CATATAAGGC AAATGAAAAC GGCAACCAAG TTAAAAGATG TGAC toxin mature polypeptide corresponding to SEQ ID NO: 46 is SEQ ID NO: 48: SEQ ID NO: 48: STCTPTDQPCPYDESCCSGSCTYKANENGNQVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 49: SEQ ID NO: 49: MNTATGFIVLLVLATVIGCISADFQGSFEPYEEEDAERIFRRSTCTPTDQPCPYHESCCS GSCTYKANENGNQVKRCD A polynucleotide encoding SEQ ID NO: 49 is SEQ ID NO: 50: SEQ ID NO: 50: ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGCATT TCTGCAGATT TTCAAGGAAG TTTCGAACCT TATGAAGAAG AAGACGCCGA AAGAATATTT CGCCGCTCAA CTTGCACTCC AACTGATCAA CCGTGCCCCT ACCACGAAAG TTGCTGCAGC GGTTCCTGTA CATATAAGGC AAATGAAAAC GGCAACCAAG TTAAAAGATG CGAC toxin of mature polypeptide corresponding to SEQ ID NO: 49 is SEQ ID NO: 51: SEQ ID NO: 51: STCTPTDQPCPYHESCCSGSCTYKANENGNQVKRCD In another embodiment, the omega-ACTX-Hvla ortholog comprises the prepropeptide sequence of SEQ ID NO: 52: SEQ ID NO: 52: MNTATGFIVLLVLATVIGCISADFQGGFEPYEEEDAERIFRRSTCTPTDQPCPYDESCCS GSCTYKANENGNQVKRCD A polynucleotide encoding SEQ ID NO: 52 is SEQ ID NO: 53: SEQ ID NO: 53: 5 ATGAATACCG CTACAGGTTT CATCGTACTT TTGGTTTTGG CGACAGTGAT CGGATGTATT TCTGCAGATT TTCAAGGAGG TTTTGAACCT TATGAAGAAG AAGACGCCGA AAGAATATTT CGCCGCTCAA CTTGCACTCC AACTGATCAA CCGTGCCCCT ACGACGAAAG TTGCTGCAGC GGTTCCTGTA CATATAAGGC AAATGAAAAC GGCAACCAAG TTAAAAGATG CGAC j_o The mature polypeptide toxin corresponding to SEQ ID NO: 52 is SEQ ID NO: 54: SEQ ID NO: 54: STCTPTDQPCPYDESCCSGSCTYKANENGNQVKRCD The invention includes isolated and purified omega-ACTX polypeptides. An "isolated" polypeptide or "Purified", or fragment thereof, is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the polypeptide is derived, or substantially free of chemical precursors or other chemicals when synthesized in a 0 chemistry The text "substantially free of cellular material" includes polypeptide preparations in which the polypeptide is separated from the cell components of the cells from which it is isolated or produced recombinantly. In this way, the polypeptide that is substantially free of cellular material includes polypeptide preparations having less than about 30%, about 20%, about 10%, or about 5% (by dry weight) of heterologous polypeptide (also referred to herein as a "contaminating polypeptide"). In one embodiment, the preparation is at least about 75% pure weight, more specifically at least about 90% pure weight, and more specifically at least about 95% pure weight. A substantially pure omega-ACTX polypeptide can be obtained, for example, by extraction from a natural source (e.g., an insect cell); by expression of a recombinant nucleic acid encoding an omega-ACTX polypeptide; or by chemically synthesizing the polypeptide > Purity can be measured by an appropriate method, for example, by column chromatography, polyacrylamide gel electrophoresis, or by high pressure liquid chromatography (HPLC) analysis. The invention also includes homologues of omega-ACTX polypeptides. "Homolog" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence that possesses a high degree of sequence relationship to a present sequence. This relationship can be quantified by determining the degree of identity and / or similarity between the sequences that are compare Within this generic term are the terms "ortholog", which means a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide in another species, and "paralogo" which means a functionally similar sequence when considered within the same species . Paralogs present in the same species or orthologs of the omega-ACTX genes in other species can be easily identified without undue experimentation, by molecular biology techniques well known in the art. As used herein, "percent homology" of two amino acid sequences, or two nucleic acids, is determined using the algorithm of Karlin and Altschul (1990) Proc. Nati Acad. Sci., USA 87: 2264-2268. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Searches of BLAST proteins with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to a reference polypeptide (for example, SEQ ID NO: 1). Search for BLAST nucleotides with the NBLAST program, score = 100, word length 12, to obtain nucleotide sequences homologous to a nucleic acid molecule. To obtain separation alignments for comparison purposes, BLAST is used with separation as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When BLAST and BLAST programs are used with 'Separation, the parameters are typically used per detect. (See http://www.ncbi.nlm.nih.gov). The related polypeptides are aligned with omega-ACTX by assigning degrees of homology to various deletions, substitutions and other modifications. Homology can be determined along the entire polypeptide or polynucleotide, or along subsets of contiguous residues. The percent identity is the percentage of amino acids or nucleotides that are identical when the two sequences are compared. The percent of similarity is the percentage of amino acids or nucleotides that are chemically similar when comparing the two sequences. Mature, homologous or omega-ACTX polypeptides are preferably greater than or equal to about 70%, specifically greater than or equal to about 75%, specifically greater than or equal to about 80%, specifically greater than or equal to about 85 %, more specifically greater than or equal to about 90%, and more specifically greater than or equal to about 95% identical. SEQ ID NO: 1 or SEQ ID NO: 2 can be used as a reference polypeptide. Where it is said that a particular polypeptide has a specific percentage of identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide. Thus, a polypeptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It can also be a polypeptide of 100 amino acids long which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. By "modification" of the primary amino acid sequence is meant that it includes "deletions" (ie, polypeptides in which one or more amino acid residues are absent), "additions" (i.e., a polypeptide having one or more additional amino acid residues compared to the specified polypeptide), "substitutions" (i.e., a polypeptide resulting from the replacement of one or more amino acid residues), and "fragments" (i.e., a polypeptide consisting of a primary sequence of amino acid that is identical to a portion of the primary sequence of the specified polypeptide). By "modification" it is also proposed to include polypeptides that are altered as a result of post-transductional events that change, for example, from the glycosylation pattern, from amidation (eg, C-terminal amidation), lipidation of lipidation, or the primary, secondary or tertiary structure of the polypeptide. N-terminal and / or C-terminal modifications are possible. The reference herein to either the nucleotide or amino acid sequence of omega-ACTX also includes reference to naturally occurring ants of these sequences. ants that do not occur naturally differ from SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49 or 52 for the prepropolypeptide , and SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54 for the mature polypeptide, and retain biological function, are also included at the moment. ants may comprise those polypeptides that have conservative amino acid changes, i.e., similarly charged or uncharged amino acid changes. Genetically encoded amino acids are generally divided into four families: (1) acids (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine). The Phenylalanine, tryptophan and tyrosine are sometimes classified together with aromatic amino acids. Since each member of a family has similar physical and chemical properties as the other members of the same family, it is reasonable to expect an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a greater effect on the binding properties of the resulting molecule. If an amino acid change results in a functional polypeptide it can be easily determined by assessing the acaricidal activity of the omega-ACTX polypeptide derivatives. The reference to omega-ACTX also refers to omega-ACTX polypeptide derivatives. As used herein, "polypeptide derivatives" includes those polypeptides that differ in length from an omega-ACTX that occurs naturally and that comprise approximately fifteen or more amino acids in the same primary order as found in omega-ACTX . The polypeptide derivatives may be longer than omega-ACTX, shorter than omega-ACTX (e.g., active fragments), while the polypeptide derivatives have acaricidal activity. Polypeptides that have substantially the same amino acid sequence as omega-ACTX but that possess Minor amino acid substitutions that do not substantially affect the acaricidal activity of omega-ACTX polypeptide derivatives are within the definition of omega-ACTX polypeptide derivatives. The omega-ACTX homologs can be identified in several ways. In one method, native mRNA sequences encoding the omega-ACTX orthologous precursors are identified by using standard molecular biology techniques to detect spider venom gland cDNA libraries for these orthologs. The amino acid sequence of the mature ortholog of omega-ACTX can be obtained from the translation of the identified cDNA sequences by noting that the endoproteolytic cleavage of the propeptide gives the mature toxin, which most likely occurs on the C-terminal side of a site of Arg-Arg processing immediately preceding the mature toxin (see second arrow in Figure 1). The mature native omega-ACTX ortholog may then be isolated by chromatographic fractionation of the venom, followed by identification and purification of a peptide toxin with a mass corresponding to that predicted from the cDNA sequence of the omega-ACTX ortholog. In another method, synthetic mature toxin can be produced by solid phase peptide synthesis of the omega-ACTX sequence followed by oxidation with cysteine to form the native disulfide isomer as described above for the production of synthetic J-atracotoxin-Hvlc (Wang et al. (2000) Nature Structural Biology 7, 505-513). In one embodiment, an omega-ACTX polypeptide is oxidized and folded into its native three-dimensional structure by incubating the lyophilized peptide, reduced in a glutathione reduction-oxidation buffer. A suitable glutathione reduction-oxidation buffer includes 3- [N-morpholino] propanesulfonic acid (MOPS) pH 7.3, 400 mM KCl, 2 mM EDTA, 4 mM reduced glutathione (GSH) and 2 mM oxidized glutathione (GSSG), although these are well-known numerous variants for those skilled in the art. This reaction mixture is incubated, for example overnight at 4 ° C, room temperature, or 37 ° C, for example, and then fractionated using reverse phase HPLC to separate the individual disulfide isomers. The fractions are collected and evaluated for acaricidal activity. In yet another method, the omega-ACTX ortholog is synthesized, either chemically or by recombinant DNA techniques, the cDNA encoding the omega-ACTX ortholog. In another method, the omega-ACTX ortholog is prepared using recombinant DNA techniques by constructing a synthetic gene encoding the omega-ACTX sequence by methods known in the art. The invention includes isolated omega-ACTX polynucleotides such as, for example, SEQ ID NOs: 8, 11, 14, 17, , 23, 26, 29, 32, 35, 38, 41, 44, 47, 50 or 53. The term "isolated polynucleotide" includes polynucleotides that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes polynucleotides that are separated from the chromosome with which the genomic DNA is naturally associated. An "isolated" polypeptide is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5 'and / or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived . For example, in various embodiments, the isolated polynucleotide may contain less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about 0.5 kb, or about 0.1 kb of the 5 'nucleotide sequences. and / or 3 'which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. In addition, an "isolated" polynucleotide, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when synthesized in a chemistry. By free of other material cellular, it is meant that an isolated polynucleotide is greater than or equal to about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% pure. "Polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of at least 5 bases in length. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of any nucleotide. The modifications include but are not limited to known substitutions of a naturally occurring base, sugar or internucleoside linkage (of structure) with a modified base such as 5-methylcytosine, a modified sugar such as 2'-methoxy sugars and 2 '-fluoro, and modified structures such as phosphorothioate and methyl phosphonate. As used herein, the term "gene" means the segment of DNA comprised in the production of a polypeptide chain; it includes regions that precede and follow the coding region (guidance and tracking) as well as intervening sequences (introns) between individual coding segments (exons). The polynucleotide may be a DNA molecule, a cDNA molecule, a genomic DNA molecule, or an RNA molecule. The polynucleotide as DNA or RNA comprises a sequence wherein T may also be U. The polynucleotide may be complementary to a polynucleotide that codes for an omega-ACTX polypeptide (eg, SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29 , 32, 35, 38, 41, 44, 47, 50 or 53), where complementary refers to the ability for precise matching between two nucleotides. For example, if a nucleotide at a certain position of a polynucleotide is capable of hydrogen bonding with a nucleotide in the same position in a DNA or RNA molecule, then the polynucleotide and the DNA or RNA molecule are complementary to each other in that position. The polynucleotide and the DNA or RNA molecule are substantially complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hybridize with each other in order to effect the desired process. As used herein, "hybridization" means hydrogen bonding, which can be hydrogen bonding of atson-Crick, Hoogsteen or reverse Hoogsteen, between complementary nucleotide or nucleoside bases. In addition, polynucleotides that are substantially identical to a polynucleotide encoding an omega-ACTX polypeptide are included (eg, SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38 , 41, 44, 47, 50 or 53) or coding for substantially identical proteins to SEQ ID N0: 1 and 2. By "substantially identical" is meant a polypeptide or polynucleotide having a sequence that is at least about 85%, specifically about 90%, and more specifically about 95% or more identical to the sequence of the amino acid sequence or reference nucleic acid. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, or specifically at least about 20 amino acids, more specifically at least about 25 amino acids, and more specifically at least about 35 amino acids. amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, specifically at least about 60 nucleotides, more specifically at least about 75 nucleotides, and more specifically about 110 nucleotides . Typically, sequences homologous to a polynucleotide can be confirmed by hybridization, where hybridization is preferred under severe conditions as described, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Using the severe hybridization summarized in Sambrook et al., (Ie, washing the nucleic acid fragments two times where each wash is at room temperature for 30 minutes with 2X sodium chloride and sodium citrate (SCC) and 0.1% sodium dodecylsulfate (SDS); followed by washing once at 50 ° C for 30 minutes with SCC 2X and 0.1% SDS; and then washed twice where each wash is at room temperature for 10 minutes with SCC 2X), homologous sequences can be identified which comprise at most about 25 to about 30% mismatches of base pairs, or about 15 to about 25% of base pair mismatches, or about 5 to about 15% of base pair mismatches. A homologous polypeptide can be produced, for example, by conventional site-directed mutagenesis of the polynucleotides (which is a way to routinely identify residues of the molecule that are functionally important, or not), by random mutation, by chemical synthesis, or by chemical or enzymatic cleavage of the polypeptides. Polynucleotides that code for omega-ACTX sequences allow the preparation of DNA sequences (or RNA) relatively short that have the ability to hybridize specifically to these gene sequences. The short nucleic acid sequences can be used as probes for detecting the presence of complementary sequences in a given sample, or they can be used as primers to detect, amplify or mutate a defined segment of the DNA sequences encoding an omega-ACTX polypeptide. A nucleic acid sequence used for hybridization studies may be greater than or equal to about 14 nucleotides in length to ensure that the fragment is of sufficient length to form a stable and selective duplex molecule. These fragments can be separated for example, by directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as PCR technology, or by cleaving selected nucleic acid fragments from recombinant plasmids containing appropriate inserts and sites. adequate restrictions. The homologous and omega-ACTX polynucleotides can be inserted into a recombinant expression vector or vectors. The term "recombinant expression vector" refers to a plasmid, virus or other means known in the art that has been manipulated by insertion or incorporation of the omega-AXTX genetic sequence. The term "plasmids" is generally designated herein by a lowercase letter p preceded and / or followed by capital letters and / or numbers, in accordance with the conventions appointment standard that are familiar to those skilled in the art. The plasmids described herein are either commercially available, publicly available in unrestricted bases, or can be constructed from available plasmids by routine application of well-known published methods. Many plasmids and other cloning and expression vectors are well known and readily available, or those skilled in the art can readily construct any number of other plasmids suitable for use. These vectors can be transformed into a suitable host cell to form a host cell vector system for the production of a polypeptide. The omega-ACTX polynucleotides can be inserted into a vector adapted for expression in a bacterial, plant, yeast, insect, amphibian or mammalian cell further comprising the regulatory elements necessary for the expression of the nucleic acid molecule in the cell bacterial, yeast, insect, amphibian, plant or mammal linked operably to the nucleic acid molecule encoding omega-ACTX. "Operationally linked" refers to a juxtaposition where the components described in this way are in a relationship that allows them to function in their proposed way. An expression control sequence linked in a way operative to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which they are operatively linked. The expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate transcription, and, as appropriate, translation of the nucleic acid sequence. In this manner, the expression control sequences may include appropriate promoters, enhancers, transcription terminators, a start codon (ie, 'atg) in front of a gene encoding the protein, splice signals for introns (if introns are present), maintaining the correct reading frame of that gene to allow proper translation of the mRNA, and codons terminators. The term "control sequences" is proposed to include, at a minimum, components whose presence may influence expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The expression control sequences may include a promoter. By "promoter" is proposed a sufficient minimum sequence to direct the transcription. Also included are those promoter elements that are sufficient to render the gene expression dependent on the controllable promoter to be cell-specific, tissue-specific, or inducible by signals or external agents; these elements can be located in the 5 'or 3' regions of the gene. Both constitutive and inducible promoters are included. If an expression vector is used to transform a plant, a promoter that has the ability to activate expression in the plant can be selected. Promoters that work in plants are well known in the art. Exemplary tissue-specific plant promoters are the corn sucrose-synthase-1 promoter, the cauliflower mosaic virus promoter (CaMV 35S), the small subunit RuBP-carboxylase promoter of S-E9, and the corn heat shock protein promoter. The choice of which expression vector, and finally to which promoter is operably linked to a peptide coding region, depends directly on the desired functional properties, for example, the location and synchronization of protein expression and the host cell that is will transform. In one embodiment, the vector used to express the polypeptide includes a selection marker that is effective in a plant cell. Transformation vectors used to transform plants and methods for making these vectors are described, for example, in U.S. Patent Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, incorporated herein by reference. The expression systems may also contain propolypeptide and signal peptide sequences that facilitate expression of the toxin gene and / or the fold of the toxin. These may be the native omega-ACTX propeptide and signal sequences, and described herein or other propeptide and / or signal sequences that serve the same purpose. Insects that are susceptible to viral infection can be a target for insect viruses. The host variety of an insect virus is determined at least in part by the wild-type variety of the unmodified wild type virus. Insect viruses are insect pathogens that occur naturally. They can be DNA viruses or RNA viruses. Many insect viruses and their variety of hosts are known in the art, including viruses that are host-specific and environmentally safe. A suitable insect virus is a DNA virus that has been traditionally used as a biological control agent in insect pests, for example, baculovirus (nucleopolyhedrovirus and granulovirus) and entomopoxvirus. For example, a baculovirus expression vector such as the type described in U.S. Patent No. 4,879,236, incorporated herein by reference, may be produced. Suitable RNA viruses include, but are not limited to, cipoviruses. Vectors useful for the expression of genes in higher plants are well known in the art and include vectors derived from the tumor inducing plasmid (Ti) from Agrobacterium tumefaciens and the transfer control vector pCaMVCN (available from Amersham Biosciences). The transformation of a host cell with an expression vector or other DNA can be carried out by techniques well known to the person skilled in the art. By "transformation" is meant a permanent or momentary genetic change induced in a cell after the incorporation of the new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cells. By "transformed cell" or "host cell" is meant a cell (eg, prokaryotic or eukaryotic) wherein a DNA molecule encoding a polypeptide of the invention (i.e., an omega-ACTX polypeptide), or a fragment of the invention has been introduced (or an ancestor of which), by means of recombinant DNA techniques. same.

When the host is a eukaryote, methods of transfection with DNA such as co-precipitation with calcium phosphate, mechanical methods such as microinjection, electroporation, insertion of a plasmid enclosed in liposomes, or virus vectors, as well as other known ones can be used. in the technique. When the host is a plant cell, other means of gene introduction into the cell can also be employed such as, for example, polyethylene glycol-mediated protoplast transformation, DNA uptake mediated by desiccation / inhibition, agitation with silicon carbide fibers, acceleration of particles coated with DNA, injection into reproductive organs, injection into immature embryos. Eukaryotic cells can also be co-transfected with DNA sequences encoding a polypeptide of this description, and a second foreign DNA molecule that codes for a selectable phenotype, such as the herpes simplex thymidine kinase gene. Suitable markers include, for example, neomycin and hygromycin, and the like, which can be taken up by mammalian cells. Resistance to the marker can be conferred by the neomycin gene or the hygromycin gene, for example, when the gene has a suitable eukaryotic promoter. Another method is to use a eukaryotic viral vector, such as a virus simiesco 40 (SV40), adenovirus, or bovine paper virus, to momentarily infect, or transform, eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). In one embodiment, a eukaryotic host is used as the host cell as described herein. The eukaryotic cell can be a yeast cell (eg, Saccharomyces cerevisiae) or it can be a mammalian cell, including a human cell. Mammalian cell systems using recombinant viruses or viral elements can be engineered to direct expression. For example, when adenovirus expression vectors are used, the nucleic acid sequences encoding a foreign protein can be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the omega-ACTX polypeptide in infected hosts. For the high-throughput and long-term production of recombinant polypeptides, stable expression is preferred. Instead of using expression vectors that contain viral origins of replication, the host cells can be transformed with the cDNA encoding an omega-ACTX fusion polypeptide controlled by appropriate expression control elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form sites, which in turn can be cloned and expanded into cell lines. For example, after the introduction of the foreign DNA, the managed cells can be allowed to grow for 1 or 2 days in an enriched medium, and then switch to a selective medium. Various screening systems can be used, including but not limited to thymidine kinase from herpes simplex virus, hypoxanthine guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase. The omega-ACTX polypeptides can also be designed to provide additional sequences, such as, for example, the addition of coding sequences for additional C-terminal or N-terminal amino acids that will facilitate purification by column overriding or use of antibodies. These brands include, for example, marks with high histidine content that allow the purification of polypeptides in nickel columns. These gene modification techniques and suitable additional sequences are well known in molecular biology techniques. The omega-ACTX proteins, polypeptides or polypeptide derivatives, can be modified by methods known in the art. These methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, crystallization, electro-focusing, preparative gel electrophoresis, and combinations comprising one or more of the methods previous Purification can be carried out according to methods known to those skilled in the art which will result in the preparation of omega-ACTX substantially free of other polypeptides and of carbohydrates, lipids, or subcellular organelles. Purity can be assessed by means known in the art, such as SDS-polyacrylamide gel electrophoresis. Also provided is an omega-ACTX fusion polypeptide, comprising an omega-ACTX polypeptide covalently linked to a polypeptide to which it would not bind in nature. The fusion polypeptides are useful for use in various assay systems. Therefore, they can be using fusion polypeptides, for example, to detect omega-ACTX expression and provide a defense mechanism for the expression of omega-ACTX when desired. For example, omega-ACTX fusion polypeptides can be used to identify proteins that interact with the omega-ACTX polypeptide and influence its function. This interaction can impart specificity to the ability of omega-ACTX to regulate other proteins, or it can increase or decrease the effect of the omega-ACTX function. Physical methods, such as protein affinity chromatography, or library-based assays for protein-protein interactions, such as phage display systems or two yeast hybrids, can be used for this purpose. These methods are well known in the art. A fusion polypeptide comprises at least two heterologous segments of polypeptide fused together by means of a peptide bond. The first polypeptide segment can comprise all or in part the contiguous amino acids of an omega-ACTX polypeptide. Where it is in part, at least about 8 contiguous amino acids of the omega-ACTX polypeptides are used, specifically at least about 10, more specifically about 15, and more specifically, at least about 20 can be employed.

The polypeptide segment can also be a full-length omega-ACTX protein. The second polypeptide segment can comprise an enzyme that will generate a detectable product, such as beta-galactosidase or other enzymes that are known in the art. Alternatively, the second polypeptide segment can include a fluorescent protein such as green fluorescent protein HcRed (Clontech) or other fluorescent proteins known in the art. Additionally, the fusion protein can be labeled with a detectable label, such as a radioactive label, a fluorescent label, a chemiluminescent label, a biotinylated label, and the like. The techniques for making fusion polypeptides, either recombinantly or by covalently linking two polypeptide segments, are well known. Recombinant DNA methods can be used to construct omega-ACTX fusion polypeptides, for example, by making a DNA construct comprising the omega-ACTX coding sequence in the appropriate reading frame with nucleotides encoding the second segment of polypeptide and expressing the construction of DNA in a host cell. The DNA construct can be operably linked to sequences that facilitate the production of protein (i.e., promoters, etc.). In addition to the fusion polypeptides, the omega- ACTX can be labeled in vitro by methods known in the art. Omega-ACTX can be conjugated to dyes such as Texas Red, rhodamine dyes, fluorescein and other dyes known in the art. Conjugation chemistries include succinimidyl ester, isothiocyanates and maleimides. Detailed information about conjugated conjugate and chemical conjugate dyes can be found in the Molecular Probes Handbook of Fluorescent Probes and Research Products. These fusion polypeptides can be used for the production of antibodies that may have greater specificity and sensitivity than those generated against short sequences of amino acids. In addition, fusion polypeptides can be used to examine their ability to influence cell survival, proliferation and differentiation in tissue culture assays. Transgenic plants expressing the omega-ACTX polypeptide or the prepolypeptide or prepropolypeptide form of the toxin can be constructed. By "transgenic plant" is meant a plant, or progeny thereof, derived from a cell or protoplast of "transformed plant", wherein the DNA of the plant (nuclear or chloroplast) contains an introduced exogenous DNA molecule not present originally in a non-transgenic native plant of the same variety. The development or regeneration of plants from either Individual plant protoplasts or several explants is well known in the art. This regeneration and growth process typically includes the selection of transformed cells, and the cultivation of these individualized cells through the usual stages of embryonic development through the rooted seedling cover. Embryos and transgenic seeds can be regenerated in a similar way. The resulting transgenic rooted shoots can then be planted in an appropriate plant growth medium such as soil. The regenerated plants can self-pollinate to provide homozygous transgenic plants. Otherwise, the pollen obtained from the regenerated plants can be crossed to plants grown from seed of congenital, agronomically important lines. In contrast, plant pollen from these important lines can be used to pollinate regenerated plants. A transgenic plant containing a desired polypeptide can be cultured using methods well known to one skilled in the art. A suitable transgenic plant includes an independent segregant that can transmit the omega-ACTX gene and its activity to its progeny. In one embodiment, a transgenic plant is homozygous for the omega-ACTX gene, and transmits that gene to all its offspring in sexual mating. The seed of a transgenic plant it can be grown in the field or in the greenhouse, and the resulting sexually mature transgenic plants self-pollinate to generate true crop plants. The progeny of these plants become the true cultivation lines that are evaluated for, as an example, increased acaricidal capacity against one or more acarines, preferably in the field, under a variety of environmental conditions. The transgenic plant can be corn, soybeans, cotton, wheat, oats, barley, other grains, vegetables, fruits, fruit trees, berries, lawns, ornamentals, shrubs and trees, and the like. Mutated acaricidal polypeptide libraries can be obtained for the purposes of detection, by evolution in vi tro of a gene for omega-ACTX-Hvla, omega-ACTX-Hv2a or a variant. Libraries can be produced using PCR prone to complete omega-ACTX error or variant gene or digestion of the omega-ACTX gene or variant with an appropriate enzyme, followed by PCR reconstruction prone to complete gene sequence error. These error prone PCR methods can be applied to the complete sequence of the prepropolypeptide gene for omega-ACTX, or a variant. The library of variant gene sequences or mutant omega-ACTX can then be used to generate a series of omega-ACTX variant antagonists. These Antagonists can then be detected by their ability to inhibit the binding of omega-ACTX, or selected variants thereof, to its molecular target. Detection can be performed, for example, by phage display of a gene library of mutants followed by selection of phage particles that bind tightly to the molecular target of omega-ACTX, or phage particles that inhibit the binding of omega-ACTX , or the selected variant thereof, to the molecular target of omega-ACTX. As will be understood by one skilled in the art, a gene library of mutant genes can also be constructed by other normal molecular biology methods such as mutagenesis of oligonucleotide cassettes or construction of synthetic genes with certain randomized nucleotide positions. The three-dimensional structure of omega-ACTX, and variants thereof, can also be used to search for libraries of structures for (or to design) either peptide or non-peptide compounds that resemble the key structural elements of omega-ACTX, particularly those regions found to be critical for the activity by mutagenesis / truncation / modification experiments. These compounds can then be tested for their ability to inhibit the binding of omega-ACTX, or the variant thereof, to its target molecular. In one embodiment, an acaricidal composition comprising a purified omega-ACTX polypeptide and an agriculturally acceptable carrier is provided. In another embodiment, an acaricide composition comprises a virus that expresses an omega-ACTX polypeptide. Infection with a virus can be achieved by conventional methods, including ingestion, inhalation, direct contact with the insect virus, and the like. The acaricidal composition may be in the form of a fluid solution or suspension such as an aqueous solution or suspension. These aqueous solutions or suspensions may be provided as a concentrated solution which is diluted before application, or alternatively, as a ready-to-apply diluted solution. In another embodiment, an acaricidal composition comprises a water dispersible granule. In yet another embodiment, an acaricidal composition comprises a wettable powder, powder, granule or colloidal concentrate. These dry forms of the acaricidal compositions can be formulated to dissolve immediately upon wetting, or alternatively, to dissolve in a controlled release manner, sustained release, or in a time dependent manner. The omega-ACTX polypeptides can be expressed in vi tro and isolate for subsequent field application. These polypeptides may be in the form of crude cell lysates, suspensions, colloids, etc., or may be further purified, refined, buffered and / or processed, prior to formulation into an active acaricidal formulation. In spite of the method of application, the amount of the active components is applied to an acaricidally effective amount, which will vary depending on factors such as, for example, the specific acarines to be controlled, the specific host or environment to be treated , the environmental conditions, and the method, speed and amount of application of the acaricidally active composition. The acaricidal compositions comprising the omega-ACTX polypeptides, polynucleotides, etc., can be formulated with an agriculturally acceptable carrier. Suitable agricultural carriers can be solid and liquid and are well known in the art. The term "agriculturally acceptable carrier" covers all adjuvants, for example, inert components, dispersants, surfactants, tackifiers, binders, etc., which are ordinarily used in the technology of insecticidal formulations; These are well known to those experts in the formulation of pesticides. The formulations are can be mixed with one or more solid or liquid adjuvants and prepared by various means, for example, by homogeneous mixing, combination and / or grinding of the acaricide composition with suitable adjuvants using conventional formulation techniques. The acaricidal compositions can be employed individually or in combination with other compounds, including and not limited to other pesticides. They can be used in conjunction with other treatments such as surfactants, detergents, polymers or time release formulations. The acaricidal compositions may comprise an acarine attractant. The acaricidal compositions can be formulated for either systemic or topical use. These agents can also be applied directly to the acarines. The omega-ACTX polypeptides are particularly useful for methods comprising the control of acarine pests. A method for controlling an acarin comprises contacting the site of an acarin with an acaricidally effective amount of an omega-ACTX polypeptide. The omega-ACTX polypeptide can be in the form of a purified polypeptide, a polynucleotide that encodes the omega-ACTX polypeptide optionally in an expression vector, a cell such as a plant cell or a bacterial cell that expresses the polypeptide of omega-ACTX, and / or a transgenic plant expressing the omega-ACTX polypeptide. The contact includes, for example, injection of the omega-ACTX polypeptide, external contact, or ingestion of the omega-ACTX polypeptide or polynucleotide or virus that expresses the omega-ACTX polypeptide. The acaricidal compositions can be applied to the site of acarus pests. The "site" of mites, or ticks, refers to the plague itself of acarines, or to the environment in which mites or ticks live or where their eggs are present, including the air that surrounds them, the food they eat, the objects or hosts with which they make contact. The resistance and duration of the acaricidal application can be adjusted with respect to specific conditions to the particular pests of acarines to be treated and to particular environmental conditions. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the acaricidal composition, as well as on the particular formulation contemplated. Subjected to the attack of ectoparasites such as ticks and acarids are the numerous livestock animals, such as cattle, sheep, pigs, goats, buffalos, water buffalos, deer, rabbits, chickens, turkeys, ducks, geese, ostriches, and the like. Horses and others Recreational animals undergo ectoparasite attack, such as mink and other animals bred by their chelation, and rats, mice and other animals used in laboratories and research establishments. Pet animals such as dogs and cats are highly subject to the attack of ectoparasites, and due to their close relationship with humans, this parasitism presents problems for humans with whom they are associated. Common practices for distributing acaricide to livestock and companion animals include direct treatment of the whole body, where the animal's body comes into contact with liquids containing the acaricide; systemic treatment, where the acaricide is allowed to circulate in the blood of the host, including oral formulations, and implantable forms, controlled release, for example; controlled release systems, for example, collars or ear tags, which usually physically bind to the animal and continuously release the pesticide over a period of weeks or months; and self-medication methods, in which an animal is attached to a device that provides a bait, eg, feed, nest building materials, etc., and which causes the animal to spray or coat with the acaricidal polypeptide. In the self-medication method, the animal either contacts the device or in some way activates the device to release the pesticide. Human beings are also potential hosts for ectoparasites such as acarinos, and in tropical areas and in areas with minimal sanitation, parasitic infections are a regular problem in medical practice. In the case of ticks, ticks can have parasites and other infectious agents that can be transmitted to humans and / or animals. Infections carried by ticks, to which humans are susceptible, include Lyme borreliosis, babesiosis, and human granulocytic anaplasmosis. Scabies, in contrast, is an infestation of the skin with the microscopic mite Sarcoptes scabei. Acaricidal polypeptides can be administered to a human by ingestion or injection, for example. In the case of disease carried by ticks, transmission to a human or animal host can be presented by a wild animal vector such as a deer or a mouse. The larvae live and feed on animals (for example, 'rodents such as mice, deer, squirrels, cattle, and any human who enters the tick's habitat) from about a week before the separation and after the moult (molt). ) anywhere from 1 week to 8 months later. The larvae then become 8-legged nymphs. The nymphs feed on the animals, flood for 3 to 11 days, detach and move about a month later (depending on the species and environmental conditions). Once the nymph dies, it becomes an adult tick (male or female). Ticks climb the grass and plants and keep their legs to "perceive" and "feel" their prey. Ticks insert their mouths, attack their prey, and devour by themselves with blood meal. During feeding, saliva from the tick can enter the bloodstream of the host's body. A tick infected with Borrelia burgdorferi, for example, can then inadvertently spread this bacteria to the host. Because the life cycle of the tick is dependent on animal vectors, a method to prevent tick borne infections in humans and animals (farm animals and companion animals) is to administer the acaricidal polypeptides to the site of an animal vector.

The polypeptides can be administered to the animal vector, to the environment of the animal vector, or to the food of the animal vector, by way of example. In one embodiment, the acaricidal composition is administered in the form of a bait composition. A bait composition may include a feed stimulant, and optionally, an attractant. An attractant is a material that is used to help put a rodent or deer, for example, near the bait. A stimulant of Feeding seduces the rodent or deer, for example, to feed and maintain feeding in the bait. A material can function as both an attractant and a food stimulant. The attractants can be a food item or "a curiosity enhancer". The bait composition can be placed, for example, in shrubs or on residential properties to reduce tick populations. In one embodiment, the acaricidal polypeptides are applied to the environment of an acarin. Suitable application techniques for treating the acarine habitat include, for example, dusting, spraying, soaking, soil injection, seed coating, seedling coating, spraying, aeration, fogging, atomization, and the like. These application methods are also well known to the person skilled in the art. Parasitic mites infect the skin, for example, of humans and animals. Scabies, for example, is an infestation of human skin with the microscopic mite Sarcoptes scabei. Mites such as those of the genus Psoroptes infect animals such as, for example, sheep and can cause scab. The acaricidal polypeptides can be used to control acarines that are fed to plants such as phytophagous acarines. The acarinos that feed on plants are among the most voracious phytophagous pests that crops. Mites that ingest plants can be controlled by applying the active compound to the plant parts from which the mites feed or inhabit. The acaricidal polypeptides can be applied in a suitable manner known in the art, for example by spraying, atomizing, vaporizing, dispersing, dusting, wetting, jetting, spraying, pouring, spraying, and the like. The dose of the acaricidal polypeptide is dependent on factors such as the type of pest, the carrier used, the method of application and climatic conditions for the application (eg, indoor, aggregate, wet, windy, cold, heat, controlled), and the type of formulation (for example, aerosol, liquid or solid). The effective dose, however, can be readily determined by persons skilled in the art. In one embodiment, phytophagous mites can be controlled by the use of transgenic plants expressing acaricidal polypeptides. Acaricidal polypeptides can control acarines that typically inhabit an inner area. Illustrative and non-limiting examples of acarines that can be controlled by using acaricidal polypeptides include Dermanyssidae such as American home dust mite (Dermatophagoides pharynx) and Dermatophagoides. pternonyssinus, Acaridae such as Lardoglyphus konoi, mite or copra o forage (Tyrophagus putrescentiae) and brown-legged grain mite (Aleuroglyphus ovatus), Glycyphagidae such as Glyciphagus privatus, Glycyphagus domesticus, grocery mite (Glycyphagus destuctor), and Chortoglyphus spp., Cheyletidae such as Chelacaropsis moorei, Chelacaropsis malaccensis, Cheyletus fortis, Cheyletus erudi tus and Chelatomorpha lepidoterorum, Macronyssidae such as Orni thonyssus bacoti, Orni thonyssus sylviarum, Dermanyssus gallinae and Dermanyssus hirundinis, Haplochthonius simplex, Pyemotidae, and Sarcoptidae, and the like. In the treatment of indoor acarus pests, the acaricidal polypeptides can be applied to an indoor environment, for example, they are sprayed on textile surfaces such as sofas, upholstered chairs, bedding, pillows, carpets, rugs, etc., which It is known that they are infested with dust mites. Although emphasis is placed on dust mites and their allergens, it should be understood that these methods are equally effective against dust particles in general and against other allergens associated with them, including pollen and animal dander. The application can be by means of a self-contained aerosol spray device. Spraying should be done carefully, ensuring that, for example, all sides are sprayed the pillows, that the spray reaches the corners and crevices, etc. The invention is further illustrated by the following non-limiting examples.

Example 1: Discovery of omega-ACTX-1 family polypeptides A female Hadronyche infénsa spider was collected from the city of Toowoomba in the state of Queensland, Australia. A female spider from Hadronyche versuta was obtained from the Blue Mountains region of New South Wales, Australia. Male and female spiders of Atrax robus tus were collected from the Sydney metropolitan area of New South Wales, Australia. The specimens were housed. in hermetic collection jugs until the extraction of the poison glands. The funnel web spiders were cooled to -20 ° C for 40 to 60 minutes. The venom glands of each specimen were dissected independently. Each pair of venom glands was independently placed in extraction buffer (Amersham Biosciences). Immediately after isolation of the venom glands, poly A + mRNA was prepared using a QuickPrep1 MicroRNA purification kit (Amersham Biosciences). The purified samples of mRNA were washed with 80% ethanol and dried with Speedvac. 10 microliters of RNA-free distilled water was obtained from a cDNA synthesis kit (Amersham Biosciences) to rehydrate the mRNA samples. The purified mRNA samples were stored at -20 ° C. Subsequently, cDNA libraries were constructed using a Marathon ™ cDNA Amplification Kit (CLONTECH). From the template adapted from mRNA, the individual strand cDNA was constructed using Superscript "III reverse transcriptase (Life Technologies, Inc.) and the Echoclonanch-2 primer, a poly (dT) anchor primer (GGGCAGGTi?) (SEQ ID NO: 58). The synthesis of the second strand was carried out according to the equipment specifications. The cDNA products were purified using Concert® rapid PCR purification equipment (GIBCO). The double-stranded cDNA was eluted with 50 μl of Tris-EDTA buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0). The Marathon1® cDNA amplification adapter (CLONTECH) then ligated to the double-stranded cDNA. The ligation reaction was allowed to settle at 16 ° C overnight. After overnight ligation, the sample was precipitated using 10 μl of a 1 to 20 dilution of glycogen, 10 μl of 3M sodium acetate, pH 5.2 and 100 μl of 100% cold ethanol. Subsequently, the sample was washed with 80% ethanol and dried for 10 minutes before of the resuspension in 200 μl of Tris-EDTA buffer. Guidance sequence information was obtained using 5 'RACE (Rapid Amplification of cDNA Ends; see Frohman et al., Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer, Proceedings of the National Academy of Sciences USA, 8993-9002, 1988). 5 'RACE is used to extend partial cDNA clones by amplifying the 5' sequence of the corresponding mRNA. 5 'RACE uses the knowledge of only a small region of sequences within the partial cDNA clone. 5 'RACE employs a first round of cDNA extension by the terminal transferase enzyme, which adds a homopolymeric extremity to the 5' end of all the template cDNAs. This synthetic tip provides a primer binding site in the 5 'direction of the unknown 5' sequence of the target mRNA. The PCR reaction is then carried out, using a general sense primer that binds to the new 5 'end and a specific antisense primer that binds to the known cDNA sequence. Redundant polymerase chain reaction (PCR) primers are designed for this technique. The redundant primers were used in conjunction with a 5 'universal adapter primer (EchoAPl) in order to obtain the unknown information from the leader sequence. Primers for 3 'RACE of the cDNA guide sequence obtained from 5' RACE were designed. The 3 'RACE primers in combination with an oligo d (T) universal adapter (CLONTECH) primer to generate gene products that have a signal sequence homologous to that of omega-ACTX-Hvla. All primers not included in equipment were constructed by PROLIGO Ltd. The 5 'RACE primers were as follows: SEQ ID NO: 55: CACCCCTAATACGACTCACTATAGG SEQ ID NO: 56: RTTNCCRTTYTCRTTYTCYTCRAA Where R = puRinas (degeneration A / G), Y = pYrimidines (C / T degeneration), and N = complete degeneration (A, G, C, or T). The 3 'RACE primers were as follows: SEQ ID NO: 57: TGCTGCAATATGAATACCGC SEQ ID NO: 58: GGGCAGGTTTTTTTTTTTTTTTTT PCR reactions were carried out using 5 μl of double-stranded cDNA, 27 μl of water Milli Q, MgCl 2 mM, lOx PCR buffer, 50x dNTP, and 5 μl of AMPLIGOLDTAQ ^ Enzyme (Perkin Elmer, AmpliTaq1® Gold with GeneAmp Kit). The PCR reactions were run in a thermal cycler using the following protocol: The amplified cDNA products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide for size verification. The verified PCR products were extracted from the agarose gel using a GIBCO gel purification kit and precipitated using Pellet Paint co-precipitation equipment (Novagen). Once precipitated, the cDNA ends were phosphorylated with kinase in preparation for cloning. The samples were ligated into the vector pSMART1 ^ and transformed into E. cloni cells (Lucigen) using the Lucone CloneSmart Direct Cloning kit. Successfully transformed clones were cultured for one hour in Terimo Broth with 50 μg / ml ampicillin, and then plated to allow overnight growth. The samples were tested for the correct size of insertion by PCR and gel electrophoresis. Samples containing the correct insertion size were submitted for DNA sequencing. They were obtained, sequentially numerous clones complete cDNA sequences coding for the omega-ACTX-Hvla preproprotein (SEQ ID N0: 1) and 16 paralogs thereof (SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49 or 52). These preproprotein sequences are summarized and aligned in Figure 1. The cleavage site of the signal peptide in these preproproteins was predicted using version 3.0 of the SignalP program (Dryl0v et al., Improved prediction of signal peptides: SignalP 3.0, Journal of Molecular Biology 340, 783-795, 2004; program available on the web at http://www.cbs.dtu.dk/services/SignalIP/). The mature toxin is predicted to result from the cleavage of the propeptide after the Arg-Arg dibasic sequence at positions 36-37, as well as for the known cleavage site of propeptide on the? -ACTX-1 toxins produced by Australian spiders from funnel web These two endoproteolytic cleavage sites are indicated by arrows in Figure 1.

Example 2: Preparation of recombinant omega-ACTX-Hyla Recombinant omega-ACTX-Hvla was prepared as described in Tedford et al., Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hvla. J. Biol. Chem. 276, 26568-26576, 2001. Briefly, BL21 cells of Escherichia coli were transformed with pHWTl for overproduction of the protein GST fusion: omega-ACTX-Hvla. The cells were cultured in the LB medium at 37 ° C to an OD600 of 0.6-0.8 before the induction of the fusion protein with 150 μM isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation 3-4 hours after induction and lysed using a French press. The recombinant fusion protein was purified from the soluble cell fraction using affinity chromatography on GSH-Sepharose (Amersham Biosciences) and excised on the column by the addition of bovine thrombin (Sigma) for about 24 hours. The unbound toxin was eluted from the column with saline buffered with Tris (150 mM NaCl, 50 mM Tris, pH 8.0) and was immediately purified using reverse phase high performance liquid chromatography (rpHPLC). The recombinant toxin and contaminants were eluted from a Vydac Cie analytical rpHPLC column (4.6x250 mm, 5 μm pore size) at a flow rate of 1 ml min "1 using a linear gradient of 12.5-20% acetonitrile during 20 minutes.An individual main peak corresponding to omega-ACTX-Hvla eluted at a retention time of approximately 11 minutes.It is noted that the recombinant toxin contains two additional N-terminal residues (Gly-Ser) in relation to the native protein. , which are a vestige of the thrombin cleavage site in the protein of fusion of GST.

Example 3: Purification of Native omega-ACTX-Hv2a Native omega-ACTX-Hv2a is purified from the venom of the Australian spider web funnel Hadronyche versuta exactly as described in Wang et al., Discovery and structure of a potent and highly specific Blocker of insect calcium channels, J. Biol. Chem. 276, 40806-40812, 2001. Briefly, the lyophilized crude venom was fractionated using an analytical reverse phase high pressure liquid chromatography (rpHPLC) column Vydac C18. The semi-pure omega-ACTX-Hv2a obtained from this initial fraction is further purified in the same column using a gradient of acetonitrile at 30-48% for about 35 minutes at a flow rate of about 1 ml / min. Once purified to more than about 98% homogeneity, the peptides are lyophilized and stored at 20 ° C until further use.

Example 4: Recombinant omega-ACTX-Hyla is lethal to ticks by injection The acaricidal activity of recombinant omega-ACTX-Hvla was determined quantitatively by direct injection of toxin into the solitary star tick Amblyomma americanum (Arachnida: Ixodida: Ixodidae). HE obtained ticks from a pathogen-free colony, bred in Amblyomma americanum laboratory maintained at 27 ± 1 ° C and 75% relative humidity in the Department of Immunology, University of Connecticut Health Center. TO . americanum is an important vector of zoonotic human pathogens in the United States. It is responsible for transmitting the causative agents of human ehrlichiosis (Ehrlichia chaffeensis and E. ewingii) and rash disease associated with southern tick (Borrelia lonestari), a clinical condition similar to Lyme disease. It can also serve as a vector for the causative agents of tularemia (Francisella tularensis) and cerebrospinal meningitis of the Rocky Mountain (Rickettsia rickettsii). Dose-response curves were constructed by injecting doses in the range of 200-1200 picomoles of toxin per gram of tick. The toxin has a molecular weight of about 4200 Daltons, so this corresponds to approximately 0.8-5 micrograms of toxin per gram of tick. The toxin was dissolved in insect saline solution (200 mM NaCl, 3.1 mM KCl, 5.4 M CaCl2, 5.0 mM MgCl2, 2 M NaHCO3, and 0.1 M NaH2P04, pH 7.2) to give a concentrated solution of sufficient concentration such that each dose required injection of no more than 2 microliters of concentrated solution. The specimens were temporarily immobilized at 4 ° C for injections. After the injection, each tick was individually housed in a flask in a modified chamber at 27 ° C. A cohort of 6-8 ticks was injected for each dose of toxin, and a cohort of 6-8 control ticks was injected with only the vehicle (insect saline solution). Injections were performed using a Hamilton microsyringe (Hamilton Co., Reno, Nevada). Death was scored at 48 hours. The severity and onset of symptoms varied with the dose and route of administration, although severe high dose effects were observed within a few minutes. The most pronounced phenotypic effect was curled from the eight legs in closed folds; This made locomotion extremely difficult although ticks can walk very slowly with the limbs bent at low doses of toxin. Ticks also lost their righting reflex, possibly due to limb weakness and the "curly" phenotype described above at higher doses of toxin (> 1000 pmol / g), the integument of the treated ticks became black , and the moribund ticks were paralyzed in the resting position. This immobilizing (paralyzing) effect of the toxin was reversible. Figure 2 shows the dose-response curve obtained from omega-ACTX-Hvla using this method. Each point represents the average of three independent measurements • made on different days. The LD50 value (ie, the dose of ega-ACTX-Hvla that kills 50% of the ticks at 48 hours after injection) was calculated by fitting the following equation to the logarithmic dose-response curve: = (a - b) / [l + (x / LD50) n] where y is the percentage of deaths in the sample population at 48 hours after injection, x is the dose of toxin in pmolg "1, n is a variable slope factor, a is the maximum response and b is the minimum response.The calculated LD50 value was 447 ± 3 prnolg "1.

Example 5: Recombinant omega-ACTX-Hyla is lethal to ticks by feeding The oral acaricidal activity of recombinant omega-ACTX-Hvla was determined quantitatively by feeding the toxin to the solitary star tick Amblyomma americanum using doses in the range of 400 -1400 picomoles of toxin per gram of tick. The toxin has a molecular weight of approximately 4200 Daltons, so this corresponds to approximately 1.7-6 micrograms of toxin per gram of tick. For the feeding experiments, the toxin was dissolved in the Roswell Park Memorial Institute (RPMI) -1640 medium and the ticks were fed with 2-3 microliters of this solution from a microliter capillary tube (WR Scientific, West Chester, PA). The RPMI medium comprises: Components g / L L-Arginine [free base] 0 .2 L-Asparagine [anhydrous] 0. .05 L-Aspartic acid 0, .02 L-Cystine "2HCl 0 .0652 L-Glutamic acid 0, .02 L-Glutamine 0, .3 Glycine 0. .01 L-Histidine [free base] 0. .015 Hydroxy-L-proline 0. .02 L-Isoleucine 0. .05 L-Leucine 0. .05 L-Lysine »HCl 0., 04 L-Methionine 0., 015 L-Phenylalanine 0. 015 L-Proline 0. 02 L-Serine 0., 03 L-Threonine 0.02 L-Tryptophan 0.005 L-Tyrosine» Na »2H20 0.02883 L-Valine 0.02 Biotin 0.0002 Components g / L Choline Chloride 0.003 Folic Acid 0.001 Mio-Inositol 0.035 Niacinamide 0.001 D-Pantothenic Acid Hemicálcico 0.00025 PABA 0.001 Pyridoxy »HCl 0.001 Riboflavin 0.0002 Thiamin» HCl 0.001 Vitamin B12 0.000005 Calcium Nitrate »H20 0.1 Magnesium Sulfate [Anhydrous] 0.04884 Potassium Chloride 0.4 Sodium Chloride 6.0 Sodium Phosphate Dibasic [Anhydrous] 0.8 D-Glucose 2.0 Glutathione, Reduced 0.001 Red Phenol «Na 0.0053 The capillary tubes were polished to give a beveled surface that was inserted over the chelicerae and hypostome, which comprise the buccal parts of the tick. Care is taken to ensure that each tick does not become dehydrated during the experiments of feeding. A cohort of 6-8 ticks was fed for each dose of toxin, and a cohort of 6-8 control ticks was fed with only the vehicle (RPMI-1640). Death was noted at 48 hours, but symptoms were seen with greater doses in a few minutes, and the ticks became completely paralyzed. The most unusual symptom of poisoning was the bending of the limbs in a closed loop. Ticks also lose their straightening reflex. The control ticks were unaffected by the ingestion of RPMI-1640. Figure 3 shows the response-dose curve obtained from feeding omega-ACTX-Hvla a, A. americanum Each point represents the average of three independent measurements taken on different days. The value of LD5o (ie, the dose of ornega-ACTX-Hvla that kills 50% of the ticks at 48 hours after the injection) was calculated by fitting the following equation to the logarithmic dose-response curve: y = (a - b) / [l + (x / LD50) n] where y is the percentage of deaths in the sample population at 48 hours after injection, x is the dose of toxin in pmolg-1, n is a variable slope factor, a is the maximum response and b is the minimum response. The LD50 value calculated for oral distribution of the toxin was 716 ± 23 p olg "1, which is less than twice as high as the LD50 obtained with direct injection of the toxin (Figure 3). Ticks fed RPMI medium bound to toxin exhibited peculiar behavior that was not evidenced by control ticks fed with toxin-free RPMI medium Ticks fed RPMI medium bound to toxin tried to unblock the capillary tube containing the medium, and consumed a smaller amount of liquid (average of 2-3 μl) during the feeding period of 60 minutes compared to control ticks (average of 4-6 μl). Additionally, the test cohort takes significantly longer to drink this smaller volume of RPMI medium (50-60 min) compared to control ticks (25-30 min). Thus, the LD50 calculated for oral administration of omega-ACTX-Hvla is almost certainly an overestimate, which implies that the toxin is virtually equipotent when distributed by this route compared to when injected into ticks. In addition, these results indicate that omega-ACTX-Hvla, in addition to being highly toxic to ticks, is also an effective anti-feeder.

Example 6: native omega-ACTX-Hv2a is lethal to ticks by injection The oral acaricidal activity of native omega-ACTX-Hv2a was determined by direct injection of a single dose of toxin (6 nmol / g, approximately 25 μg / g) in a cohort of five uncrowded males or five females crammed with A. ameri canum. The toxin was dissolved in insect saline solution to give a concentrated solution of sufficient concentration so that the injection volume was less than 2 microliters. A cohort of five ticks was injected with only the vehicle (insect saline solution). The specimens were temporarily immobilized at 4 ° C for injections, then each tick was individually housed in a flask in a humidified chamber at 27 ° C. In the ticks injected with toxin, symptoms were seen in the space of a few minutes, and the ticks quickly became irreversibly paralyzed. The most unusual symptom of poisoning was the bending of the limbs in a closed loop. The ticks also lost their righting reflex. There was 100% mortality in both female and male tick populations at 48 hours. In contrast, the control ticks were unaffected by the injection of insect saline solution. As we have seen in the present, the plagues of acarines such as ticks can be effectively controlled using acaricidal polypeptides. The polypeptides may comprise the families of omega-ACTX-1 and omega-ACTX-2 that previously showed to have insecticidal activity. Since the insects and ticks are not closely related, the acaricidal activity of the polypeptides was unexpected. The terms "first", "second", and the like, do not denote any order, amount, or importance in the present, but rather are used to distinguish one element from another, and the terms "a" and "an" do not denote herein a quantity limitation, but rather denotes the presence of at least one of the referenced articles. All ranges described herein are inclusive and combinable. While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and the equivalents may be replaced by elements thereof without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is proposed that the invention is not limited to the particular mode described as the best mode contemplated to carry out this invention, but the invention will include all modalities that fall within the scope of the appended claims. All of the patents cited, patent applications, and other references are incorporated herein by reference in their entirety. It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.

Claims (20)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for controlling acarus pests, characterized in that it comprises applying to the site of the acarin plates, an isolated polypeptide toxin, wherein the polypeptide toxin has acaricidal activity.
2. 2. Method according to claim 1, characterized in that the polypeptide toxin comprises three intrachain chain disulfide bonds.
3. Method according to claim 1, characterized in that the polypeptide toxin is a component of a venom of an Australian funnel web spider of the genus Atrax or Hadronyche.
4. 4. Method according to claim 3, characterized in that the polypeptide is an omega-ACTX-Hvla peptide having greater than or equal to 70% sequence identity to SEQ ID NO: 1.
5. 5. Method according to claim 3, characterized in that the polypeptide is an omega-ACTX-Hv2a peptide having greater than or equal to 70% sequence identity to SEQ ID NO: 2.
6. 6. Method according to claim 1, characterized in that the toxin polypeptide comprises a prepropolypeptide.
7. Method according to claim 6, characterized in that the prepropolypeptide comprises any of SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49 or 52.
8. 8. Method according to claim 1, characterized in that the polypeptide toxin comprises a mature polypeptide.
9. 9. Method according to claim 8, characterized in that the mature polypeptide comprises any of SEQ ID NO: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54.
10. Method according to claim 1, characterized in that the polypeptide toxin is administered in the form of a polynucleotide encoding the polypeptide toxin operably linked to expression control sequences.
11. Method according to claim 1, characterized in that the application to the environment comprises applying to an animal vector of an acarin pest.
12. Method according to claim 11, characterized in that the animal vector comprises a deer or a rodent.
13. 13. Method according to claim 11, characterized in that the polypeptide toxin is in the form of a bait composition.
14. Method according to claim 1, characterized in that the acariño pest comprises a phytophagous mite and applying the polypeptide toxin to a part of the plant that the mites eat or inhabit.
15. 15. Method according to claim 1, characterized in that the acariño pest comprises a phytophagous mite and the application comprises exposing the phytophagous mite to a transgenic plant expressing the polypeptide toxin.
16. Method according to claim 1, characterized in that the acariño pest is a tick capable of carrying a pathogen that infects humans, farm animals, or companion animals.
17. Method according to claim 1, characterized in that the acariño pest is a mite that is parasitic to humans or animals.
18. Method according to claim 1, characterized in that the acariño pest is a powder mite and the application comprises the application to an indoor environment.
19. 19. A method for inhibiting acarus pest infection in a farm animal, a companion animal, or an animal vector, characterized in that it comprises applying a polypeptide toxin to the site of acarus pests. isolated, wherein the polypeptide toxin has acaricidal activity.
20. 20. Isolated polypeptide toxin, characterized in that it comprises any of SEQ ID NO: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54.