AU2012300197B2

AU2012300197B2 - Pest-controlling agents isolated from spider venom and uses thereof

Info

Publication number: AU2012300197B2
Application number: AU2012300197A
Authority: AU
Inventors: Margaret Carter HARDY; Glenn Frederick KING
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 2011-08-24
Filing date: 2012-08-24
Publication date: 2018-01-18
Anticipated expiration: 2032-08-24
Also published as: NZ621552A; WO2013026105A1; AU2012300197A1

Abstract

The invention relates to proteinaceous agents derived from the venom of Theraphosidae spiders and their use in controlling pest infestations, particularly insect pest infestations. The invention also relates to nucleic acid molecules encoding the proteinaceous agents as well as constructs, host cells, genetically modified plants and insect pathogens comprising those nucleic acid molecules.

Description

H:\kyg\Jmerwoven\NRPorlbl\DCC\KYG\6044960_l.doc

2) February 2014

2012300197 21 Feb 2014

TITLE OF THE INVENTION “Pest-controlling Agents Isolated from Spider Venom and Uses Therefor”

FIELD OF THE INVENTION [0001] This invention relates generally to agents for controlling pest 5 infestation. More particularly, the present invention relates to proteinaceous agents including peptides that increase mortality, stimulate paralysis, inhibit the development or growth rate or prevent feeding of pests such as insects as well as to compositions containing such molecules and their use in methods for controlling insects and other pests. The invention also relates to nucleic acid molecules encoding the proteinaceous agents as well as constructs, host cells, genetically modified plants and insect pathogens comprising those nucleic acid molecules.

BACKGROUND OF THE INVENTION [0002] Although only a small number of arthropods are classified as pests, they destroy around 15% of the world’s food supply and transmit a diverse array of human and animal pathogens. In Australia, insect pests cause over $3 billion of damage to crops annually. Furthermore, insect pests are responsible for the spread of diseases such as dengue fever, malaria, Chagas disease and African sleeping sickness.

[0003] Chemical insecticides have been the primary method of controlling insect pests since the advent of synthetic insecticides in the 1940s with the commercialization of DDT. In contrast with the pharmaceutical industry, however, there are few validated insecticide targets and thus few classes of chemical insecticides. A major consequence of this paucity of insecticides is that their continued use over many decades has led to the evolution of widespread resistance in many insect populations. Moreover, the poor phyletic specificity of many insecticides has negatively impacted on non-target species, including natural enemies of the targeted insect pest.

[0004] Furthermore, regulatory authorities have become increasingly concerned with the potential adverse effects of chemical insecticides on human health. Thus there has been a global push to revoke registrations for those chemical insecticides considered to have the most adverse impacts on the environment and human health. For example, over the four years from 2005 to 2009, the US Environmental Agency (EPA)

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PCT/AU2012/001000 cancelled registrations for 169 insecticidal active ingredients (AIs) whereas only nine new AIs were registered during this period.

[0005] Over the past decade, several “environmentally friendly” bioinsecticide strategies have been proposed to combat highly resistant insect pests. One recently introduced, and thus far highly successful, approach is the production of transgenic crops that express insecticidal toxins, such as engineered potato, com, and cotton crops that express delta-endotoxins from the soil bacterium Bacillus thuringiensis. An alternative bioinsecticide strategy that has been successfully fieldtrialed,.and which obviates the problem of introducing a foreign protein into the food supply, is the release of insect-specific viruses that have been engineered to express insecticidal peptide neurotoxins. A related approach is the engineering of entomopathogenic fungi to express insecticidal neurotoxins.

[0006] A number of investigators have recognized spider venoms as a possible source of insect-specific toxins for agricultural applications. A class of peptide toxins known as the omega-atracotoxins are disclosed in U.S. Pat. No. 5,763,568 as being isolated from Australian funnel-web spiders by screening the venom for “anticotton bollworm” activity. One of these compounds, designated omega-ACTX-Hvla, has been shown to selectively inhibit insect, as opposed to mammalian, voltage-gated calcium channel currents. A second, unrelated family of insect-specific peptidic calcium channel blockers is disclosed as being isolated from the same family of spiders in U.S. Pat. No. 6,583,264.

[0007] While several insecticidal peptide toxins isolated from scorpions and spiders appear to be promising leads for the development of insecticides, there still remains a significant need for compounds that act quickly and with high potency against insects, but which display a differential toxicity between insects and vertebrates.

SUMMARY OF THE INVENTION [0008] The present invention is predicated in part on the discovery of pestcontrolling peptides from the venom of Theraphosidae spiders (e.g., tarantula species including Australian tarantulas illustrative examples of which include Selenotypus species such as S', plumipes). In specific embodiments, these peptides are orally active when fed or otherwise administered to pests such as insects, and are effective in increasing pest mortality, stimulating pest paralysis, inhibiting pest developrnent or

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PCT/AU2012/001000 growth rate, or preventing pests from feeding. These discoveries have been reduced to practice in novel molecules, compositions and methods for treating or controlling pests, as described hereafter.

[0009] Accordingly, in one aspect, the present invention provides isolated or purified proteinaceous molecules for treating or controlling pests such as insects. These molecules generally comprise, consist or consist essentially of an amino acid sequence corresponding to a mature peptide or mature peptide together with an amidation signal, wherein the amino acid sequence is selected from the group consisting of:

[0010] (a) an amino acid sequence selected from:

DCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVRVGR [SEQ ID NO.4, Orally Active Insecticidal Peptide (OAIP)-1 mature peptide and amidation signal (M + A)]; DCLGQWASCEPKNSKCCPNYACTWKYPWCRYRAGK [SEQ ID NO: 12, OAIP-2 mature peptide and amidation signal (M + A)]; ECGGLMTRCDGKTTFCCSGMNCSP TWKWCVYAPGRR [SEQ ID NO:20, OAIP-3 mature peptide and amidation signal (M + A)]; YCQKWMWTCDAERKCCEDMACELWCKKRLG [SEQ ID NO:28,

OAIP-4 mature peptide]; FECVLKCDIQYNGKNCKGKGENKCSGGWRCRFKLCLK I [SEQ ID NO:36, OAIP-5 mature peptide]; FECVLKCDIKYDGKNCKGKGEKKCSG GWRCRFKLCLKI [SEQ ID NO:54, OAIP-5 Homolog-1 (Hl) mature peptide]; DCGH LHDPCPNDRPGHRTCCIGLQCRYGKCLVRV [SEQ IDNO:57, OAIP-1 mature peptide]; DCLGQWASCEPKNSKCCPNYACTWKYPWCRYRA [SEQ ID NO:58, OA IP-2 mature peptide]; and ECGGLMTRCDGKTTFCCSGMNCSPTWKWCVYAP [SE Q ID NO:59, OAIP-3 mature peptide];

[0011] (b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in any one of SEQ ID NO: 4, 12, 20, 28, 36, 54, 57, 58 or 59;

[0012J (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in any one of SEQ ID NO:3 (nucleotide sequence encoding OAIP-1 mature peptide and amidation signal), SEQ ID NO:11 (nucleotide sequence encoding OAIP-2 ' · A?

mature peptide and amidation signal), SEQ ID NO: 19 (nucleotide sequence encoding

OAIP-3 mature peptide and amidation signal), SEQ ID NO:27 (nucleotide sequence encoding OAIP-4 mature peptide) or SEQ ID NO:35 (nucleotide sequence encoding

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OAIP-5 mature peptide); SEQ ID NO:53 (nucleotide sequence encoding OAIP-5 Hl mature peptide); SEQ ID NO:60 (nucleotide sequence encoding OAIP-1 mature peptide); SEQ ID NO: 61 (nucleotide sequence encoding OAIP-2 mature peptide); or SEQ ID NO:62 (nucleotide sequence encoding OAIP-3 mature peptide);

[0013] (d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 3, 11, 19,27, 35, 53, 60, 61 or 62, or a complement thereof; or [0014] (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 3,11,19, 27, 35, 53, 60, 61 or 62, or a complement thereof, [0015] wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0016] In some embodiments, the proteinaceous molecules further comprise an amino acid sequence corresponding to a propeptide region, wherein the amino acid sequence is selected from the group consisting of:

[0017] (a) an amino acid sequence selected from:

DTEDADLMEMVQLSRPFFNPIIRAVELVELREERQR [SEQ ID NO:6, OAIP-1 propeptide region]; SEMKERSSFNEVLSEFFAADEPQER [SEQ ID NO: 14, OAIP-2 propeptide region]; VELEETGR [SEQ ID NO:22, OAIP-3 propeptide region]; EDQFA SPNELLKSMFVESTHELTPEVEGR [SEQ ID NO:30, OAIP-4 propeptide region]; and

EELEAKDVIESKALATLDEER [SEQ ID NO:38, OAIP-5 and OAIP-5 Hl propeptide region];

[0018] (b) an amino acid sequence that shares at least 70% (and at least 71% }

to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in any one of SEQ ID NO: 6, 14, 22, 30 or 38; or [0019] (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in any one of SEQ ID NO :5 (nucleotide sequence encoding OAIP-1 propeptide

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PCT/AU2012/001000 region), SEQ ID NO: 13 (nucleotide sequence encoding OAIP-2 propeptide region),

SEQ ID NO:21 (nucleotide sequence encoding OAIP-3 propeptide region), SEQ ID NO:29 (nucleotide sequence encoding OAIP-4 propeptide region) or SEQ ID NO:37 (nucleotide sequence encoding OAIP-5 and OAIP-5 Hl propeptide region);

[0020] (d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 5,13, 21,29 or 37, or a complement thereof; or [0021] (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 5, 13,21,29 or 37, or a complement thereof, [0022] wherein the proteinaceous molecule further comprising the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0023] In some embodiments, the proteinaceous molecules further comprise an amino acid sequence corresponding to a signal peptide, wherein the amino acid sequence is selected from the group consisting of:

[0024] (a) an amino acid sequence selected from:

MIFLLPSIISVMLLAEPVLMLG [SEQ ID NO:8, OAIP-1 signal peptide]; MRVLFIIA GLALLSVVCYT [SEQ ID NO: 16, OAIP-2 signal peptide]; MKTSVLFAILGLALLFC LSFG [SEQ ID NO:24, OAIP-3 signal peptide]; MKASLFAVIFGLVVLCACSFA [SEQ ID NO:32, OAIP-4 signal peptide region]; and MLIVILTCALLVIYHAAAA [SEQ ID NO:40, OAIP-5 and OAIP-5 Hl signal peptide];

[0025] (b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in any one of SEQ ID NO: 8,16, 24, 32 or 40; or [0026] (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in any one of SEQ ID NO:7 (nucleotide sequence encoding OAIP-1 signal peptide), SEQ ID NO: 15 (nucleotide sequence encoding OAIP-2 signal peptide), SEQ

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ID NO:23 (nucleotide sequence encoding OAIP-3 signal peptide), SEQ ID NO:31 (nucleotide sequence encoding OAIP-4 signal peptide region) or SEQ ID NO:39 (nucleotide sequence encoding OAIP-5 and OAIP-5 Hl signal region);

[0027] (d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 7, 15, 23, 31 or 39, or a complement thereof; or ' [0028] (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 7,15,23, 31 or 39, or a complement thereof, [0029] wherein the proteinaceous molecule further comprising the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

. [0030] In specific embodiments, the proteinaceous molecules comprise, consist or consist essentially of an amino acid sequence corresponding to a precursor peptide including the propeptide region sequence (P) plus the mature peptide sequence (M) or mature peptide sequence together with an amidation signal (M + A), wherein the amino acid sequence is selected from the group consisting of:

[0031] (a) an amino acid sequence selected from:

DTEDADLMEMVQLSRPFFNPIIRAVELVELREERQRDCGHLHDPCPNDRPGHRT CCIGLQCRYGKCLVRVGR [SEQ ID NO:42, OAIP-1 P + M + A]; SEMKERSSFNE VLSEFFAADEPQERDCLGQWASCEPKNSKCCPNYACTWKYPWCRYRAGK [SEQ ID NO:44, OAIP-2 P + M + A]; VELEETGRECGGLMTRCDGKTTFCCSGMN

CSPTWKWCVYAPGRR [SEQ ID NO:46, OAIP-3 P + M + A]; EDQFASPNELLKS MFVESTHELTPEVEGRYCQKWMWTCDAERKCCEDMACELWCKKRLG [SEQ ID NO:48, OAIP-4 P + M]; EELEAKDVIESKALATLDEERFECVLKCDIQYNGKNC KGKGENKCSGGWRCRFKLCLKI [SEQ ID NO:50, OAIP-5 P + M]; EELEAKDVIE

SKALATLDEERFECVLKCDIKYDGKNCKGKGEKKCSGGWRCRFKLCLKI [SEQ

ID NO:56, OAIP-5 Hl P + M]; DTEDADLMEMVQLSRPFFNPIIRAVELVELREERQ RDCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVRV [SEQ ID NO:63, OAIP-1

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P + Μ]; SEMKERSSFNEVLSEFF AADEPQERDCLGQWASCEPKNSKCCPNY ACT WKYPWCRYRA [SEQ ID NO:64, OAIP-2 P + M]; and VELEETGRECGGLMTRCD GKTTFCCSGMNCSPTWKWCVYAP [SEQ ID NO:65, OAIP-3 P + M];

[0032] (b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in any one of SEQ ID NO: 42,44,46,48, 50, 56,

63,64 or 65; or [0033] (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in any one of SEQ ID NO:41 (nucleotide sequence encoding OAIP-1 P + M +

A), SEQ ID NO:43 (nucleotide sequence encoding OAIP-2 P + M + A), SEQ ID NO:45 (nucleotide sequence encoding OAIP-3 P + M + A), SEQ ID NO:47 (nucleotide sequence encoding OAIP-4 P + M), SEQ ID NO:49 (nucleotide sequence encoding OAIP-5 P + M) SEQ ID NO:55 (nucleotide sequence encoding OAIP-5 Hl P + M);

SEQ ID NO:66 (nucleotide sequence encoding OAIP-1 P + M); SEQ ID NO:67 (nucleotide sequence encoding OAIP-2 P + M) or SEQ ID NO:68 (nucleotide sequence encoding OAIP-3 P + M);

[0034] (d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in _zbetween) sequence identity with the sequence set forth in any one of SEQ ID NO: 41,

43,45,47,49, 55,66,67 or 68 or a complement thereof; or [0035] (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 41,43,45, 47,49, 55, 66, 67 or 68, or a complement thereof, [0036] wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0037] In other embodiments, the proteinaceous molecules comprise, consist or consist essentially of an amino acid sequence corresponding to a prepropeptide that includes the signal peptide (S) plus the propeptide region (P) plus the mature peptide

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PCT/AU2012/001000 (M) or the mature peptide together with an amidation sequence, wherein the amino acid sequence is selected from the group consisting of:

[0038] (a) an amino acid sequence selected from:

MIFLLPSIISVMLLAEPVLMLGDTEDADLMEMVQLSRPFFNPIIRAVELVELREE RQRDCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVRVGR [SEQ ID NO:2, OAIP-1 S + P + M + A]; MRVLFIIAGLALLSVVCYTSEMKERSSFNEVLSEFFAAD EPQERDCLGQWASCEPKNSKCCPNYACTWKYPWCRYRAGK [SEQ ID NO: 10, OAIP-2 S + P + M + A]; MKTSVLFAILGLALLFCLSFGVELEETGRECGGLMTRC DGKTTFCCSGMNCSPTWKWCVYAPGRR [SEQ ID NO: 18, OAIP-3 S + P + M + A]; MKASLFAVIFGLVVLCACSFAEDQFASPNELLKSMFVESTHELTPEVEGRYC QKWMWTCDAERKCCEDMACELWCKKRLG [SEQ ID NO:26, OAIP-4 S + P +

M]; MLIVILTCALLVIYHAAAAEELEAKDVIESKALATLDEERFECVLKCDIQYN GKNCKGKGENKCSGGWRCRFKLCLKI [SEQ ID NO:34, OAIP-5 S + P + M]; MLI VILTCALLVIYHAAAAEELEAKDVIESKALATLDEERFECVLKCDIKYDGKNCK GKGEKKCSGGWRCRFKLCLKI [SEQ ID NO:52, OAIP-5 Hl S + P + M]; MIFLLPS IISVMLLAEPVLMLGDTEDADLMEMVQLSRPFFNPIIRAVELVELREERQRDCG HLHDPCPNDRPGHRTCCIGLQCRYGKCLVRV [SEQ ID NO:69, OAIP-1 S + P + M]; MRVLFIIAGLALLSVVCYTSEMKERSSFNEVLSEFFAADEPQERDCLGQWA SCEPKNSKCCPNYACTWKYPWCRYRA [SEQ ID NO:70, OAIP-2 S + P + M]; and MKTSVLFAILGLALLFCLSFGVELEETGRECGGLMTRCDGKTTFCCSGMNCSPT WKWCVYAP [SEQ ID NO:71, OAIP-3 S + P + M];

[0039] (b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in any one of SEQ ID NO: 2,10, 18,26, 34, 52, 69, 70 or 71; or [0040] (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in any one of SEQ ID NO:1 (nucleotide sequence encoding OAIP-1 S + P + M + A), SEQ ID NO:9 (nucleotide sequence encoding OAIP-2 S + P + M + A), SEQ ID NO: 17 (nucleotide sequence encoding OAIP-3 S + P + M + A), SEQ ID NO:25 (nucleotide sequence encoding OAIP-4 S + P + M) SEQ ID NO:33 (nucleotide sequence encoding OAIP-5 S + P + M); SEQ ID NO:51 (nucleotide sequence encoding OAIP-5 Hl S + P + M); SEQ ID NO:72 (nucleotide sequence encoding OAIP-1 S + P +

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PCT/AU2012/001000

M); SEQ ID NO:73 (nucleotide sequence encoding OAIP-2 S + P + M) or SEQ ID NO:74 (nucleotide sequence encoding OAIP-3 S + P + M);

[0041J (d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 9, 17,25, 33, 51 72, 73 or 74, or a complement thereof; or . [0042] (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 1,9, 17, 25, 33, 51, 72, 73 or 74 or a complement thereof, [0043] wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0044] Another aspect of the present invention provides isolated nucleic acid molecules that comprise, consist or consist essentially of a nucleotide sequence encoding the amino acid sequence of a proteinaceous molecule as broadly defined above. In some embodiments, the nucleic acid molecules comprise, consist or consist essentially of a nucleotide sequence encoding an amino acid sequence corresponding to a mature peptide or mature peptide together with an amidation signal, wherein the nucleotide sequence is selected from the group consisting of:

[0045] (a) a nucleotide sequence selected from: gactgtggtcacctgcacgatccatgtc ctaatgatcgtcctggacaccgtacgtgctgcataggactccagtgcagatacggtaaatgcctcgtgcgggttggtcgc [SEQ ID NO:3, nucleotide sequence encoding OAIP-1 mature peptide and amidation

5 signal]; gactgtctaggacagtgggccagttgtgaacctaagaacagcaagtgctgccccaactatgcatgtacttggaaat acccttggtgcagatatcgcgctggtaaatag [SEQ ID NO:11, nucleotide sequence encoding OAIP-2 mature peptide and amidation signal]; gagtgtgggggactaatgacccgctgtgatggaaagac aacgttttgctgttcaggtatgaattgttctccaacgtggaaatggtgtgtctatgctcctggacgccgttga [SEQ ID NO: 19, nucleotide sequence encoding OAIP-3 mature peptide and amidation signal]; tattgcc aaaaatggatgtggacctgtgatgcagaaagaaaatgctgcgaagacatggcttgcgaactgtggtgcaaaaagagactcgg a [SEQ ID NO:27, nucleotide sequence encoding OAIP-4 mature peptide]; ttcgaatgtgtttt gaaatgcgacattcaatacaatgggaaaaattgtaagggcaaaggagagaacaaatgttcaggaggatggagatgccgtttta

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PCT/AU2012/001000 aattgtgtctgaaaatataa [SEQ ID NO:35, nucleotide sequence encoding OAIP-5 mature peptide]; ttcgaatgtgttttgaaatgcgacattaaatacgatgggaaaaattgtaagggcaaaggagagaagaaatgttca ggaggatggagatgccgttttaaattgtgtctgaaaata [SEQ ID NO:53, nucleotide sequence encoding OAIP-5 Hl mature peptide]; gactgtggtcacctgcacgatccatgtcctaatgatcgtcctggacaccgtacgtg ctgcataggactccagtgcagatacggtaaatgcctcgtgcgggtt [SEQ ID NO:60, nucleotide sequence encoding OAIP-1 mature peptide]; gactgtctaggacagtgggccagttgtgaacctaagaacagcaagtgct gccccaactatgcatgtacttggaaatacccttggtgcagatatcgcgct [SEQ ID NO;61, nucleotide sequence encoding OAIP-2 mature peptide]; and gagtgtgggggactaatgacccgctgtgatggaaag acaacgttttgctgttcaggtatgaattgttctccaacgtggaaatggtgtgtctatgctcct [SEQ ID NO:62, nucleotide sequence encoding OAIP-3 mature peptide];

[0046] (b) a nucleotide sequence that shares at least 70% (and at least 71 % to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 3,11,19, 27, 35, 53, 60, 61 or 62, or a complement thereof;

[0047] (c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 3,11,

19,27,35, 53,60, 61 or 62, or a complement thereof, [0048] wherein the amino acid sequence encoded by the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of:

being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0049] In some embodiments, the nucleic acid molecules further comprise a nucleotide sequence encoding an amino acid sequence corresponding to a propeptide region, wherein the nucleotide sequence is selected from the group consisting of:

[0050] (a) a nucleotide sequence selected from: gataccgaagatgcagatttgatggaga tggttcagttgtctagaccatttttcaatcccattatccgagctgttgaacttgtggaactacgtgaagaaagacaaaga [SEQ ID NO;5, nucleotide sequence encoding OAIP-1 propeptide region]; tccgagatgaaggagcg aagctcatttaatgaagtgctttcggagttttttgctgccgacgagcctcaggaaaga [SEQ ID NO: 13, nucleotide sequence encoding OAIP-2 propeptide region]; gttgaattggaagagaccggaagg [SEQ ID

NO:21, nucleotide sequence encoding OAIP-3 propeptide region]; gaagatcaatttgcttcgcct aatgaactgctgaaatcaatgtttgtggagagtacacatgaactcacacctgaagtggaaggaaga [SEQ ID NO:29,

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PCT/AU2012/001000 nucleotide sequence encoding OAIP-4 propeptide region]; and gaggaacttgaagcaaaagatgt gatagaatctaaagcactagcaactctggatgaagaaaga [SEQ ID NO:37, nucleotide sequence encoding OAIP-5 and OAIP-5 Hl propeptide region];

[0051] (b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 5, 13, 21, 29 or 37, or a complement thereof;

[0052] (c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 5,13,

21,29 or 37, or a complement thereof, [0053] wherein the amino acid sequence encoded by the nucleic acid molecule further comprising the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0054] In some embodiments, the nucleic acid molecules further comprise a nucleotide sequence encoding an amino acid sequence corresponding to a signal peptide, wherein the nucleotide sequence is selected from the group consisting of: .

[0055] (a) a nucleotide sequence selected from: atgatatttctactaccttcgatcatttctgt tatgcttttggccgagcctgtcctaatgcttgga [SEQ ID NO:7, nucleotide sequence encoding

OAIP-1 signal peptide]; atgagggttctgttcatcattgccggattagccctgctttccgttgtttgctacact [SEQ ID NO: 15, nucleotide sequence encoding OAIP-2 signal peptide]; atgaagacatcagttttattcg ccatcttgggattggctctgcttttctgcctttcatttgga [SEQ ID NO:23, nucleotide sequence encoding OAIP-3 signal peptide]; atgaaggcttcactattcgctgtcatatttggattggttgtgttgtgcgcctgctcctttgcc [SEQ ID NO:31, nucleotide sequence encoding OAIP-4 signal peptide]; and atgttgattgtc attctgacatgtgctctgttggttatttatcacgccgcagcagcg [SEQ ID NO:39, nucleotide sequence encoding OAIP-5 and OAIP-5 Hl signal peptide];

[0056] (b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 7,15, 23, 31 or 39, or a complement thereof;

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PCT/AU2012/001000 [0057] (c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 7, 15,

23, 31 or 39, or a complement thereof, [0058] wherein the amino acid sequence encoded by the nucleic acid 5 molecule further comprising the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0059] In other embodiments, the nucleic acid molecules comprise, consist or 10 consist essentially of a nucleotide sequence encoding an amino acid sequence corresponding to a precursor peptide including the propeptide region (P) and the mature peptide (M) or the mature peptide together with an amidation signal, wherein the nucleotide sequence is selected from the group consisting of: · (a) a nucleotide sequence selected from: gataccgaagatgcagatttgatggagatggttcagttgtctagac 15 catttttcaatcccattatccgagctgttgaacttgtggaactacgtgaagaaagacaaagagactgtggtcacctgcacgatcc atgtcctaatgatcgtcctggacaccgtacgtgctgcataggactccagtgcagatacggtaaatgcctcgtgcgggttggtcg ctag [SEQ ID NO:41, nucleotide sequence encoding OAIP-1 P +M + A]; tccgagatgaagg agcgaagctcatttaatgaagtgctttcggagttttttgctgccgacgagcctcaggaaagagactgtctaggacagtgggcca gttgtgaacctaagaacagcaagtgctgccccaactatgcatgtacttggaaatacccttggtgcagatatcgcgctggtaaata g [SEQ ID NO:43, nucleotide sequence encoding OAIP-2 P +M + A]; gttgaattggaagaga ccggaagggagtgtgggggactaatgacccgctgtgatggaaagacaacgttttgctgttcaggtatgaattgttctccaacgt ggaaatggtgtgtctatgctcctggacgccgttga [SEQ ID NO:45, nucleotide sequence encoding OAIP-3 P +M + A]; gaagatcaatttgcttcgcctaatgaactgctgaaatcaatgtttgtggagagtacacatgaactc acacctgaagtggaaggaagatattgccaaaaatggatgtggacctgtgatgcagaaagaaaatgctgcgaagacatggctt gcgaactgtggtgcaaaaagagactcggataa [SEQ ID NO:47, nucleotide sequence encoding

OAIP-4 P + M]; and gaggaacttgaagcaaaagatgtgatagaatctaaagcaCtagcaactctggatgaagaaag attcgaatgtgttttgaaatgcgacattcaatacaatgggaaaaattgtaagggcaaaggagagaacaaatgttcaggaggatg gagatgccgttttaaattgtgtctgaaaatataa [SEQ ID NO:49, nucleotide sequence encoding OAIP-5 P +M] ;gaggaacttgaagcaaaagatgtgatagaatctaaagcactagcaactctggatgaagaaagattcga atgtgttttgaaatgcgacattaaatacgatgggaaaaattgtaagggcaaaggagagaagaaatgttcaggaggatggagat gccgttttaaattgtgtctgaaaata [SEQ ID NO:55, nucleotide sequence encoding OAIP-5 Hl P + M]; gataccgaagatgcagatttgatggagatggttcagttgtctagaccatttttcaatcccattatccgagctgttgaac

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PCT/AU2012/001000 ttgtggaactacgtgaagaaagacaaagagactgtggtcacctgcacgatccatgtcctaatgatcgtcctggacaccgtacgt gctgcataggactccagtgcagatacggtaaatgcctcgtgcgggtt [SEQ ID NO:66, nucleotide sequence encoding OAIP-1 P + M]; tccgagatgaaggagcgaagctcatttaatgaagtgctttcggagttttttgctgccga cgagcctcaggaaagagactgtctaggacagtgggccagttgtgaacctaagaacagcaagtgctgccccaactatgcatgt acttggaaatacccttggtgcagatatcgcgct [SEQ ID NO:67, nucleotide sequence encoding

OAIP-2 P + M]; and gttgaattggaagagaccggaagggagtgtgggggactaatgacccgctgtgatggaaaga caacgttttgctgttcaggtatgaattgttctccaacgtggaaatggtgtgtctatgctcct [SEQ ID NO:68, nucleoti de sequence encoding OAIP-3 P + M];

[0060] (b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 41, 43,45,47, 49, 55,66, 67 or 68, or a complement thereof;

[0061] (c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 41,43,

45,47,49, 55, 66, 67 or 68, or a complement thereof, [0062] wherein the amino acid sequence encoded by the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of: being orally active against pests such as insects, increasing mortality of pests, stimulating paralysis of pests, inhibiting development or growth rate of pests, or preventing pests from feeding.

[0063] In other embodiments, the nucleic acid molecules comprise, consist or consist essentially of a nucleotide sequence encoding an amino acid sequence corresponding to a prepropeptide including the signal peptide (S), propeptide region (P) and mature peptide (M) or mature peptide together with an amidation signal, wherein the nucleotide sequence is selected from the group consisting of: ^: [0064] (a) a nucleotide sequence selected from: atgatatttctactaccttcgatcatttctgt tatgcttttggccgagcctgtcctaatgcttggagataccgaagatgcagatttgatggagatggttcagttgtctagaccatttttc aatcccattatccgagctgttgaacttgtggaactacgtgaagaaagacaaagagactgtggtcacctgcacgatccatgtcct aatgatcgtcctggacaccgtacgtgctgcataggactccagtgcagatacggtaaatgcctcgtgcgggttggtcgctag [SEQ ID NO: 1, nucleotide sequence encoding OAIP-1 S + P + M + A]; atgagggttctgttca tcattgccggattagccctgctttccgttgtttgctacacttccgagatgaaggagcgaagctcatttaatgaagtgctttcggagt tttttgctgccgacgagcctcaggaaagagactgtctaggacagtgggccagttgtgaacctaagaacagcaagtgctgcccc

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PCT/AU2012/001000 aactatgcatgtacttggaaatacccttggtgcagatatcgcgctggtaaatag [SEQ ID N0:9, nucleotide sequence encoding OAIP-2 S + P + M + A]; atgaagacatcagttttattcgccatcttgggattggctctgc ttttctgcctttcatttggagttgaattggaagagaccggaagggagtgtgggggactaatgacccgctgtgatggaaagacaa cgttttgctgttcaggtatgaattgttctccaacgtggaaatggtgtgtctatgctcctggacgccgttga [SEQ ID

NO: 17, nucleotide sequence encoding OAIP-3 S + P + M + A]; atgaaggcttcactattcgctgtc atatttggattggttgtgttgtgcgcctgctcctttgccgaagatcaatttgcttcgcctaatgaactgctgaaatcaatgtttgtgga gagtacacatgaactcacacctgaagtggaaggaagatattgccaaaaatggatgtggacctgtgatgcagaaagaaaatgct gcgaagacatggcttgcgaactgtggtgcaaaaagagactcggataa [SEQ ID NO:25, nucleotide sequenc e encoding OAIP-4 S + P + M]; atgttgattgtcattctgacatgtgctctgttggttatttatcacgccgcagcagc ggaggaacttgaagcaaaagatgtgatagaatctaaagcactagcaactctggatgaagaaagattcgaatgtgttttgaaatg cgacattcaatacaatgggaaaaattgtaagggcaaaggagagaacaaatgttcaggaggatggagatgccgttttaaattgt gtctgaaaatataa [SEQ ID NO:33, nucleotide sequence encoding OAIP-5 S + P + M]; atgttgattgtcattctgacatgtgctctgttggttatttatcacgccgcagcagcggaggaacttgaagcaaaagatgtgataga atctaaagcactagcaactctggatgaagaaagattcgaatgtgttttgaaatgcgacattaaatacgatgggaaaaattgtaag ggcaaaggagagaagaaatgttcaggaggatggagatgccgttttaaattgtgtctgaaaata [SEQ ID NO: 51, nucleotide sequence encoding OAIP-5 Hl S + P + M]; atgatatttctactaccttcgatcatttctgttat gcttttggccgagcctgtcctaatgcttggagataccgaagatgcagatttgatggagatggttcagttgtctagaccatttttcaa tcccattatccgagctgttgaacttgtggaactacgtgaagaaagacaaagagactgtggtcacctgcacgatccatgtcctaat gatcgtcctggacaccgtacgtgctgcataggactccagtgcagatacggtaaatgcctcgtgcgggtt [SEQ ID

NO:72, nucleotide sequence encoding OAIP-1 S + P + M]; atgagggttctgttcatcattgccggatt agccctgctttccgttgtttgctacacttccgagatgaaggagcgaagctcatttaatgaagtgctttcggagttttttgctgccga cgagcctcaggaaagagactgtctaggacagtgggccagttgtgaacctaagaacagcaagtgctgccccaactatgcatgt acttggaaatacccttggtgcagatatcgcgct [SEQ ID NO:73, nucleotide sequence encoding OAIP-2 S + P + M]; and atgaagacatcagttttattcgccatcttgggattggctctgcttttctgcctttcatttggagt tgaattggaagagaccggaagggagtgtgggggactaatgacccgctgtgatggaaagacaacgttttgctgttcaggtatga attgttctccaacgtggaaatggtgtgtctatgctcct [SEQ ID NO:74, nucleotide sequence encoding OAIP-3 S + P + M];

[0065] (b) a nucleotide sequence that shares at least 70% (and at least 71 % to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1,9, 17, 25, 33, 51, 72, 73 or 74, or a complement thereof;

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PCT/AU2012/001000 [0066] (c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in any one of SEQ ID NO: 1,9, 17, 25, 33,51, 72, 73 or 74, or a complement thereof, [0067] wherein the amino acid sequence encoded by the nucleotide sequence 5 of (a), (b) or (c) has any one or more activities selected from the group consisting of:

[0068] The nucleic acid molecules as broadly described above may be used to 10 design specific oligonucleotide probes and primers for detecting and isolating homologous or orthologous nucleic acid molecules. Thus, in yet another aspect, the present invention provides probes for interrogating nucleic acid for the presence of a nucleic acid molecule as broadly described above. These probes generally comprise, consist or consist essentially of a nucleotide sequence that hybridizes under at least medium or high stringency conditions to a nucleic acid molecule as broadly described above. In some embodiments, the probes consist essentially of a nucleic acid sequence which corresponds or is complementary to at least a portion of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 2,4, 6, 8, 10, 12, 14,16,18,20, 22,24, 26,28, 30, 32, 34, 36, 38,40,42, 44,46, 48, 50, 52, 54, 56, 57,

58, 59,63, 64, 65,69, 70 or 71, wherein the portion is at least 15 nucleotides in length.

In illustrative examples of this type, the probes comprise, consist or consist essentially of a nucleotide sequence that is capable of hybridizing to at least a portion of any one of SEQ ID NO: 1,3,5, 7, 9, 11,13, 15, 17, 19,21,23,25, 28,29,31,33, 35,37, 39,41,

43,45,47, 49, 51, 53, 55, 60, 61, 62, 66, 67, 68, 72, 73 or 74 under at least medium or high stringency conditions, wherein the portion is at least 15 nucleotides in length.

[0069] The present invention also contemplates antigen-binding molecules that are immuno-reactive with the proteinaceous molecules of the invention, which can suitably used to screen organisms, especially theraphosids, for structurally and/or functionally related pest-controlling peptides and polypeptides. Accordingly, in still another aspect, the present invention provides antigen-binding molecules that are immuno-reactive with a proteinaceous molecule as broadly described above.

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PCT/AU2012/001000 [0070] In specific embodiments according to any of the foregoing aspects, the pests are insects (e.g,, crickets, flies, mealworms, mosquitoes, termites, etc.) or arachnids (e.g., ticks and mites).

[0071] Still another aspect of the present invention provides constructs for expressing the nucleic acid molecules broadly described above (e.g., for making recombinant proteinaceous molecules in commercial quantities or for expressing the nucleic acid molecules in microbial or plant hosts for controlling insects). These constructs generally comprise a nucleic acid molecule as broadly described above operably connected to a regulatory sequence. The constructs may be introduced into insects (e.g., via insect vectors such as baculovirus and entomopoxvirus), into microorganisms known to inhabit the habitat of insects (e.g., bacteria, algae, and fungi) or into plant cells (e.g., for increasing resistance of plants, including crops such as cotton, tomato, green bean, sweet com, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, sunflower, and field lupins, to infestation by insects).

[0072] In a related aspect, the present invention provides viruses or host cells that contain a construct as broadly described above.

[0073] In another aspect, the present invention provides fusion proteins comprising a proteinaceous molecule of the invention and a non-theraphosid peptide or polypeptide, especially wherein the non-TPC peptide or polypeptide is Galanthus nivalis agglutinin which improves oral activity of the proteinaceous molecule of the invention.

[0074] The proteinaceous molecules, nucleic acid molecules, constructs, viruses, fusion proteins and host cells of the present invention (also referred to collectively herein as “theraphosid pest-controlling agents” or “TPC agents”) useful for controlling harmful, annoying or undesired pests (e.g., insects). They may be used singularly or in combination with other pest-controlling agents, including the TPC agents of the present invention. Thus, in another aspect, the present invention provides compositions comprising at least one theraphosid pest-controlling agent as broadly described above and optionally an agriculturally acceptable carrier, diluent and/or excipient. In specific embodiments, the compositions are formulated for oral delivery to pests such as insects. In illustrative examples of this type, the at least one TPC agent is in intimate admixture with an insect food. In some embodiments, the at least one TPC

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PCT/AU2012/001000 agent is formulated with an attractant for attracting the insects to the composition. In some embodiments, the at least one TPC agent is formulated with another pesticide or an agent that enhances the activity of the TPC agent.

[0075] In a related aspect, the present invention provides methods for controlling pests, including combating or eradicating infestations of plants, plant products, land and waterways by pests such as insects. These methods generally comprise administering to a plant or plant part, product or site having or at risk of developing an pest infestation an effective amount of a theraphosid pest-controlling agent as broadly described above optionally in combination with other pesticides.

[0076] In another aspect of the present invention there is provided a method for controlling ectoparasite pests, including fleas, ticks and mites. The method generally comprising administering to the dermis of an animal having or at risk of developing an ectoparasite infestation, an effective amount of a theraphosid pestcontrolling agent as broadly described above, optionally in combination with other pesticides.

Table 1

Brief Description of the Sequences

SEQUENCE ID NUMBER	SEQUENCE	LENGTH
SEQ ID NO:1	Nucleotide sequence from S. plumipes, which encodes OAIP-1 prepropeptide and amidation signal (S + P + M + A)	285 nts
SEQ ID NO:2	Peptide encoded by SEQ ID NO: 1	94 aa
SEQ ID NO:3	Nucleotide sequence from S. plumipes, which encodes OAIP-1 mature peptide and amidation signal (M + A)	108 nts
SEQ ID NO:4	Peptide encoded by SEQ ID NO:3	36 aa
SEQ ID NO:5	Nucleotide sequence from 5. plumipes, which encodes OAIP-1 propeptide region (P)	108 nts
SEQ ID NO:6	Peptide encoded by SEQ ID NO:5	36 aa
SEQ ID NO:7	Nucleotide sequence from S. plumipes, which encodes OAIP-1 signal peptide (S)	66 nts .

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PCT/AU2012/001000

SEQUENCE ID NUMBER	SEQUENCE	LENGTH
SEQ ID NO:8	Peptide encoded by SEQ ID NO:7	22 aa
SEQ ID NO:9	Nucleotide sequence from 5. plumipes, which encodes OAIP-2 prepropeptide and amidation signal (S + P + M + A)	240 nts
SEQ ID NO: 10	Peptide encoded by SEQ ID NO:9	79 aa
SEQ ID NO: 11	Nucleotide sequence from 5. plumipes, which encodes OAIP-2 mature peptide and amidation signal (M + A)	108 nts
SEQ ID NO: 12	Peptide encoded by SEQ ID NO: 11	35 aa
SEQ ID NO: 13	Nucleotide sequence from S. plumipes, which encodes OAIP-2 propeptide region (P)	75 nts
SEQ ID NO: 14	Peptide encoded by SEQ ID NO: 13	25 aa
SEQ ID NO: 15	Nucleotide sequence from S', plumipes, which encodes OAIP-2 signal peptide (S)	57 nts
SEQ ID NO: 16	Peptide encoded by SEQ ID NO: 15	19 aa
SEQ ID NO: 17	Nucleotide sequence from S. plumipes, which encodes OAIP-3 prepropeptide and amidation signal (S + P + M + A)	198 nts
SEQ ID NO: 18	Peptide encoded by SEQ ID NO: 17	65 aa
SEQ ID NO: 19	Nucleotide sequence from S. plumipes, which encodes OAIP-3 mature peptide and amidation signal (M + A)	111 nts
SEQ ID NO:20	Peptide encoded by SEQ ID NO: 19	36 aa
SEQ ID NO:21	Nucleotide sequence from S. plumipes, which encodes OAIP-3 propeptide region (P)	24 nts
SEQ ID NO:22	Peptide encoded by SEQ ID NO:21	8 aa
SEQ ID NO:23	Nucleotide sequence from S. plumipes, which encodes OAIP-3 signal peptide (S)	63 nts
SEQ ID NO:24	Peptide encoded by SEQ ID NO:23	21 aa
SEQ ID NO:25	Nucleotide sequence from S. plumipes, which encodes OAIP-4 prepropeptide (S + P + M)	243 nts

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PCT/AU2012/001000

SEQUENCE ID NUMBER	SEQUENCE	LENGTH
SEQ ID NO:26	Peptide encoded by SEQ ID NO:25	80 aa
SEQ ID NO:27	Nucleotide sequence from S. plumipes, which encodes OAIP-4 mature peptide (M)	90 nts
SEQ ID NO:28	Peptide encoded by SEQ ID NO:27	30 aa
SEQ ID NO:29	Nucleotide sequence from S. plumipes, which encodes OAIP-4 propeptide region (P)	87 nts
SEQ ID NO:30	Peptide encoded by SEQ ID NO:29	29 aa
SEQ IDNO:31	Nucleotide sequence from S. plumipes, which encodes OAIP-4 signal peptide (S)	63 nts
SEQ ID NO:32	Peptide encoded by SEQ ID NO:31	21 aa
SEQ ID NO:33	Nucleotide sequence from S. plumipes, which encodes OAIP-5 prepropeptide (S + P + M)	237 nts
SEQ ID NO:34	Peptide encoded by SEQ ID NO:33	78 aa
SEQ ID NO:35	Nucleotide sequence from 5¹. plumipes, which encodes OAIP-5 mature peptide (M)	117 nts
SEQ ID NO:36	Peptide encoded by SEQ ID NO:35	38 aa
SEQ ID NO:37	Nucleotide sequence from S', plumipes, which encodes OAIP-5 and OAIP-5 Hl propeptide region (P)	63 nts
SEQ IDNO:38	Peptide encoded by SEQ ID NO:37	21 aa
SEQ ID NO:39	Nucleotide sequence from S. plumipes, which encodes OAIP-5 and OAIP-5 Hl signal peptide (S)	57 nts
SEQ IDNO:40	Peptide encoded by SEQ ID NO:39	19 aa
SEQ ID N0:41	Nucleotide sequence from S. plumipes, which encodes OAIP-1 propeptide region and mature peptide and amidation signal (P + M + A)	219 nts
SEQIDNO:42	Peptide encoded by SEQ ID NO:41	72 aa
SEQ ID NO:43	Nucleotide sequence from S. plumipes, which encodes OAIP-2 propeptide region and mature peptide and amidation signal (P + M + A)	183 nts

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PCT/AU2012/001000

SEQUENCE ID NUMBER	SEQUENCE	LENGTH
SEQ ID NO:44	Peptide encoded by SEQ ID NO:43	60 aa
SEQ ID NO:45	Nucleotide sequence from 5¹. plumipes, which encodes OAIP-3 propeptide region and mature peptide and amidation signal (P + M + A)	135 nts
SEQ ID NO:46	Peptide encoded by SEQ ID NO:45	44 aa
SEQ ID NO:47	Nucleotide sequence from S. plumipes, which encodes OAIP-4 propeptide region and mature peptide (P + M)	180 nts
SEQ ID NO-.48	Peptide encoded by SEQ ID NO:47	59 aa
SEQ ID NO:49	Nucleotide sequence from 5. plumipes, which encodes OAIP-5 propeptide region and mature peptide (P + M)	180 nts
SEQ ID NO:50	Peptide encoded by SEQ ID NO:49	59 aa
SEQ ID N0:51	Nucleotide sequence from S. plumipes, which encodes OAIP-5 Homolog-1 prepropeptide (S + P + M)	234 nts
SEQ ID NO:52	Peptide encoded by SEQ ID NO:51	78 aa
SEQIDNO:53	Nucleotide sequence from 5¹. plumipes, which encodes OAIP-5 Homolog-1 mature peptide (M)	114 nts
SEQ ID NO:54	Peptide encoded by SEQ ID NO:53	38 aa
SEQ ID NO:55	Nucleotide sequence from & plumipes, which encodes OAIP-5 Homolog-1 propeptide region and mature peptide (P + M)	177 nts
SEQ ID NO:56	Peptide encoded by SEQ ID NO:55	59 aa
SEQIDNO:57	Peptide encoded by.SEQ ID NO:60	34 aa
SEQ ID NO:58	Peptide encoded by SEQ ID NO:61	33 aa
SEQ ID NO:59	Peptide encoded by SEQ ID NO:62	33 aa
SEQ ID NO:60	Nucleotide sequence from 5. plumipes, which encodes OAIP-1 mature peptide (M)	102 nts
SEQ ID N0:61	Nucleotide sequence from S. plumipes, which encodes OAIP-2 mature peptide (M)	99 nts
SEQ ID NO:62	Nucleotide sequence from S. plumipes, which encodes	99 nts

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PCT/AU2012/001000

SEQUENCE ID NUMBER	SEQUENCE	LENGTH
	OAIP-3 mature peptide (M)
SEQ IDNO:63	Peptide encoded by SEQ ID NO :66	70 aa
SEQ ID NO:64	Peptide encoded by SEQ ID NO:67	58 aa
SEQ ID NO:65	Peptide encoded by SEQ ID NO :68	41 aa
SEQ ID NO:66	Nucleotide sequence from S. plumipes, which encodes OAIP-1 propeptide region and mature peptide (P +M)	210 nts
SEQ ID NO:67	Nucleotide sequence from S', plumipes, which encodes OAIP-2 propeptide region and mature peptide (P +M)	174 nts
SEQ ID NO:68	Nucleotide sequence from S. plumipes, which encodes OAIP-3 propeptide region and mature peptide (P +M)	123 nts
SEQ ID NO:69	Peptide encoded by SEQ ID NO:72	92 aa
SEQ ID NO:70	Peptide encoded by SEQ ID NO:73	77 aa
SEQIDN0:71;	Peptide encoded by SEQ ID NO:74	62 aa
SEQ ID NO:72	Nucleotide sequence from S. plumipes, which encodes OAIP-1 prepropeptide (S +P +M)	276 nts
SEQ ID NO:73	Nucleotide sequence from S. plumipes, which encodes OAIP-2 prepropeptide (S +P +M)	231 nts
SEQ IF NO:74	Nucleotide sequence from S. plumipes, which encodes OAIP-3 prepropeptide (S +P +M)	186 nts
SEQ ID NO:75	Peptide OAIP-3 produced recombinantly	34 aa

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PCT/AU2012/001000

DETAILED DESCRIPTION OF THE INVENTION

1. Abbreviations [0077] The following abbreviations are used throughout the application:

nt =nucleotide nts =nucleotides aa =amino acid(s) kb =kilobase(s) or kilobase pair(s) kDa =kilodalton(s) ^;

2. Definitions [0078] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below, [0079] The articles “a” and “a«” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0080] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 15,14,13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0081] By “agriculturally acceptable carrier, excipient or diluent” is meant adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in agricultural formulation technology. Generally, the agriculturally acceptable carrier, excipient or diluent is not deleterious to the other ingredients of the composition and is not deleterious to the plant, plant part, land or waterway recipient thereof. In the context of the other ingredients of the composition, “not deleterious” means that the carrier, excipient or diluent will not react with or degrade the other

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PCT/AU2012/001000 ingredients or otherwise interfere with their efficacy. Interference with the efficacy of an ingredient does not encompass mere dilution of the ingredient.

[0082] By “antigen-binding molecule is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.

[0083] As used herein, the term “binds specifically, “specifically immunoreactive and the like refers to antigen-binding molecules that bind or are immunoreactive with the polypeptide or polypeptide portions of the invention but do not significantly bind to homologous prior art polypeptides.

[0084] The term “biologically active fragment, as applied to fragments of a reference or full-length polynucleotide or polypeptide sequence, refers to a fragment that has at least about 0.1, 0.5, 1, 2, 5, 10, 12, 14, 16,18, 20, 22, 24, 26,28, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% of the activity of a reference sequence. Included within the scope of the present invention are biologically active fragments of at least about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30,40, 50, 60, 70, 80, 90, 100, 120, 140,160, 180,200 nucleotides or residues in length, which comprise or encode an activity of a reference polynucleotide or polypeptide. Representative biologically active fragments of a theraphosid pest-controlling proteinaceous molecule include peptides comprising amino acid sequences with

Sufficient similarity or identity to or derived from the amino acid sequence of the pestcontrolling proteinaceous molecules of the present invention, as for example set forth in SEQ ID NO: 2, 4, 10, 12, 18, 24, 30, 36,42,44,46 or 48 and comprise at least one activity selected from being orally active against insects; increasing mortality of insects, stimulating paralysis of insects, or inhibiting growth rate of insects and the like. In specific embodiments, the biologically active fragment comprise six cysteine residues.

In illustrative examples of this type, pairs of cysteine resides form individual disulfide bonds. Suitably, the biologically active fragments comprise three intrachain disulfide bonds. In particular embodiments, the connectivity of the six cysteine residues is cysteine-1 —> cysteine-4, cysteine-2 —> cysteine-5 and cysteine-3 —> cysteine-6 thereby forming three disulfide bonds. In some embodiments, the three interchain disulfide bonds form an inhibitor cystine knot motif as defined in King et al. (2002) Structure and

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PCT/AU2012/001000 function of insecticidal neurotoxins from Australian funnel-web spiders. Toxin Reviews 21,361-389.

[0085] By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene. .

[0086] The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”

Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

· [0087] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of' is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0088] By “controlling” is meant increasing mortality, stimulating paralysis or inhibiting the growth rate of insects. In certain embodiments, “control” or “controlling” means that a desired/selected activity (e.g., insect mortality-enhancing

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PCT/AU2012/001000 activity, insect paralysis-stimulating activity or insect growth rate-inhibiting activity) is more efficient (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), more rapid (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more), greater in magnitude (e.g, at least 10%, 20%, 30%, 40%, 50%, 60% or more), and/or more easily induced (e.g, at least 10%, 20%, 30%, 40%, 50%, 60% or more) than in the absence of a theraphosid pest-controlling agent of the invention.

[0089] The term preventing from feeding as used herein is meant that upon exposure to the pest-controlling proteinaceous molecules of the invention, the pests stop feeding or feed less than untreated pests. In particular, upon ingestion, the pests appear to lose their appetite and/or stop feeding.

[0090] By “corresponds to” or “corresponding to” is meant a peptide which comprises an amino acid sequence that displays substantial sequence similarity or identity to an amino acid sequence in a reference peptide. In general the peptide will display at least about 70, 71, 72, 73,74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97,

88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to at least a portion of the reference peptide.

[0091] By “effective amount,” in the context of controlling pests including insects, is meant the administration of an amount of TPC agent to a plant, plant part, plant product or site having, or at risk of developing, an insect infestation, either in a single dose or as part of a series, that is effective for controlling one or more species of insect. An effective amount will typically result in at least one pest-controlling activity including, for example, killing or paralyzing insect, or inhibiting insect development or growth or preventing insects from feeding in such a manner, for example, that in the case of agricultural applications, the insects provide less damage to a plant and plant yield is not significantly adversely affected. The effective amount will vary depending upon the application, the taxonomic group of insects to be controlled, the formulation of the composition comprising the theraphosid pest-controlling agent and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. TPC agents having pest-controlling activity are also referred to as toxic to insects. Pest-controlling specificity is the specificity of a TPC agent for one or more insect species. The LD50 is the dose of a TPC agent that results in the death of 50% of the insects tested.

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PCT/AU2012/001000 [0092] The terms “theraphosidpest-controlling agents,''’ “TPC agents,”

TPCs” and the like, as used herein encompasses, without limitation, pest-controlling proteinaceous molecules (e.g. , peptides, polypeptides etc.) comprising an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity or similarity with the sequence set forth in any one of SEQ ID NOs: 2,4,10,12,18, 24, 30, 36, 42, 44,46 or 48, including wildtype (or naturally-occurring) peptides derived, for example, from spiders, such as from Theraphosidae spiders, which include, but are not limited to, the following genera: Acanthopelma (e.g.,Acanthopelma beccarii, Acanthopelma rufescens), Acanthoscurria (e.g., Acanthoscurria brocklehursti, Acanthoscurria geniculata, Acanthoscurria theraphosoides), Ami, Annandaliella (e.g., Annandaliella travancorica), Antilles (e.g., Antillespinktoe), Aphonopelma (e.g., Aphonopelma chalcodes, Aphonopelma hentzi, Aphonopelma pallidum, Aphonopelma seemanni, Aphonopelma smithi), Avicularia (e.g., Avicularia affinis, Avicularia alticeps, Avicularia ancylochira, Avicularia anthracina, Avicularia aurantiaca, Avicularia avicularia, Avicularia bicegoi,

Avicularia metallica, Avicularia urticans), Brachypelma (e.g., Brachypelma albiseps, Brachypelma albopilosum, Brachypelma auratum, Brachypelma boehmei, Brachypelma emilia, Brachypelma klaas, Brachypelma smithi, Brachypelma vagans), Ceratogyrus (e.g., Ceratogyrus brachycephalus, Ceratogyrus darlingi), Chromatopelma (e.g.,

Chromatopelma cyaneopubescens), Cubanana (e.g., Cubanana cristinae, Cyclosternum (e.g., Cyclosternumfasciatum), Cyriocosmus (e.g., Cyriocosmus elegans), Euathlus, Eupalaestrus (e.g., Eupalaestrus campestratus), Ephebopus (e.g., Ephebopus murinus), Grammostola (e.g., Grammostola actaeon, Grammostolapulchra, Grammostola pulchripes, Grammostola rosed), Haplopelma (e.g., Haplopelma albostriatum,

Haplopelma hainanum, Haplopelma lividum, Haplopelma scmidti), Harpactira (e.g., Harpactira gigas), Harpactirinae, Hysterocrates (e.g., Hysterocratesgigas), Lampropelma (e g., Lampropelma violaceopes), Lasiodora (e.g., Lasiodora klugi, Lasiodoraparahybana), Nhandu (e.g.,Nhandu chromatus), Ornithoctoninae, Ornithoctonus, Pamphobeteus (e.g., Pamphobeteus nigricolor), Pelinobius (e.g.,

Pelinobius muticus), Phormictopus (e.g., Phormictopus canCerides), Phlogius (e.g.,

Phlogius crassipes), Poecilotheria (e.g., Poecilotheria metallica, Poecilotheria regalis), Psalmopoeus (e.g., Psalmopoeus cambridgei), Pterinochilus (e.g., Pterinochilus murinus), Selenocosmia, Selenotypus (e.g., Selenotypusplumipes, Selenotypus sp. 2,

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PCT/AU2012/001000

Selenotypus sp. 3, Selenotypus sp. 4, Selenotypus sp. 5, Selentoypus sp. 10, Selenotypus sp. plumebo, Selenotypus nebo, Selenotypus sp. gold, Selenotypus sp. woodstock, Selenotypus sp. gemfields, Selenotypus sp. tahnee, Selenotypus sp. dwarf, Selenotypus sp. Wallaces birdspider), Theraphosa (e.g., Theraphosa apophysis, Theraphosa blondi),

Xenesthis (e.g.,Xenesthis immanis). The terms “theraphosid pest-controlling agents,” “TPC agents,” TPCs” and the like further encompass natural allelic variation of theraphosid pest-controlling molecules that may exist and occur from one organism to another. Also, the degree and location of glycosylation or other post-translation modifications (e.g., amidation) may vary depending on the chosen host and the nature of the host cellular environment. The above terms are also intended to encompass TPC peptide in their precursor form (e.g., prepropeptide, propeptide region plus mature peptide etc.), as well as those that have been processed to yield their respective bioactive forms. It further encompasses TPC peptides that have either been chemically modified relative to a reference or naturally-occurring TPC and/or contain one or more amino acid sequence alterations relative to a reference or naturally-occurring TPC and/or contain truncated amino acid sequences relative to a reference or naturallyoccurring full-length or precursor TPC. Alternatively, or in addition, TPCs may exhibit different properties relative to a reference or naturally-occurring TPC, including stability, altered specific activity selected from oral activity against insects, insect .

mortality-enhancing activity, insect paralysis-stimulating activity, insect growth rateinhibiting activity or insect anti-feedant activity, and the like. The above terms also encompass proteinaceous molecules with a slightly modified amino acid sequence, for instance, peptides having a modified N-terminal end including N-terminal amino acid deletions or additions, and/or peptides that have been chemically modified relative to a reference or naturally-occurring TPC. TPC agents also encompass proteinaceous molecules exhibiting substantially the same or better bioactivity than a reference or naturally-occurring TPC, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to a reference or naturally-occurring TPC. They also include, without limitation, peptides having an amino acid sequence that differs from the sequence of a reference or naturally-occurring TPC peptide by insertion, deletion, or substitution of one or more amino acids and in illustrative examples, encompass proteinaceous molecules that exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%,

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PCT/AU2012/001000

70%, 80%, 90%, 100%, 110%, 120%, and 130% of the specific activity of a reference or naturally-occurring TPC peptide (e.g., that has been produced in the same cell type).

[0093] By “gene” is meant a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5’ and 3’ untranslated sequences).

[0094] The term “host cell” includes an individual cell or cell culture which can be or has been a recipient of any recombinant construct(s)/vectors or isolated nucleic acid molecules of the invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transformed, transfected or infected in vivo or in vitro with a recombinant construct or a nucleic acid molecule of the invention. A host cell which comprises a recombinant construct of the invention is a recombinant host cell.

[0095] “Hybridization” is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G. In this regard, the terms “match” and “mismatch” as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.

[0096] By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolatedpolynucleotide,” as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g, a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolatedpeptide” or an “isolatedpolypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other

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PCT/AU2012/001000 components of the cell, i.e., it is not associated with in vivo substances. Similarly, an “isolated' or “purified' proteinaceous molecule (e.g., peptide, polypeptide, protein etc.) is substantially free of cellular material or other contaminating molecules from the cell or tissue source from which the proteinaceous molecule is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.

“Substantially free means that a preparation of TPC proteinaceous molecule is at least 10,15,20, 25, 30, 35,40,45, 50, 55,60, 65, 70, 75, 80, 85,90,91,92,93,94,95,96, 97,98 or 99% pure. Purity can be measured by any appropriate method, eg., by column chromatography, polyacrylamide gel electrophoresis, mass spectrometry, or by high pressure liquid chromatography (HPLC) analysis. In a specific embodiment, the preparation of TPC proteinaceous molecule has less than about 30,25,20, 15, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1% (by dry weight), of non-TPCs (also referred to herein as a “contaminating molecules”), or of chemical precursors or non-TPC chemicals. When the TPC is recombinantly produced, it is also desirably substantially free of culture medium, i.e., culture medium represents less than about 50,40; 30,20,15,10, 5,4, 3, 2, 1% of the volume of the TPC preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1,1.0, and 10 milligrams in dry weight.

[0097] By “obtainedfrom is meant that a sample such as, for example, a polynucleotide extract hr polypeptide extract is isolated from, or derived from, a particular source.

[0098] The term “operably connected' or “operably linked' as used herein means placing a structural gene under the regulatory control of a regulatory element including but not limited to a promoter, which then controls the transcription and optionally translation of the gene. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter, at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting; i.e. the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting; i.e. the genes from which it is derived.

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PCT/AU2012/001000 [0099] The term “oligonucleotide as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via . phosphodiester bonds (or related structural variants or synthetic analogues thereof).

Thus, while the term “oligonucleotide” typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of any length, although the term “polynucleotide” or “nucleic acid” is typically used for large oligonucleotides.

[0100] The term “pest” as used herein, refers to pests that may cause crop damage, structural damage or disease. Illustrative examples of pests include arthropods such as insects, arachnids, centipedes and millipedes; helminths such as cestodes, nematodes and trematodes and molluscs such as snails and slugs. Jn particular embodiments, the pests are insects, ticks, mites, snails, slugs and helminths, especially insects, ticks and mites, more especially insects.

[0101] The term “polynucleotide” or “nucleic acid' as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or ' a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0102] The terms “polynucleotide variant” and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well

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PCT/AU2012/001000 understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. The terms “polynucleotide variant” and “variant” also include naturally occurring allelic variants.

[0103] ''Polypeptide ” 'peptide,'' '‘protein’’’ and 'proteinaceous molecule” are used interchangeably herein to refer to molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.

[0104] The terms ‘peptide variant” and 'polypeptide variant” and the like refer to peptides and polypeptides that are distinguished from a reference peptide or polypeptide by the addition, deletion or substitution of at least one amino acid residue.

In certain embodiments, a peptide or polypeptide variant is distinguished from a reference peptide or polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, the peptide or polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the peptide or polypeptide. Peptide and polypeptide variants also encompass peptides and polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.

[0105] By 'primer” is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotide

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PCT/AU2012/001000 residues, although it can contain fewer nucleotide residues. Primers can be large polynucleotides, such as from about 200 nucleotide residues to several kilobases or more. Primers can be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By ‘‘'substantially complementary,” it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide. Suitably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotide residues can be attached to the 5’ end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of noncomplementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.

[0106] Probe” refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the “target polynucleotide”, through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly.

[0107] The term recombinant polynucleotide” as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.

[0108] By recombinant polypeptide” is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant polynucleotide.

[0109] By regulatory element” or regulatory sequence” is meant nucleic 30 acid sequences (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The regulatory sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a eft-acting sequence

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PCT/AU2012/001000 such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or trailing sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.

[0110] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G; I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr,

Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The present invention contemplates the use in the methods and systems of the present invention of full-length TPC peptides as well as their biologically active fragments. Typically, biologically active fragments of a full-length TPC peptide may participate in an interaction, for example, an intra-molecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). Biologically active fragments of a full-length TPC peptide include peptides comprising amino acid sequences sufficiently similar to or derived from the amino acid sequences of a (putative) full-length TPC polypeptide. Typically, biologically active fragments comprise a domain or motif with at least one activity selected from: oral activity against pests, pest mortality-enhancing activity, pest paralysis-stimulating activity, pest growth rate-inhibiting activity or pest anti-feedant activity, and the like. A biologically active fragment of a full-length TPC peptide can be a polypeptide which is, for example, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27,

28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90 or more amino acid residues in length. Suitably, the biologically-active fragment has no

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PCT/AU2012/001000 less than about 1%, 10%, 25% 50% of an activity of the full-length peptide from which it is derived.

[0111] “Similarity” refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Tables 2 and 3 infra.

Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research 12:387-395,1984). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

i ' [0112] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window”, “sequence identity,” “percentage of sequence identity” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50. to about 100, in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage similarity over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al.,

Nucl. Acids Res. 25:3389-3402,1997. A detailed discussion of sequence analysis can - 34 WO 2013/026105

PCT/AU2012/001000 be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0113] “Sfr/Mge/icj’” as used herein, refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization and washing procedures. The higher the stringency, the higher will be the degree of complementarity between immobilized target nucleotide sequences and the labeled probe polynucleotide sequences that remain hybridized to the target after washing. The term high stringency” refers to temperature and ionic conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization. Generally, stringent conditions are selected to be about 10 to 20° C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.

[0114] By vector” is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra 25 chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

The choice of the vector will typically depend on the compatibility of the vector with the'host cell into which the vector is to be introduced. In the present case, the vector is preferably a bacterial or fungal-derived vector. The vector can also include a selection -35WO 2013/026105

PCT/AU2012/001000 marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.

[0115] The terms “wild-type” and “naturally occurring” are used interchangeably to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.

3. Theraphosidpest-controlling agents [0116] The present inventors have identified several peptides from the venom of the Australian tarantula, Selenotypus plumipes, which are highly toxic to and orally active against pests, for example, both adult and/or larval insects. These peptides may be in the form of a mature peptide, a precursor peptide (i.e., a peptide containing both a propeptide region sequence followed by a mature peptide sequence) or a prepropeptide (i.e., a peptide containing a signal sequence, followed by a propeptide region sequence, followed by a mature peptide sequence). The mature peptides each consist of between about 30 and about 38 amino acid residues, may be C-terminally amidated or may have a free C-terminal carboxylic acid and are capable of forming three intra-chain disulfide . bonds. Without wishing to be limited to any one theory or mode of operation, it is believed that the biologically active form of the peptides is produced by posttranslational proteolytic processing (e.g., cleavage) of the prepropeptide precursor to produce the mature pest-controlling active peptide. Cleavage includes, for example, endoproteolytic cleavage of the prepropeptide by a protease that recognizes a particular amino acid sequence motif in the prepropeptide. The “pre” portion of the prepropeptide refers to the signal peptide portion of the prepropeptide which, without being held to theory, is believed to be responsible for targeting the prepropeptide to, as well as its translocation across, the endoplasmic reticulum membrane in cells that produce the peptides. In some embodiments, the signal peptide sequence is selected from any one of SEQ ID NO: 8,16,24, 32 or 40. Other signal sequences that function in a similar manner may also be employed. The “pro” portion of the prepropeptide may be selected

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PCT/AU2012/001000 from any one of SEQ ID NO: 6, 14,22, 30 or 38; or other sequences covalently attached upstream of a mature peptide of the invention. Possible roles for the precursor peptide sequence include facilitating peptide export from the endoplasmic reticulum^ assisting enzyme-catalyzed oxidative folding of the mature peptide, and signaling enzymes involved in proteolytic processing and post-translational modification. An isolated or purified peptide of the present invention will suitably comprise an amino acid sequence corresponding to the mature peptide and optionally one or both of a signal peptide sequence and a propeptide region sequence. The prepropeptide architecture of the peptide of the present invention appear similar to that determined by the inventors for other toxins (e.g., κ-HXTX-Hvlc and co-ACTX-Hv2a toxin peptides) expressed in the venom gland of Australian funnel-web spiders.

[0117] In view of the close structural similarity observed between toxin peptides from different species of spiders (e.g., the structural similarity observed between κ-HXTX-Hvlc and oa-ACTX-Hv2a toxin peptides from different funnel-web spiders), the present inventors propose the existence of S. plumipes peptide homologs in other spider species, including those of the family Theraphosidae, which will have analogous pest-controlling activities. The present inventors thus consider that these peptides, their encoding-nucleic acid molecules as well as constructs, vectors and hosts cells that are capable of expressing those nucleic acid molecules (“theraphosid pest20 controlling” or “TPC” agents or “TPCs”) will be useful in increasing the mortality of pests, stimulating the paralysis of pests, inhibiting the development or growth rate of pests and/or preventing pests from feeding.

[0118] Accordingly, the present invention provides TPCs in methods and compositions for controlling pests, including combating or eradicating infestations of plants, plant products, land and waterways by pests. When included in compositions, the TPCs are suitably combined with an agriculturally acceptable carrier, excipient and/or diluent. The TPCs of the present invention can be delivered to the pests such as insects, plant, plant part or site, which has or is at risk of developing pest infestation, by any suitable route including, for example, by foliar spray or vector delivery.

[0119] In some embodiments, the TPCs are isolated, purified or otherwise obtained from the venom of a theraphosid, non-limiting examples of which include theraphosids from the family Theraphosidae, representative examples of which include

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PCT/AU2012/001000 theraphosids from the genera: Acanthopelma (e.g., Acanthopelma beccarii, Acanthopelma rufescens), Acanthoscurria (e.g., Acanthoscurria brocklehursti, Acanthoscurria geniculata, Acanthoscurria theraphosoides), Ami, Annandaliella (e.g., Annandaliella travancorica), Antilles (e.g., Antillespinktoe), Aphonopelma (e.g.,

Aphonopelma chalcodes, Aphonopelma heritzi, Aphonopelma pallidum, Aphonopelma seemanni, Aphonopelma smithi), Avicularia (e.g., Avicularia affinis, Avicularia alticeps, Avicularia ancylochira, Avicularia anthracina, Avicularia aurantiaca, Avicularia avicularia, Avicularia bicegoi, Avicularia metallica, Avicularia urticans), Brachypelma (e.g, Brachypelma albiseps, Brachypelma albopilosum, Brachypelma auratum,

Brachypelma boehmei, Brachypelma emilia, Brachypelma klaas, Brachypelma smithi, Brachypelma vagans), Ceratogyrus (e.g., Ceratogyrus brachycephalus, Ceratogyrus darlingi), Chromatopelma (e.g., Chromatopelma cyaneopubescens), Cubanana (e.g., Cubanana cristinae, Cyclostemum (e.g., Cyclosternum fasciatum), Cyriocosmus (e.g., Cyriocosmus elegans), Euathlus, Eupalaestrus (e.g., Eupalaestrus campestratus),

Ephebopus (e.g., Ephebopus murinus), Grammostola (e.g., Grammostola actaeon, Grammostola pulchra, Grammostola pulchripes, Grammostola rosea), Haplopelma. (e.g., Haplopelma albostriatum, Haplopelma hainanum, Haplopelma lividum, Haplopelma scmidti), Harpactira (e.g., Harpactira gigas), Harpactirinae, Hysterocrates (e.g., Hysterocrates gigas), Lampropelma (e.g., Lampropelma violaceopes), Lasiodora (e.g·, Lasiodora klugi, Lasiodora parahybana), Nhandu (e.g., Nhandu chromatus),

Omithoctoninae, Omithoctonus, Pamphobeteus (e.g., Pamphobeteus nigricolor), Pelinobius (e.g., Pelinobius muticus), Phormictopus (e.g., Phormictopus cancerides), Phlogius (e.g., Phlogius crassipes), Poecilotheria (e.g., Poecilotheria metallica, Poecilotheria regalis), Psalmopoeus (e.g., Psalmopoeus cambridgei), Pterinochilus (e.g., Pterinochilus murinus), Selenocosmia, Selenotypus (e.g., Selenotypusplumipes,

Selenotypus sp. 2, Selenotypus sp. 3, Selenotypus sp. 4, Selenotypus sp. 5, Selentoypus sp. 10, Selenotypus sp. plumebo, Selenotypus nebo, Selenotypus sp. gold, Selenotypus sp. woodstock, Selenotypus sp. gemfields, Selenotypus sp. tahnee, Selenotypus sp. dwarf, Selenotypus sp, Wallaces birdspider), Theraphosa (e.g.,Theraphosa apophysis,

Theraphosa blondi), Xenesthis (e.g., Xenesthis immanis). In other embodiments, the TPCs are isolated, purified or otherwise obtained from the venom of a non-theraphosid spider, including any genus within the Order Araneae. In specific embodiments, the TPCs are isolated, purified or otherwise obtained from the genus Selenotypus. In other

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PCT/AU2012/001000 embodiments, the TPCs are produced by recombinant DNA techniques. Alternatively, they may be derived by chemical synthesis and oxidizing/folding the peptide using similar techniques to those described previously for producing synthetic spider toxins (See, Atkinson et al., Insecticidal toxins derived from funnel web spider (Atrax or

Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HVl, from the venom of an Australian funnel web spider. Nature Struct. Biol. 4:559-566,1997; Wang et al., Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bond. Nature Struct. Biol., 7:505-513, 2000, which are incorporated herein by reference in their entirety).

(0120] TPC peptides of the present invention include peptides which arise as a result of the existence of alternative translational and post-translational events. The TPCs can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when the TPC is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

(0121] In some embodiments, a TPC agent has any one or more of the following characteristics: (a) is orally active against pests such as arthropods, helminths or molluscs; (b) increases mortality of pests such as arthropods, helminths or molluscs;

(c) stimulates paralysis of pests such as arthropods, helminths or molluscs; (d). inhibits development of pests such as arthropods, helminths or molluscs; (e) inhibits growth rate of pests such as arthropods, helminths or molluscs; (f) prevents feeding by pests such as arthropods, helminths or molluscs.

[0122] The present invention contemplates TPC prepropeptides as well as their biologically active fragments (e.g., mature peptide). Typically, biologically active fragments of TPC prepropeptides may participate in an interaction, for example, an intramolecular or an inter-molecular interaction and/or may display any one or more of activities (a) to (f) noted above. Such biologically active fragments include peptides comprising amino acid sequences sufficiently similar to or derived from the amino acid sequences of a TPC prepropeptide, for example, the amino acid sequences shown in SEQ ID NO: 4,12,20,28, 36,42,44,46,48, 50, 54, 56, 57, 58, 59, 63, 64 and 65, which include less amino acids than a TPC prepropeptide, and exhibit at least one

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PCT/AU2012/001000 activity selected from (a) to (f) defined above. In specific embodiments, the biologically active fragment comprises six cysteine residues, pairs of which are capable of forming intrachain disulfide bonds. Suitably, the biologically active fragment has or is capable of forming three intrachain disulfide bonds, especially where the connectivity of the interchain disulfide bonds is cysteine-1 —> cysteine-4, cysteine-2 —> cysteine-5 and cysteine-3 —> cysteine-6. In some embodiments, the three interchain disulfide bonds form an inhibitor cystine knot.

[0123] A biologically active fragment of a TPC prepropeptide can be a peptide/polypeptide which is, for example, 28,29, 30, 31, 32, 33, 34,35, 36, 37, 38,39,

40,41, 42,43,44,45, 46,47,48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91,92, or more amino acid residues in length. Suitably, the biologically-active fragment has no less than about 1%, 10%, 25% 50% of an activity of the parent peptide/polypeptide from which it is derived.

[0124] The present invention also contemplates TPC peptides that are variants of wild-type or naturally-occurring TPCs or their fragments. Such “variant” peptides or polypeptides include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Non-limiting examples of such variant TPCs include processed forms of a precursor TPC, including but not limited to peptides or polypeptides in which the signal peptide domain and/or pro region have been removed from the precursor form.

[0125] Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.

[0126] TPC variants can be identified in several ways. In some embodiments, native mRNA sequences encoding the precursors of TPC orthologs can be identified using standard molecular biology techniques to screen theraphosid venom-gland cDNA libraries for such orthologs. The amino acid sequence of the mature ortholog can be

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PCT/AU2012/001000 obtained from translation of the identified cDNA sequences by noting that endoproteolytic cleavage of the propeptide to give the mature toxin most likely occurs on the C-terminal side of an Xaa-Arg processing site that immediately precedes the mature toxin especially where Xaa is Glu, Gin, Gly or Lys. Native mature TPC .orthologs can then be isolated by chromatographic fractionation of the venom, followed by identification and purification of a peptide toxin with a mass matching that predicted from the TPC ortholog cDNA sequence. TPC peptides including variants that can be purified 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, electrofocusing, preparative gel electrophoresis, and combinations comprising one or more of the foregoing methods. Purification may be performed according to methods known to those of skill in the art that will result in a preparation of TPCs substantially free from other polypeptides and from carbohydrates, lipids, or subcellular organelles. Purity may be assessed by means known in the art, such as SDS-polyacrylamide gel electrophoresis or mass spectrometry.

[0127] In other embodiments, synthetic mature toxin can be produced by solid-phase peptide synthesis of the TPC sequence followed by cysteine oxidation to form the native disulfide isomer as described previously for production of synthetic J-atracotoxin-Hvlc (Wang et al., Nature Struct. Biol. 7:505-513,2000). The TPC can be chemically synthesized in toto, or as fragments that are subsequently joined by native chemical ligation to produce the full-length peptide, as described previously for synthesis of the sea anemone toxin APETx2 (Jensen et al., Chemical synthesis and folding of APETx2, a potent and selective blocker of acid sensing ion channel 3. Toxicon, 54:56-61, 2009). A TPC polypeptide may be oxidized and folded into its native three-dimensional structure by incubating the reduced, lyophilized peptide in a glutathione or other redox buffer. A suitable glutathione redox buffer includes 200 mM

3-[N-morpholino]propanesulphonic acid (MOPS) pH 7.3,400 mM KC1, 2 mM EDTA, mM reduced glutathione (GSH) and 2 mM oxidized glutathione (GSSG), although numerous variants are well known to those practiced in the art. This reaction mixture is incubated overnight at 4° C, room temperature, or 37° C, for example, and then fractionated using reverse-phase HPLC to separate individual disulfide isomers. Fractions may be collected and assayed for pest-controlling activity.

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PCT/AU2012/001000 [0128] In other embodiments, the TPC ortholog can be synthesized, chemically or by recombinant DNA techniques, from cDNA encoding the TPC ortholog. In still other embodiments, the TPC ortholog can be prepared using recombinant DNA techniques by constructing a synthetic gene encoding the TPC sequence by methods known in the art. In some embodiments, TPC orthologs are detected and isolated using antigen-binding molecules (e.g., antibodies) that are specifically immuno-reactive with TPC peptides and the like or related proteins of the present invention. Antigen-binding molecules include polyclonal and monoclonal antibodies, which may be produced using standard immunological techniques, as ' described for example in Coligan et al., CURRENT PROTOCOLS IN

IMMUNOLOGY, (John Wiley & Sons, Inc, 1991), and Ausubel et al. (1994-1998, supra), in particular Section III of Chapter 11. Alternatively, the antigen-binding molecules can be selected from Fv, Fab, Fab' and F(ab')2 immunoglobulin fragments as well as synthetic stabilized Fv fragments, minibodies, diabodies and the like. The present invention thus encompasses TPC peptide and polypeptides that cross-react with antigen-binding molecules that are specifically immuno-reactive with a TPC peptide as set forth, for example, in SEQ ID NO: 4,12,20,28, 36, 42,44,46, 48, 50, 54, 56, 57, 58, 59, 63, 64 and 65.

[0129] Anti-TPC antigen-binding molecules also have other useful applications including use in immunoassays for determining the amount or presence of a TPC peptide/polypeptide or related protein in a biological sample. Such assays are also useful in quality-controlled production of compositions containing one or more of the proteins of the present invention or related proteins. In addition, the antigen-binding molecules can be used to assess the efficacy of recombinant production of one or more of the TPC peptides/polypeptides or related proteins, as well as for screening expression libraries for the presence of a nucleotide sequence encoding one or more of the TPC proteins of the present invention or related protein coding sequences. Anti-TPC antigenbinding molecules are useful also as affinity ligands for purifying and/or isolating any one or more of the proteins of the present invention and related proteins. The TPC peptides/polypeptides and proteins containing related antigenic epitopes may be obtained by overexpressing full or partial lengths of a sequence encoding all or part of a TPC peptides/polypeptides in a suitable host cell.

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PCT/AU2012/001000 [0130] A TPC peptide or polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of TPC peptides or polypeptides can be prepared by mutations in the DNA.

Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel et al. (Proc. Natl. Acad. Sci. USA. 82:488-492, 1985), Kunkel et al. (Methods Enzymol., 154:367-382, 1987), U.S. Pat. No. 4,873,192, Watson et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.). Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of TPC peptides or polypeptides. Libraries or fragments e.g., N-terminal, C-terminal, or internal fragments, of a TPC coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a reference TPC. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify TPC variants (Arkin and Yourvan, Proc. Natl. Acad. Sci. USA 89:7811-7815, 1992; Delgrave etal., Protein Engineering, 6:327-331,1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.

[0131] Variant TPC peptides or polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to a parent (e.g., naturally-occurring or reference) TPC amino acid sequence. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

[0132] Acidic: The residue has a negative charge due to loss of a hydrogen ion (“proton”) at physiological pH and the residue is attracted by aqueous solution so as -43WO 2013/026105

PCT/AU2012/001000 to seek, the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

[0133] Basic: The residue has a positive charge due to association with a hydrogen ion ^'proton”) at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in . aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

[0134] Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

[0135] Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline and tryptophan.

[0136] Neutral/polar: The residues are not charged at physiological pH, but the residue is sufficiently attracted by aqueous solutions so that it would seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, histidine, serine and threonine.

[0137] This description also characterizes certain amino acids as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, “small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for -44WO 2013/026105

PCT/AU2012/001000 example by Dayhoff et al. (A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, 5:345-358, National Biomedical Research Foundation, Washington DC, 1978; and by Gonnet et al. (Science, 256:1443-1445,1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a “small” as well as a “hydrophobic” amino acid.

[0138] The degree of attraction or repulsion required for classification as polar or non-polar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.

[0139] Amino acid residues can be further sub-classified as aromatic or nonaromatic, which is self-explanatory with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in Table 2.

Table 2

Amino acid sub-classification

Sd^CLASSBS	Amino acids
Acidic	Aspartic acid, Glutamic acid
Basic	Arginine, Lysine, Histidine
Charged	Aspartic acid, Glutamic acid, Arginine, Lysine, Histidine
Small	Glycine, Serine, Alanine, Threonine, Proline \
Polar/neutral	Asparagine, Histidine, Glutamine, Cysteine, Serine, Threonine
Hydrophobic	Tyrosine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan, Alanine, Proline
Aromatic	Tryptophan, Tyrosine, Phenylalanine
Residues that influence chain orientation ,	Glycine and Proline

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PCT/AU2012/001000 [0140] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatichydroxyl side chains is serine and threonine; a group of amino acids having amide5 containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that 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 major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional TPC peptide/polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table 3 under the heading of exemplary and preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulk of the side chain, or (d) the disulfide bond connectivity. After the substitutions are introduced, the variants are screened for biological activity.

Table 3

Exemplary and Preferred Amino Acid Substitutions

OWCaWALRESWWE	Exemplary Substitutions	^REFERREDSUBSTITUTIONS
Ala	Val, Leu, Ile	Val
Arg	Lys, Gln, Asn	Lys
Asn	Gln, His, Lys, Arg, Asp	Gln
Asp	Glu, Asn	Glu
Cys	Ser	Ser
Gln	Asn, His, Lys, Glu	Asn
Glu	Asp, Gln	Asp
' Gly	. Ala, Pro	Ala
His	Asn, Gln, Lys, Arg	Arg

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GRiG&iAE Residue .	Exemplary Substitutions ;	Inferred Substitutions
He	Leu, Val, Met, Ala, Phe, Norleu	Leu
Leu	Norleu, lie, Val, Met, Ala, Phe	He
Lys	Arg, Gln, Asn	Arg
Met	Leu, Ile, Phe	Leu
Phe	Tyr, Leu, Val, Ile, Ala	Tyr
Pro	Gly	Gly
Ser	Thr	Thr
Thr	Ser	Ser
Trp	i Tyr	Tyr
Tyr	Trp, Phe, Thr, Ser	Phe
Val	Ile, Leu, Met, Phe, Ala, Norleu	Leu

[0141] Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).

[0142] Thus, a predicted non-essential amino acid residue in a TPC peptide or polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a TPC gene coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide, as described for example herein, to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide or polypeptide can be expressed recombinantly and its activity determined. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment peptide or polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. By contrast, an “essential” amino acid residue is a residue that, when altered from the wild-type

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PCT/AU2012/001000 sequence of a reference TPC peptide or polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present. For example, such essential amino acid residues include those that are conserved in TPC peptides or polypeptides e.g., the six cysteine residues that are capable of forming the three intrachain disulfide bonds.

[0143] Accordingly, the present invention also contemplates as TPC peptides or polypeptides, variants of the naturally-occurring TPC polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturallyoccurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity to a parent or reference TPC peptide or polypeptide sequence as, for example, set forth in SEQ ID NO: 2, 4, 6, 8,10,12,14,16, 17,18,20,22,24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44,

46,48, 50, 57, 58, 59,63,64, 65, 69, 70 or 71, as determined by sequence alignment programs described elsewhere herein using default parameters. Desirably, variants will have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a parent TPC peptide or polypeptide sequence as, for example, set forth in SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 17, 18, 20, 22, 24, 26,28,

30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 57, 58, 59, 63, 64, 65, 69, 70 or 71, as determined by sequence alignment programs described elsewhere herein using default parameters. Variants of a wild-type TPC peptide or polypeptide, which fall within the scope of a variant polypeptide, may differ from the wild-type molecule generally by as much 50, 49,48,47, 46,45, 44,43,42,41,40, 39, 38, 37, 36, 35, 34,33, 32, 31, 30, 29, . 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, or 11 amino acid residues or suitably by as few as 10, 9, 8, 7, 6, 5 4, 3, 2, or 1 amino acid residue(s). In some embodiments, a variant polypeptide differs from the corresponding sequences in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16,17, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40,

42, 44,46,48, 50, 57, 58, 59,63, 64,65, 69, 70 or 71 by at least 1 but by less than or equal to 25,24,23,22,21, 20,19,18,17, 16,15,14,13,12,11,10, 9, 8, 7, 6, 5,4, 3 or 2 amino acid residues. In other embodiments, it differs from the corresponding sequence in any one of SEQ ID NO: 2, 4, 6, 8,10, 12, 14,16, 17,18,20, 22,24,26, 28,

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30, 32, 34, 36, 38, 40,42,44, 46,48, 50, 57, 58, 59, 63, 64, 65,69, 70 or 71 by at least 1% but less than or equal to 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% of the residues. If the sequence comparison requires alignment, the sequences are typically aligned for maximum similarity or identity. “Looped” out sequences from deletions or insertions, or mismatches, are generally considered differences. The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution, as discussed in more detail below.

[0144] In some embodiments, calculations of sequence similarity or sequence 10 identity between sequences are performed as follows:

[0145] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, usually at least 40%, more usually at least 50%, 60%, and even more usually at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. For amino acid sequence comparison, when a position in the first sequence is occupied by the same or similar amino acid residue (i.e., conservative substitution) at the corresponding position in the second sequence, then the molecules are similar at that position.

[0146] The percent identity between the two sequences is a function of the number of identical amino acid residues shared by the sequences at individual positions, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. By contrast, the percent similarity between the two sequences is a function of the number of identical and similar amino acid residues shared by the sequences at individual positions, taking into

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PCT/AU2012/001000 account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. .

[0147] The comparison of sequences and determination of percent identity or percent similarity between sequences can be accomplished using a mathematical algorithm. In certain embodiments, the percent identity or similarity between amino acid sequences is determined using the Needleman and Wiinsch (J. Mol. Biol. 48:444453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6. In specific embodiments, the percent identity between nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3,4, 5, or 6. A non-limiting set of parameters (and the one that should be used unless otherwise specified) includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0148] In some embodiments, the percent identity or similarity between amino acid or nucleotide sequences can be determined using the algorithm of Meyers and Miller (Cabios, 4:11-17,1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0149] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (J. Mol.

Biol. 215:403-410, 1990). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to 53010 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to 53010 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402,1997). When utilizing

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PCT/AU2012/001000

BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0150] The TPC peptides and polypeptides of the present invention may be prepared by any suitable procedure known to those of skill in the art. For example, the

TPC peptides or polypeptides may be produced by any convenient method such as by purifying the peptides or polypeptides from naturally-occurring reservoirs including theraphosids. Methods of purification include size exclusion, affinity or ion exchange chromatography/separation. The identity and purity of derived TPC is determined for example by SDS-polyacrylamide electrophoresis or chromatographically such as by high performance liquid chromatography (HPLC). Alternatively, the TPC peptides or polypeptides may be synthesized by chemical synthesis, e.g., using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (supra) and in Roberge et al. (Science, 269:202-204, 1995).

[0151] In some embodiments, the TPC peptides or polypeptides are prepared by recombinant techniques. For example, the TPC peptides or polypeptides of the invention may be prepared by a procedure including the steps of: (a) preparing a construct comprising a polynucleotide sequence that encodes a TPC peptide or polypeptide and that is operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the polynucleotide sequence to thereby produce the encoded TPC peptide or polypeptide; and (d) isolating the TPC peptide or polypeptide from the host cell. In illustrative examples, the nucleotide sequence encodes at least a biologically active portion of the sequences set forth in SEQ ID NO: 2, 4,10,12,18,20, 26,28, 34, 36, 42, 44,46,48, 50, 52, 54, 57, 58, 59, 70, 71 or 72, or a variant thereof. Recombinant TPC peptides or polypeptides can be conveniently prepared using standard protocols as described for example in Sambrook et al. (1989, supra), in particular Sections 16 and 17; Ausubel et al. (1994, supra), in particular Chapters 10 and 16; and Coligan et al., Current Protocols in Protein • Science (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.

[0152] The TPCs of the present invention also encompass TPC peptide or

30· polypeptides comprising modifications, illustrative examples of which include peptides or polypeptides that are altered as a result of post-translational events which change, for example, the glycosylation, amidation (e.g., C-terminal amidation), lipidation pattern, or

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PCT/AU2012/001000 the primary, secondary, or tertiary structure of the polypeptide. N-terminal and/or Cterminal modifications are also possible. Other illustrative modifications include TPC peptides or polypeptides comprising amino acids with modified side chains, incorporation of unnatural amino acid residues and/or their derivatives during peptide, polypeptide or protein synthesis and the use of cross-linkers and other methods which impose conformational constraints on the peptides, portions and variants of the invention. Examples of side chain modifications include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate;

carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal5-phosphate followed by reduction with NaBH₄; reductive alkylation by reaction with an aldehyde followed by reduction with NaBkU; and trinitrobenzylation of amino groups with 2,4, 6-trinitrobenzene sulfonic acid (TNBS).

[0153] The carboxyl group may be modified by carbodiimide activation via

O-acylisourea formation followed by subsequent derivatization, by way of example, to a corresponding amide.

[0154] The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[0155] Sulfhydryl groups not participating in disulfide bonds in the mature peptide sequence may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate; 2-chloromercuri-4-nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulfides with other thiol compounds;

reaction with maleimide, maleic anhydride or other substituted maleimide;

carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

[0156] Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides or by oxidation with N-bromosuccinimide.

[0157] Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

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PCT/AU2012/001000 [0158] The imidazole ring of a histidine residue may be modified by N-carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

[0159] Examples of incorporating unnatural amino acids and derivatives 5 during peptide synthesis include but are not limited to, use of 4-amino butyric acid,

6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3hydroxy-6-methylheptanoic acid, /-butylglycine, norleucine, norvaline, phenylglycine, ' ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated by the present invention is shown in Table 4.

Table 4

Non-Conventional Amino acids

Non-Conventional Amino Adds
α-aminobutyric acid	L-N-methylalanine
a-amino-a-methylbutyrate	L-N-methylarginine
aminocyclopropane-carboxylate	L-N-methylasparagine
aminoisobutyric acid	L-N-methylaspartic acid
aminonorbomyl-carboxy late	L-N-methylcysteine '
cyclohexylalanine	L-N-methylglutamine
cyclopentylalanine	L-N-methylglutamic acid
L-N-methylisoleucine	L-N-methylhistidine
D-alanine	L-N-methylleucine
D-arginine	L-N-methyllysine
D-aspartic acid	L-N-methylmethionine
D-cysteine	L-N-methylnorleucine
D-glutamate	L-N-methylnorvaline
D-glutamic acid	L-N-methylomithine
D-histidine	L-N-methylphenylalanine
D-isoleucine	L-N-methylproline _t
D-leucine	L-N-medlylserine
D-lysine	L-N-methylthreonine
D-methionine	L-N-methyltryptophan
D-omithine	L-N-methyltyrosine

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PCT/AU2012/001000

Non-Conventional Amino Acids
D-phenylalanine	L-N-methyl valine
D-proline	L-N-methylethylglycine
D-serine	L-N-methyl-t-butylglycine
D-threonine	L-norleucine
D-tryptophan	L-norvaline
D-tyrosine	a-methyl-aminoisobutyrate
D-valine	a-methyl-y-aminobutyrate
D-a-methylalanine	a-methylcyclohexylalanine
D-a-methylarginine	a-methylcylcopentylalanine
D-a-methylasparagine	a-methyl-a-napthylalanine
D-a-methylaspartate	a-methylpenicillamine
D-a-methylcysteine	N-(4-aminobutyl)glycine
D-a-methylglutamine	N-(2-aminoethyI)glycine
D-a-methylhistidine	N-(3-aminopropyl)glycine
D-a-methylisoleucine	N-amino-a-methylbutyrate
D-a-methylleucine	a-napthylalanine
D-a-methyllysine	N-benzylglycine
D-a-methylmethionine	N-(2-carbamylediyl)glycine
D-a-methylomithiine	N-(carbamylmethyl)glycine
D-a-methylphenylalanine	N-(2-carboxyethyl)glycine
D-a-methylproline	N-(carboxymethyl)glycine
D-a-methylserine	N-cyclobutylglycine
D-a-methylthreonine	N-cycloheptylglycine
D-a-methyltryptophan	N-cyclohexylglycine
D-a-methyltyrosine	N-cyclodecylglycine
L-a-methylleucine	L-a-methyllysine
L-a-methylmethionine	L-a-methylnorleucine
L-a-methylnorvatine	L-a-methylomithine
L-a-methylphenylalanine	L-a-methylproline
L-a-methylserine	L-a-methylthreonine
L-a-methyltryptophan	L-a-methyltyrosine
L-a-methylvaline	L-N-methylhomophenylalanine

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Non-Conventional Amino Acids
N-(N-(2,2-diphenylethyl carbamylmethyl)glycine	N-(N-(3,3 -diphenylpropy 1 carbamylmethyl)glycine
1 -carboxy-1 -(2,2-diphenyl-ethyl amino)cyclopropane

[0160] The present invention also contemplates the use of TPC chimeric or fusion proteins. As used herein, a TPC “chimeric protein or “fusion protein includes a TPC peptide or polypeptide linked to a non-TPC peptide or polypeptide. A “non-TPC peptide or polypeptide” refers to a peptide or polypeptide having an amino acid sequence corresponding to a protein which is different from a TPC and which is derived from the same or a different organism. The TPC peptide or polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a TPC polypeptide amino acid sequence. In a specific embodiment, a TPC fusion protein includes at least one biologically active portion of a TPC polypeptide. The non-TPC ' peptide or polypeptide can be fused to the N-terminus or C-terminus of the TPC peptide or polypeptide.

[0161] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-TPC fusion protein in which the

TPC sequence is fused to the C-terminus of the glutathione S-transferase (GST) sequence. Such fusion proteins can facilitate the purification of a recombinant TPC peptide or polypeptide. Alternatively, the fusion protein can be TPC protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of TPC peptides or polypeptides can be increased through use of a heterologous signal sequence. In some embodiments, fusion proteins may include all or a part of a serum protein, e.g., an IgG constant region, or a serum albumin.

[0162] In another embodiment, the fusion protein could be a protein that is designed to improve the oral activity of the TPC peptide, including but not limited to

Galanthus nivalis agglutinin (GNA, also known as snowdrop lectin). The oral insecticidal activity of arachnid toxins has previously been demonstrated to be enhanced by N- or C-terminal fusion to GNA (e.g., Fitches et al., Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal

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PCT/AU2012/001000 spider venom toxin to insect haemolymph following oral ingestion. J. Insect Physiol. 50:61-71,2004; Down et al., Insecticidal spider venom toxin fused to snowdrop lectin as toxic to the peach-potato aphid, Myzus persicae (Hemiptera:Aphidae) and the rice brown planthopper, Nilaparrata lugens (Hemiptera:Delphacidae), Pest Manag. Sci., 62:77-85, 2006; Fitches et al., Insecticidal activity of scorpion toxin (ButalT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders. Pest Manag. Sci. 66:74-83,2010; Fitches et al., Fusion to snowdrop lectin magnifies the oral activity of insecticidal omega-hexatoxin-Hvla peptide by enabling its delivery to the central nervous system. PLoS ONE 7, e39389, 2012).

[0163] The TPCs of the present invention also include peptides and polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially medium or high stringency conditions, to TPCencoding polynucleotide sequences, or the non-coding strands thereof, as described below. Illustrative TPC polynucleotide sequences are set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15,17,19,21,23,25,27, 29,31,33,35,37, 39,41,43, 45,47,49, 51,53,55, 60, 61, 62, 66, 67, 68, 72, 73 and 74, or their complements.

[0164] Exemplary nucleotide sequences that encode the TPC peptides and polypeptides of the invention encompass full-length TPC genes as well as portions of the full-length or substantially full-length nucleotide sequences of the TPC genes or their transcripts or DNA copies of these transcripts. Portions of a TPC nucleotide sequence may encode polypeptide portions or segments that retain the biological activity of the native polypeptide. A portion of a TPC nucleotide sequence that encodes a biologically active fragment of a TPC polypeptide may encode at least about 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40,41, 42,43,44,45,46, 47,48, 49, 50, 51, 52,

53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75,

76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or more contiguous amino acid residues, or almost up to the total number of amino acids present in a TPC prepropeptide.

[0165] The invention also contemplates variants of the TPC nucleotide sequences. Nucleic acid variants can be naturally-occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally-occurring. Naturally-occurring nucleic acid variants (also referred to

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PCT/AU2012/001000 herein as polynucleotide variants) such as these can be identified with the use of wellknown molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as known in the art. Non-naturally occurring polynucleotide variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a reference TPC peptide or polypeptide. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a TPC peptide or polypeptide. Generally, variants of a particular TPC nucleotide sequence will have at least about 70%, 71%, 72%, 73%,

74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters. In some embodiments, the TPC nucleotide sequence displays at least about 70%, 71%, 72%, 73%, 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleotide sequence selected from any one of SEQ ID NO: 1, 3, 5, 7,9,11, 13, 15, 17, 19,21,23, 25,27, 29, 31, 33, 35, 37, 39,41, 43,45, 47, 49, 51, 53, 55, 60, 61, 62, 66,

67, 68, 72, 73 and 74, or their complements.

[0166] TPC nucleotide sequences can be used to isolate corresponding sequences and alleles from other organisms, particularly other theraphosids. Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other organisms may be isolated according to well known techniques based on their sequence identity with the coding sequences set forth herein. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other TPC-coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism (e.g., a theraphosid). The TPC gene sequences of the present invention allow • -57WO 2013/026105

PCT/AU2012/001000 for the preparation of relatively short DNA (or RNA) sequences, which have the ability to specifically hybridize to such gene sequences. The short nucleic acid sequences may be used as probes for detecting the presence of complementary sequences in a given sample, or may be used as primers to detect, amplify or mutate a defined segment of the

DNA sequences encoding a TPC peptide or polypeptide. A nucleic acid sequence employed for hybridization studies may be greater than or equal to about 15 nucleotides in length to ensure that the fragment is of sufficient length to form a stable and selective duplex molecule. Such fragments may be prepared, for example, by directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as PCR technology, or by excising selected nucleic acid fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.

[0167] Accordingly, the present invention also contemplates polynucleotides that hybridize to reference TPC nucleotide sequences, or to their complements, (e.g.,

SEQ ID NO: 1,3,5,7,9,11,13, 15,17,19, 21,23,25, 27, 29, 31,33,35, 37, 39,41, 43, 45, 47,49, 51, 53, 55, 60, 61, 62, 66, 67, 68, 72, 73 and 74, or their complements) under stringency conditions described below. As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al. (1998, supra),

Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used. Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1

M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x sodium chloride/sodium citrate (SSC), 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6 x SSC at about 45° C, followed by two washes in 0.2 x SSC, 0.1% SDS at least at 50° C (the temperature of the washes can be increased to 55° C for low stringency conditions). Medium stringency conditions include and encompass from at

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PCT/AU2012/001000 least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C. Medium stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, ,0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 60-65° C. One embodiment of medium stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 60° C. High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C, and about 0.01 M to about 0.02 M salt for washing at 55° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. One embodiment of high stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65° C.

[0168] In certain embodiments, a TPC peptide or polypeptide is encoded by a polynucleotide that hybridizes to a disclosed nucleotide sequence under very high stringency conditions. One embodiment of very high stringency conditions includes hybridizing in 0.5 M sodium phosphate, 7% SDS at 65° C, followed by one or more washes at 0.2 x SSC, 1% SDS at 65° C.

[0169] Other stringency conditions are well known in the art and a skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to 1.104.

[0170] While stringent washes are typically carried out at temperatures from about 42° C to 68° C, one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rate typically occurs at about 20° C to 25° C below the T_m for formation of a DNA-DNA hybrid. It is well

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PCT/AU2012/001000 known in the art that the T_m is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating T_m are well known in the art (see Ausubel et al., supra at page 2.10.8). In general, the T_m of a perfectly matched duplex of DNA may be predicted as an approximation by the formula:

[0171] T_m= 81.5 + 16.6 (log,₀ M) + 0.41 (%G+C) - 0.63 (% formamide)(600/length) [0172] wherein: M is the concentration of Na⁺, preferably in the range of 0.01 molar to 0.4 molar; %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C;

% formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex. The T_m of a duplex DNA decreases by approximately 1° C with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at T_m - 15° C for high stringency, or Tm - 30° C for moderate stringency.

[0173] In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 42° C in a hybridization buffer (50% deionized formamide, 5 x SSC, 5 x Denhardt’s solution (0.1% ficoll, 0.1% polyvinylpyrrolidone and 0.1% bovine serum albumin), 0.1 % SDS and 200 mg/mL denatured salmon sperm DNA) containing labeled probe. The membrane is then subjected to two sequential medium stringency washes (/.e., 2 x SSC, 0.1% SDS for 15 min at 45° C, followed by 2 x SSC, 0.1% SDS for 15 min at 50° C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55° C followed by 0.2 x SSC and 0.1% SDS solution for

12 min at 65-68° C.

[0174] The TPC toxin peptide-encoding nucleotide sequences of the present invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular or extracellular production and maintenance of the pesticide. With suitable microbial hosts, the microbes can be applied to the sites of the insect, where they will proliferate and be ingested by, or make contact with, the insect. The result is a control of the insect

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PCT/AU2012/001000 exhibited by reduced plant damage, increased plant yield, decreased prevalence of the plant insect in the general local environment of the transgenic organism expressing the toxin protein(s), and the death or stunted growth of the plant pest, generally without any additional impact on the microbial flora surrounding the plant or transgenic organism expressing the toxin protein(s), and without any additional impact on the environment in general. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.

[0175] Expression of TPC nucleic acid molecules is typically achieved by operably linking a TPC nucleotide sequence to a regulatory element (e.g., a promoter, which may be either constitutive or a temporally, spatially, chemically, photosynthetically, thermally, or artificially regulated or inducible promoter), suitably incorporating the construct into an expression vector, and introducing the vector into a suitable virus or host cell. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, enhancers and promoters useful for regulation of the expression of the particular nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors may be suitable for replication and integration in prokaryotes, eukaryotes, or both. See, Giliman and Smith, Gene 8:81-97, 1979; Roberts et al., Nature 328:731-734,1987; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods Enzymol., 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989), MOLECULAR CLONING - A LABORATORY MANUAL (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y., (Sambrook); and F. M. Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). The regulatory element(s) employed will generally be operable for expression of the TPC nucleotide sequence in the host cell of interest (e.g., a bacterial, fungal, insect, amphibian, plant or mammalian cell).

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PCT/AU2012/001000 [0176] In some embodiments, the expression vector is used to transform a microbe. In illustrative examples of this type, the vector is designed for genetic transformation of prokaryotic cells. A variety of prokaryotic expression vectors may be used, non-limiting examples of which include chromosomal vector (e.g., a bacteriophage such as bacteriophage λ), extrachromosomal vectors (e.g., a plasmid or a cosmid expression vector). The expression vector will also typically contain an origin of replication, which allows autonomous replication of the vector, and one or more genes that allow phenotypic selection of the transformed cells. Any of a number of suitable promoter sequences, including constitutive and inducible promoter sequences, may be used in the expression vector (see e.g., Bitter, et al., Methods Enzymol., 153:516-544,

1987). For example, inducible promoters such as pL of bacteriophage γ, plac, ptrp, ptac ptrp-lac hybrid promoter and the like may be used. The nucleic acid construct may then be used to transform the desired prokaryotic host cell to produce a recombinant prokaryotic host cell, e.g., for producing a TPC peptide/polypeptide.

[0177] The invention also contemplates eukaryotic host-expression vectors including for example yeast transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, Vaccinia virus), or transformed animal cell systems engineered for stable expression.

[0178] If an expression vector is used to transform a plant, a promoter may be selected that has the ability to drive expression in the plant. Numerous promoters that are active in plant cells have been described in the literature, illustrative examples of which include the nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter (which is carried on tumour-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter and the CaMV 35S promoter, the figwort mosaic virus 35S-promoter, the light-inducible promoter from the small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, the GST-II-27 gene promoter, the chlorophyll a/b binding protein gene promoter, the Arabidopsis sucrose-H⁺ symporter promoter (SUC2), etc.

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PCT/AU2012/001000 [0179] The expression vector may also contain signal peptide and propeptide region sequences that facilitate expression of the TPC gene and/or folding of the toxin peptide/polypeptide. These could be the naturally-occurring TPC signal and propeptide region sequences disclosed herein or other signal and/or propeptide region sequences that serve the same purpose. For example, the vector may specifically include a host signal sequence to enable extracellular export of the expressed TCP.

[0180] In other embodiments, the expression vector is introduced into an insect virus. Insect viruses are naturally occurring insect pathogens. Insects that are susceptible to viral infection can be a target for insect viruses. Insect viruses may be

DNA viruses or RNA viruses, which can be replicated and expressed inside a host insect once the virus infects the host insect. Many insect viruses and their host range are known in the art, including viruses that are host-specific and environmentally safe. The insecticidal efficacy of an insect virus can be enhanced by incorporation of a gene encoding an insect toxin into its genome, using method similar to those disclosed in

U.S. Pat. No. 6,096,304. A suitable insect virus is a DNA virus that has been traditionally used as a biological control agent on insect pests, such as baculovirus (nucleopolyhedrovirus and granulovirus), and entomopoxvirus. Another example of a suitable DNA virus is the mosquito-specific baculovirus disclosed in U.S. Pat. No. 6,521,454. Suitable RNA viruses include, but are not limited to, cypovirus.

[0181] In another embodiment, the expression vector is introduced into an entomopathogenic fungus, including the genera Metarhizium (e.g., M. acridum and M. anisopliae) and Beauveria (e.g., B. bassiana). The insecticidal activity of entomopathogenic fungi can be enhanced by incorporation of genes encoding insecticidal arachnid toxins as described by Wang and St Leger, Nature Biotech.

25:1455-1456,2007.

[0182] Plant-colonizing or root-colonizing microorganisms may also be employed as host cells for the production of one or more of TPC peptides/polypeptide of the present invention or related proteins. Exemplary microorganism hosts for TPC genes include the plant-colonizing microbe Clavibacter xyli as described by Turner et al. (Endophytes: an alternative genome for crop improvement; International Crop Science Congress, Ames, Iowa, USA, 14-22 Jul. 1992, 555-560, 1993) and root-63WO 2013/026105

PCT/AU2012/001000 colonizing pseudomonad strains, as described by Obukowicz et al. (U.S. Pat. No. 5,229,112).

[0183] Where the toxin gene of the present invention or a related nucleotide coding sequence is introduced by means of a suitable vector into a microbial host, and the host is applied to the environment in a living state, it is advantageous to use certain host microbes. For example, microorganism hosts can be selected which are known to occupy the insect’s habitat. Microorganism hosts may also live symbiotically with a specific species of insect. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

[0184] A large number of microorganisms are known to inhabit the habitat of insects. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria (e.g., the genera Bacillus, Escherichia,

Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes) and fungi (e.g., generaMetarhizium, Beauveria, Saccharomyces, Cryptococcus,

Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium). A wide variety of means are available for introducing a TPC gene encoding into a microorganism host under conditions that allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867.

[0185] Generally, transformation of a host cell with an expression vector or other DNA may be carried out by techniques well known to those skilled in the art. By . transformation” is meant a permanent or transient genetic change induced in a cell following incorporation of 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 • k of the DNA into the genome of the cell. By transformed cell” or host cell” is meant a cell (e.g., prokaryotic or eukaryotic) into which (or into an ancestor of which) has been

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PCT/AU2012/001000 introduced, by means of recombinant DNA techniques, a DNA molecule encoding a polypeptide of the invention (i.e., a TPC peptide or polypeptide).

[0186] When the host is a eukaryote, methods of transfection with DNA such as calcium phosphate co-precipitates, mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors, as well as others known in the art, may be used. When the host is a plant cell, other means of gene introduction into the cell may also be employed such as, for example, polyethylene glycol-mediated transformation of protoplasts, desiccation/inhibition-mediated DNA uptake, agitation with silicon carbide fibers, acceleration of DNA coated particles, injection into reproductive organs, injection into immature embryos, and Agrobacterium tumefaciens-mediated transformation.

[0187] Eukaryotic cells can also be co-transfected with DNA sequences encoding a polypeptide of this disclosure, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.

Suitable markers include, for example, neomycin and hygromycin, and the like, that 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 simian virus 40 (SV40), adenovirus, or bovine papilloma virus, to transiently 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 utilized as the host cell as described herein. The eukaryotic cell may be a yeast cell (e.g., Saccharomyces cerevisiae) or may be a mammalian cell, including a human cell.

[0188] Mammalian cell systems that utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the nucleic acid sequences encoding a foreign protein may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the TPC peptide/polypeptide in infected hosts (e.g., Logan & Shenk,

Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659,1984).

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PCT/AU2012/001000 [0189j The present invention also extends to pest-controlling composition. In some embodiments, the pest-controlling composition comprises a microbe (e.g., a virus, bacterium or fungi) expressing a TPG peptide or polypeptide. The microbe may be delivered to the insect by ingestion, inhalation or direct contact with the insect or insect larvae. The composition may also be formulated for application to land, plants, animals including human, and waterways, wherever an infestation is occurring.

[0190] In other embodiments, the pest-controlling composition comprising at least one TPC agent and optionally an agriculturally acceptable carrier, diluent and/or excipient. The pest-controlling composition may be in the form of flowable solution or suspension such as an aqueous solution or suspension. Such aqueous solutions or suspensions may be provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready-to-apply. In other embodiments, the pest-controlling composition comprises a water dispersible granule. In yet other embodiments, the pest-controlling composition comprises a wettable powder, dust, pellet, or colloidal concentrate. Such dry forms of the insecticidal compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a . controlled-release, sustained-release, or other time-dependent manner.

[0191] When the TPC peptide or polypeptide or a fusion protein comprising the TPC peptide or polypeptide, is expressed by a microorganism (e.g., an insect virus, bacterium or fungi), the microorganism expressing the TPC peptide or polypeptide can be applied to the crop to be protected. The microorganism may be engineered to express a TPC peptide or polypeptide, either alone or in combination with one or several other TPC peptides or polypeptides, or in combination with other pesticides such as insecticides including other insecticidal polypeptide toxins that may result in enhanced or synergistic insecticidal activity, or in combination with a protein that enhances the activity of the TPC peptide when ingested by insects, such as GNA.

[0192] When the insecticidal compositions comprise intact cells (e.g., bacterial or fungal cells) expressing a TPC peptide or polypeptide or a fusion protein comprising the TPC peptide or polypeptide, such cells may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut

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PCT/AU2012/001000 shells, and the like), and combinations comprising one or more of the foregoing materials. The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, surfactants, and combinations comprising one or more of the foregoing additives. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, suspensions, emulsifiable concentrates, and the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, polymers, liposomes, and combinations comprising one or more of the foregoing ingredients.

[0193] Alternatively, the TPC peptide or polypeptide or a fusion protein comprising the TPC peptide or polypeptide, may be expressed in vitro and isolated for subsequent field application. Such peptides or polypeptides may be in the form of crude cell lysates, suspensions, colloids, etc., or may be purified, refined, buffered, and/or further processed, before formulating in an active insecticidal formulation.

[0194] Regardless of the method of application, the amount of the active component(s) is applied at an pest controlling-effective amount, which will vary depending on such factors as, for example, the specific insects to be controlled, the ’ specific plant or crop to be treated, the environmental conditions, and the method, rate, and quantity of application of the pest controlling-active composition.

[0195] In some embodiments, pest-controlling compositions comprising TPC peptides/polypeptides, polynucleotides, cells, constructs, vectors, fusion proteins, etc., can be formulated with an agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, .25 or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with another other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term “agriculturally-acceptable carrier” covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously

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PCT/AU2012/001000 mixing, blending and/or grinding the insecticidal composition with suitable adjuvants using conventional formulation techniques.

[0196] The pest-controlling compositions may be applied to the environment of the target pest, for example onto the foliage of the plant or crop to be protected, by conventional methods, suitably by spraying. The strength and duration of pestcontrolling application may be set with regard to conditions specific to the particular pest(s), crop(s) to be treated and particular environmental conditions. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the pesticidal composition, as well as the particular formulation contemplated.

[0197] Other application techniques, e.g., dipping, dusting, sprinkling, soaking, soil injection, seed coating, seedling coating, spraying, aerating, misting, atomizing, and the like, are also feasible and may be required under certain circumstances such as, for example, insects that cause root or stalk infestation, or for application to delicate vegetation or ornamental plants. These application procedures are also well-known to those of skill in the art.

[0198] The pest-controlling compositions may be employed singly or in combination with other compounds, including and not limited to other pesticides or agents that enhance the activity of the TPC agent. They may be used in conjunction with other treatments such as surfactants, detergents, polymers or time-release formulations. The pest-controlling compositions may comprise a pest attractant. The pest-controlling compositions may be formulated for either systemic or topical use. In some embodiments, the pest-controlling compositions may be formulated as baits, for example, where the TPC agent is mixed with a food source, such as a sugar or protein source or food pulp, leading to ingestion of the TPC agent by the pest. Such agents may also be applied to pests directly.

[0199] Other pesticides that may be used in combination with the TPC agent include, but are not limited to, neonicotinoids such as acetamiprid, clothiandin, imidacoprid, nitenpyram, nithiazine, thiacloprid and thiamethoxam; pyrethroids such as allethrin, bifenthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyfluthrin, deltamethrin, etofenprox, fenvalerate, permethrin, phenothrin, prallethrin, resmethrin, tetramethrin, tralamethrin and trasfluthrin; organochlorides such as aldrin, chlordane,

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PCT/AU2012/001000 chlordecone, dieldrin, endosulfan, endrin, heptachlor, hexachlorobenzine, lindane, methoxychlor, mirex and pentachlorophenol; organophosphates such as acephate, azinphos-methyl, bensulide, chlorethoxyfos, chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorfos, dicrotophos, dimetoate, disulfoton, ethoprop, fenamiphos, fenitrothion, fenthion, fosthiazate, malathion, methamidophos, methidathion, mevinphos, monocrotophos, naled, omethoate, oxydemeton-methyl, parathion, parathion-methyl, phorate, phosalone, phosmet, phostebuprim, phoxim, pirimiphosmethyl, profenofos, terbufos, tetrachlorvinphos, tribufos and trichlorfon; carbamates such as aldicarb, bendiocarb, carbofuran, carbaryl, dioxacarb, fenobucarb, fenobucarb, fenoxycarb, isoprocarb, methomyl and 2-(l-methylpropyl)phenyl methylcarbamate; insect growth regulators such as benzoylureas including diflubenzuron and flufenoxuron, methoprene, hydroprene and tebufenozide; plant derived pesticides such as anabasine, anethole, annonin, asimina, azidirachtin, caffeine, carapa, cinnamaldehyde, citral, deguelin, eugenol, linalool, myristicin and pyrethrin; and other insecticides such as fipronil. Other pesticides include bacterially-derived insecticides such as spinosads, avermectins, Bacillus strains and Cry proteins. In particular embodiments, the other pesticide is a neonicotinoid insecticide such as imidacloprid. In some embodiments, the combination may have a synergistic effect.

' » ' .

[0200] The concentration of the pest-controlling composition that is used for environmental, systemic, or foliar application may vary depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of pesticidal activity. .

[0201] Alternatively, a plant (e.g. a plant crop or ornamental plant) may be engineered to express a TPC peptide or polypeptide, either alone, or in combination with other pest-controlling peptide/polypeptide toxins that may result in enhanced or synergistic insecticidal activity. Illustrative crops for which this approach would be useful include, but are not limited to, cotton, tomato, sweet com, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, sunflower, and field lupins.

[0202] The present invention extends to the use of the TPC agents of the present invention for controlling pests, In some embodiments, the pests are selected from arthropods such as insects, arachnids, centipedes and millipedes. Arthropods of suitable agricultural, household and/or medical/veterinary importance for treatment with

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PCT/AU2012/001000 the pest-controlling TPC agents include, for example, flies, aphids, fruit flies, thrips, and other leaf or fruit eating insects, termites and other wood boring insects, locusts, cockroaches, wireworms, blowflies, mealworms, moths, mosquitoes, ants, weevils, fleas, ticks and mites such as companion animal fleas, ticks and mites, livestock lice, mites, fleas and ticks and flies such as cattle lice, buffalo flies and sheep blowflies. Exemplary pests include members of the classes and orders: Coleoptera such as the American bean weevil Acanthoscelides obtectus, the leaf beetle Agelastica alni, click beetles (Agriotes lineatus, Agriotes obscurus, Agriotes bicolor), the grain beetle Ahasverus advena, the summer schafer Amphimallon solstitialis, the furniture beetle Anobium punctatum, Anthonomus spp. (weevils), the Pygmy mangold beetle Atomaria linearis, carpet beetles (Anthrenus spp., Attagenus spp.), the cowpea weevil Callosobruchus maculatus, the fried fruit beetle Carpophilus hemipterus, the cabbage seedpod weevil Ceutorhynchus assimilis, the rape winter stem weevil Ceutorhynchus picitarsis, the wireworms Conoderus vespertinus and Conoderus falli, the banana weevil Cosmopolites sordidus, the New Zealand grass grub Costelytra zealandica, the June beetle Cotinis nitida, the sunflower stem weevil Cylindrocopturus adspersus, the larder beetle Dermestes lardarius, the com rootworms Diabrotica virgifera, Diabrotica virgifera virgifera, and Diabrotica barberi, the Mexican bean beetle Epilachna varivestis, the old house borer Hylotropes bajulus, the lucerne weevil Hypera postica, the shiny spider beetle Gibbium psylloides, the cigarette beetle Lasioderma serricorne, the Colorado potato beetle Leptinotarsa decemlineata, Lyctus beetles (Lyctus spp.), the pollen beetle Meligethes aeneus, the common cockshafer Melolontha melolontha, the American spider beetle Mezium americanum, the golden spider beetle Niptus hololeucus, the grain beetles Oryzaephilus surinamensis and Oryzaephilus mercator, the black vine weevil Otiorhynchus sulcatus, the mustard beetle Phaedon cochleariae, the crucifer flea beetle Phyllotreta cruciferae, the striped flea beetle Phyllotreta striolata, the cabbage steam flea beetle Psylliodes chrysocephala, Ptinus spp. (spider beetles), the lesser grain borer Rhizopertha dominica, the pea and been weevil Sitona lineatus, the rice and granary beetles Sitophilus oryzae and Sitophilus granarius, the Mango pulp weevil Sternochaetus frigidus, the red sunflower seed weevil Smicronyx fulvus, the drugstore beetle Stegobium paniceum, the yellow mealworm beetle Tenebrio molitor, the flour beetles Tribolium castaneum, and Tribolium confusum, warehouse and cabinet beetles (Trogoderma spp.), and the sunflower beetle Zygogramma exclamationis;

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PCT/AU2012/001000

Dermanptera (earwigs) such as the European earwig Forflcula auricularia and the striped earwig Labidura riparia·, Dictyoptera such as the oriental cockroach Blatta orientalis, the German cockroach Blatella germanica, the Madeira cockroach Leucophaea maderae, the American cockroach Periplaneta americana, and the smokybrown cockroach Periplaneta fuliginosa·, Diplopoda such as the spotted snake millipede Blaniulus guttulatus, the flat-back millipede Brachydesmus superus, and the greenhouse millipede Oxidus gracilis·, Diptera such as the African tumbu fly (Cordylobia anthropophaga), biting midges (Culicoides spp.), bee louse (Braula spp.), the beet fly Pegomyia betae, black flies (Cnephia spp., Eusimulium spp., Simulium spp.), hot flies (Cuterebra spp., Gastrophilus spp., Oestrus spp.), craneflies (Tipula spp.), eye gnats (Hippelates spp.), filth-breeding flies (Calliphora spp., Fannia spp., Hermetia spp., Lucilia spp., Musca spp., Muscina spp., Phoenicia spp., Phormia spp.), flesh flies (Sarcophaga spp., Wohlfahrtia spp.); the frit fly Oscinella fnt,fruitflies (Dacus spp., BaCtrocera spp., Drosophila spp.) including Bactrocera tryoni, the mediterranean fruit fly Cerititis capitata, the melon fly Bactrocera Cucurbitae, the papaya fruit fly Bactrocera papayae, head and carion flies (Hydrotea spp.), the hessian fly Mayetiola destructor, horn and buffalo flies (Haematobia spp.), horse and deer flies (Chrysops spp., Haematopota spp., Tabanus spp.), louse flies (Lipoptena spp., Lynchia spp., and Pseudolynchia spp.), medflies (Ceratitus spp.), mosquitoes (Aedes spp.,

Anopheles spp., Culex spp., Psorophora spp.), sandflies (Phlebotomus spp., Lutzomyia spp.), screw-worm flies (Chrysomya bezziana and Cochliomyia hominivorax), sheep keds (Melophagus spp.); stable flies (Stomoxys spp.), tsetse flies (Glossina spp.), ash white fly (Siphoninus phillyreae, mango leaf gall midge (Procontarinia pustulata), silver leaf white fly (Bemisia tabaci), spiraling white fly (Aleurodicus dispersus) and warble flies (Hypoderma spp.); Isoptera (termites) including species from the familes Hodotermitidae, Kalotermitidae, Mastotermitidae, Rhinotermitidae, Serritermitidae, Termitidae, Termopsidae·, Heteroptera such as the bed bug Cimex lectularius, the cotton stainer Dysdercus intermedius, the Sunn pest Eurygaster integriceps, the tarnished plant bug Lygus lineolaris, the green stink bug Nezara antennata, the southern green stink bug Nezara viridula, and the triatomid bugs Panstrogylus megistus, Rhodnius ecuadoriensis, Rhodnius pallescans, Rhodnius prolixus, Rhodnius robustus, Triatoma dimidiata, Triatoma infestans, and Triatoma sordida·, Homoptera such as the California red scale Aonidiella aurantii, the black bean aphid Aphis fabae, the cotton or melon

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PCT/AU2012/001000 aphid Aphis gossypii, the green apple aphid Aphis pomi, the citrus spiny whitefly Aleurocanthus spiniferus, the oleander scale Aspidiotus hederae, the sweet potato whitefly Bemesia tabaci, the cabbage aphid Brevicoryne brassicae, the pear psylla Cacopsylla pyricola, the currant aphid Cryptomyzus ribis, the grape phylloxera

Daktulosphaira vitifoliae, the citrus psylla Diaphorina citri or Trioza erytreae, Mango leaf hopper (idioscopus clypealis and idioscopus nitidulus), the potato leafhopper Empoasca fabae, the bean leafhopper Empoasca solana, the vine leafhopper Empoasca vitis, the woolly aphid Eriosoma lanigerum, the European fruit scale Eulecanium corni, the mealy plum aphid Hyalopterus arundinis, the small brown planthopper Laodelphax striatellus, the potato aphid Macrosiphum euphorbiae, the green peach aphid Myzus persicae, the green rice leafhopper Nephotettix cinticeps, the brown planthopper Nilaparvata lugens, gall-forming aphids (Pemphigus spp.), the hop aphid Phorodon ₍ humuli, the bird-cherry aphid Rhopalosiphum padi, the black scale Saissetia oleae, the greenbug Schizaphis graminum, the grain aphid Sitobion avenae, and the greenhouse whitefly Trialeurodes vaporariorum; Isopoda such as the common pillbug

Armadillidium vulgare and the common woodlouse Oniscus asellus; Hymenoptera such as Anaplolepis gracilipes (yellow crazy ant), Solenopsis invicta (fire ants) and Wasmannia auropunctata (electric ants); Lepidoptera such as Adoxophyes orana (summer fruit tortrix moth), Agrotis ipsolon (black cutworm), Amyelois transitella (navel orangeworm^rcA/pj¹ podana (fruit tree tortrix moth), Bucculatrix pyrivorella (pear leafininer), Bucculatrix thurberiella (cotton leaf perforator), Bupalus piniarius (pine looper), Carpocapsa pomonella (codling moth), Chilo suppressalis (striped rice borer), Citripestis sagitiferella (citrus fruit borer), Choristoneura fumiferana (eastern spruce budworm), Cochylis hospes (banded sunflower moth), Diatraea grandiosella (southwestern com borer), Deanolis sublimbalis (red banded mango caterpillar), Earis insulana (Egyptian bollworm), Euphestia kuehniella (Mediterranean flour moth), Eupoecilia ambiguella (European grape berry moth), Euproctis chrysorrhoea (browntail moth), Euproctis subflava (oriental tussock moth), Galleria mellonella (greater wax moth), Helicoverpa armigera (cotton bollworm), Helicoverpa zea (cotton bollworm),

Heliothis virescens (tobacco budworm), Hofinannophila pseudopretella (brown house moth), Homeosoma electellum (sunflower moth), Homona magnanima (oriental tea tree tortrix moth), Lithocolletis blancardella (spotted tentiform leafininer), Lymantria dispar (gypsy moth), Malacosoma neustria (tent caterpillar), Mamestra brassicae (cabbage

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PCT/AU2012/001000 armyworm), Mamestra configurata (Bertha army worm), the homworms Manduca sexta and Manuduca quinquemaculata, Operophtera brumata (winter moth), Ostrinia nubilalis (European com borer), Panolis flammea (pine beauty moth), Pectinophora gossypiella (pink bollworm), Phyllocnistis citrella (citrus leafininer), Pieris brassicae (cabbage white butterfly), Plutella xylostella (diamondback moth), Rachiplusia ni (soybean looper), Spilosoma virginica (yellow bear moth), Spodoptera exigua (beet armyworm), Spodoptera frugiperda (fall armyworm), Spodoptera littoralis (cotton leafworm), Spodoptera litura (common cutworm), Spodoptera praefica (yellowstriped armyworm), Sylepta derogata (cotton leaf roller), Tineola bisselliella (webbing clothes moth), Tineola pellionella (case-making clothes moth), Tortrix viridana (European oak leafroller), Trichoplusia ni (cabbage looper), Yponomeuta padella (small ermine moth); Orthoptera such as the common cricket Acheta domesticus, tree locusts (Anacridium spp.), the migratory locust Locusta migratoria, the twostriped grasshopper Melanoplus bivittatus, the differential grasshopper Melanoplus differentialis, the redlegged grasshopper Melanoplus femurrubrum, the migratory grasshopper Melanoplus sanguinipes, the northern mole cricket Neocurtillahexadectyla, the red locust Nomadacris septemfasciata, the shortwinged mole cricket Scapteriscus abbreviatus, the southern mole cricket Scapteriscus borellii, the tawny mole cricket Scapteriscus vicinus, and the desert locust Schistocerca gregaria', Phthiraptera such as the cattle biting louse

Bovicola bovis, biting lice (Damalinia spp.), the cat louse Felicola subrostrata, the shortnosed cattle louse Haematopinus eurysternus, the tail-switch louse Haematopinus quadripertussus, the hog louse Haematopinus suis, the face louse Linognathus ovillus, the foot louse Linognathus pedalis, the dog sucking louse Linognathus setosus, the long-nosed cattle louse Linognathus vituli, the chicken body louse Menacanthus stramineus, the poultry shaft louse Menopon gallinae, the human body louse Pediculus humanus, the pubic louse Phthirus pubis, the little blue cattle louse Solenopotes capillatus, and the dog biting louse Trichodectes canis·, Psocoptera such as the booklice Liposcelis bostrychophila, Liposcelis decolor, Liposcelis entomophila, and Trogium pulsatorium¹, Siphonaptera such as the bird flea Ceratophyllus gallinae, the dog flea

Ctenocephalides canis, the cat flea Ctenocephalides felis, the human flea Pulex irritans, and the oriental rat ReaXenopsylla cheopis·, Symphyla such as the garden symphylan Scutigerella immaculata', Thysanura such as the gray silverfish Ctenolepisma longicaudata, the four-lined silverfish Ctenolepisma quadriseriata, the common

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PCT/AU2012/001000 silverfish Lepisma saccharina, and the firebrat Thermobia domestica; Thysanoptera such as the tobacco thrips Frankliniella fusca, the flower thrips Frankliniella intonsa, the western flower thrips Frankliniella occidentalis, the cotton bud thrips Frankliniella schultzei, the banded greenhouse thrips Hercinothrips femoralis, the soybean thrips

Neohydatothrips variabilis, Kelly's citrus thrips Pezothrips kellyanus, the avocado thrips Scirtothrips perseae, South African citrus thrips (Scirtothrips aurantii) the melon thrips Thrips palmi, and the onion thrips Thrips tabaci·, and the like, Acarina spp. such as mites including southern red mite (Olgonychus ilicis), and combinations comprising one or more of the foregoing arthropods.

[0203] In some embodiments, the pest-controlling compositions comprising the TPC peptide/polypeptides, polynucleotides, cells, constructs, vectors, etc., can be employed to treat ectoparasites. Ectoparasites include, for example, fleas, ticks, mange, mites, mosquitoes, nuisance and biting flies, lice, and combinations comprising one or more of the foregoing ectoparasites. The term fleas includes the usual or accidental species of parasitic flea of the order Siphonaptera, and in particular the species

Ctenocephalides, in particular C. felis and C. canis, rat fleas (Xenopsylla cheopis) and human fleas (Pulex irritans). Ectoparasites on farm animals (e.g., cattle), companion animals (e.g., cats and dogs), and human may be treated. In the case of farm and domestic animals, treatment may include impregnation in a collar or topical application to a localized region followed by diffusion through the animal's dermis, for example, spot treatments. In the case of a human, treatment may include a composition suitable for the treatment of lice in humans. Such a composition may be suitable for application to a human scalp such as a shampoo or a conditioner.

[0204] The TPC agents and compositions of the invention may be applied to a surface of material or article of manufacture such as soil, timber, buildings or physical barriers by, for example, spraying, painting or coating, or may be applied by impregnating a matrix such as soil, sand, sawdust, wood or timber products.

Impregnated soil or sand may be applied in a band or furrow around a potential site of infestation, such as a building or may be mixed with a layer of soil at the site of application. Material such as wood, timber or physical barriers may be impregnated, coated or laminated with the compounds or compositions of the invention.

Alternatively, plants from which the wood is derived may be engineered to express a transgene encoding a TPC peptide. Such materials or articles of manufacture are thereby -74WO 2013/026105

PCT/AU2012/001000 resistant to wood-associated pest damage. For example, timber may be treated before, during, or after it is incorporated into a structure or building, thereby protecting it against damage from wood-associated insect pests or combating an already existing wood-associated insect pest infestation. For timber treatment, the TPC agent containing compositions may optionally contain a penetrant, such as, for example, parafinic hydrocarbons, 2-ethoxyethanol, or methyl isobutyl ketone, and/or an anti-bloom agent, such as, for example, dibutyl phthalate or o-dichlorobenzene. Timber treatment compositions may also optionally contain fungicides, other insecticides, and/or pigments. Application of the compounds of the present invention onto the surface or into the matrix of the wood or timber can be accomplished using conventional techniques such as immersion of the timber or wood into a liquid composition, painting by spraying or brushing, dipping, or injecting the composition into the timber or incorporation into particle board or laminates. For such applications, the concentration of the TPC agent in the composition should be sufficient to provide an effective amount of the compound in or on the timber.

[0205] Furthermore the TPC agents and their compositions may be applied to pest shields and used in pest-proofing systems. Pest shields include metal shields incorporated during building of the structure to protect areas particularly susceptible to wood-associated pest attack, such as window sills, wooden steps, porches and verandas and lattice work. For example, suitable termite proofing systems include those described in U.S. Patent No. 6,397,518.

[0206] In other embodiments, the TPC agents and their compositions may be applied to an environment such as a household, industrial or outdoor environment. By household environment is meant an indoor environment to control pests such as flies, moths and the like or pests that damage stored food. Household environment includes domestic gardens and garden plants that suffer or are at risk of suffering pest infestation. Industrial environments include factories and storage facilities, such as food storage facilities suffering pest infestation or at risk of pest infestation. Outdoor environment includes parks and non-cropping farms that are suffering or at risk of suffering pest infestation or wet environments such as lakes, pools, ponds, puddles or where water collects that may provide a breeding ground for pests such as mosquitoes.

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BRIEF DESCRIPTION OF THE FIGURES [0207] Figure 1 is a graphical representation of the toxicity of each of SEQ ID NOs: 57, 58, 59,28 and 36 (OAIP 1,2, 3,4, 5) when fed to termites at a dose of 350 nmoles/gram. Each column represents the mean ± standard deviation (SD) of three replicates of 10 insects.

[0208] Figure 2 is a graphical representation of the toxicity of each of SEQ ID NOs: 57, 58, 59,28 and 36 (OAIP 1, 2, 3, 4, 5) when injected into mealworm larvae at a dose of 3 pmoles/gram. Each column represents the mean ± SD of three replicates of 10 insects.

[0209] Figure 3A is a reverse-phase (RP) HPLC chromatogram showing fractionation of SEQ ID NO:57 (sOAIP-1), produced by solid-phase peptide synthesis, after folding overnight in a redox buffer. The solid black line shows the peptide absorbance at 214 nm while the dotted line shows the gradient of solvent B. Correctly folded sOAIP-1 elutes as a single peak at approximately 25% Solvent B. The inset shows the m/z (mass) profile for the peak with a retention time of ~20 minutes, which corresponds to the mass of correctly folded sOAIP-1 (3718.7 Da).

[0210] Figure 3B is a RP-HPLC chromatogram showing elution of synthetic SEQ ID NO:57 (sOAIP-1) (dashed line) and co-elution of equal amounts of sOAIP-1 and native OAIP-1 purified from venom (black line).

[0211] Figure 4 is a pictorial representation of the synthetic SEQ ID NO: 57 (sOAIP-1 j structure. The peptide backbone is shown as a white tube except for the two β-sheets which are shown as white arrows located near the C-terminus of the molecule.. The three disulfide bonds are shown as black tubes and the cysteine connectivities are labelled. The N-terminus and C-terminus of the protein are also labelled.

[0212] Figure 5 shows the stability of SEQ ID NO:57 (sOAIP-1) in insect hemolymph. A series of RP-HPLC chromatograms of hemolymph from H armigera at various times following addition of 30 pg sOAIP-1 (highlighted in the solid box in

Figure 3A). Immediately before the purification, 30 pg of ω-HXTX-Hvla was added to each sample as a positive control (dashed box); the identity of both toxins was confirmed using mass spectrometry. RP-HPLC chromatograms were obtained after incubation of OAIP-1 in hemolymph for 0 hours, 1 day, 3 days, and 7 days. A plot showing the percent of sOAIP-1 remaining at each time point is provided in Figure 3B. -76WO 2013/026105

PCT/AU2012/001000 [0213] Figure 6 is a graphical representation of mortality curves for mealworms injected (A) and fed (B) with synthetic SEQ ID NO:57 (OAIP-1).

[0214] Figure 7 is a graphical representation of the dose-response curve resulting from feeding cotton bollworms (i.e., larval Helicoverpa armigera) an agar5 based diet containing synthetic SEQ ID NO:57 (sOAIP-1). The calculated LD50 was 104.2 pmol/g.

[0215] Figure 8 is a graphical representation of the mortality observed at 48 hours after feeding cotton bollworms with 100 pmoles imidacloprid, 100 pmoles synthetic SEQ ID NO:57 (sOAIP-1), or a 50:50 mixture of imidacloprid and sOAIP-1 (i.e., 50 pmoles of each compound). Each data point is the mean ± SEM of three replicates of 10 individuals per dose.

[0216] Figure 9 is a graphical representation of mortality of mealworms (Tenebrio molitor larvae) determined 48 h after insects were simultaneously offered toxin-treated and untreated agar. The toxin concentration in the treated agar ranged from

1 mmol to 1 pmol, and the data represent the mean and SEM of three replicates of 10 individuals for each dose. The data correlate well with the oral toxicity of synthetic SEQ ID NO:57 (sOAIP-1) in a non-choice test; the mortality at the same dose in the choice test is approximately the same as that observed in the non-choice test. Mortality at all but the lowest two doses (10 and 1 pmol) was significantly greater than the untreated agar control (P < 0.01). Columns represent the mean ± SD for three replicates of 10 insects for each dose.

[0217] Figure 10 is a graphical representation of phenotypic response of mealworms (Tenebrio molitor larvae) following injection of SEQ ID NO:57 (OAIP-1). Larvae were monitored 5, 30, and 60-min post-injection (horizontally striped, diagonally striped, and black bars, respectively). The response was scored relative to the control as excitatory (prolonged muscle spasms), excitation to the point of paralysis (spasms so severe the insect was unable to move independently), or death/moribund (dead or, if alive, the insect was unable to right itself when turned on its back).

[0218] Figure 11 is a RP-HPLC chromatogram showing the high purity of recombinant OAIP-3 (SEQ ID NO:75). The upper and lower traces correspond to absorbance at 214 nm and 280 nm, respectively (left ordinate axis, arbitray units). The

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PCT/AU2012/001000 gradient of solvent B (0.05% TFA in 90% acetonitrile) is also shown (right ordinate axis, %).

[0219] Figure 12 is a two-dimensional ’H-^1SN heteronuclear single-quantum coherence (HSQC) spectrum of uniformly ¹⁵N-labeled recombinant OAIP-3 (SEQ ID

NO:75) produced in E. coli. The spectrum reveals the expected number of backbone *H^l5N connectivities (31) as well as peaks for the sidechain amide groups of the two Trp residues (boxed and labeled “W-SC”), a pair of sidechain *H-^1SN connectivities for the single Asn residue (connected by a dashed line and labelled “N-SC”) and a single peak for the sidechain amide group of the single Asn residue (labelled “R-SC”). The excellent chemical shift dispersion in both the *H and ^l5N frequency dimensions is characteristic of a protein with a stable tertiary fold.

[0220] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

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EXAMPLES

EXAMPLE 1

Venom Fractionation (0221] Venom peptides were isolated by fractionating 500 pL of a 10-fold dilution of crude spider venom using a Vydac Cig analytical RP-HPLC column (5 pm,

4.6 x 250 mm). The crude venom was obtained by milking adult specimens of the Australian tarantula Selenotypus plumipes Pocock (Araneae:Theraphosidae) via electrostimulation applied to the base of the chelicerae. Solvent A for RP-HPLC was 0.1% trifluoroacetic acid (TFA) in water and Solvent B was 0.1% TFA in acetonitrile. Peptides were eluted at a constant flow rate of 1.0 mL/min using a gradient of 5% Solvent B for 5 min, 5-25% Solvent B over 20 min, then 25-50% Solvent B over 48 min. Individual fractions were lyophilized, resuspended in 100 pL of water, and further purified using cation exchange chromatography using an Amersham MonoS HR5/5 column (50 x 100 mm). Buffer A was 0.1 M NaCl (pH 5.5) and Buffer B was 2 M NaCl (pH 5.5); the gradient used was 5% Buffer B for 15 min followed by 5-45% Buffer B over 40 min. Peptides were desalted using RP-HPLC then lyophilized and stored at -20° C until further use.

EXAMPLE 2

Insecticidal Activity in termites [0222] In order to determine which venom fractions had oral insecticidal activity, individual lyophilized protein fractions were fed to termites (Coptotermes acinaciformis (Froggatt), Isoptera: Rhinotermitidae) collected from colonies maintained by the Department of Primary Industries and Fisheries at Long Pocket, Indooroopilly, Queensland, Australia. Termites were fed a 20% α-cellulose matrix (Sigma Aldrich, MO, USA) mixed with water; the toxin was dissolved in water, and 20 pL was added to the cellulose matrix to a final concentration of 350 nmol/g, and then pipetted into Petri dishes. After the cellulose matrix had dried (to prevent the termites from drowning in the wet bait), nine workers and one soldier termite were added to each dish; each toxin dose was replicated three times. Five venom peptides purified using ion exchange chromatography were found to be orally active in termites and these were designated orally active insecticidal peptide (OAIP) 1 through 5.

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PCT/AU2012/001000 [0223] The toxicity of each of purified OAIP 1 to 5 when fed to termites at a dose of 350 nmoles/gram is shown in Figure 1.

EXAMPLE 3

Insecticidal Activity in Mealworms [0224] The insecticidal activity of each OAIP was also examined by injection into mealworms (larvae of Tenebrio molitor Linnaeus, Coleoptera: Tenebrionidae). Mealworms of mass ~ 180 mg/individual were purchased from Pisces Enterprises (Kenmore, Queensland, Australia). Insects between 3 ^rd and 4^th instar were used. For each mealworm, 2.6 pL of OAIP diluted in ultrapure water was injected into the metathoracic plurite to give a final OAIP concentration of 3 pmoles/gram. Injections were performed using a 29.5 gauge insulin syringe (B-D Ultra-Fine, Terumo Medical Corporation, Elkton, Maryland, USA). Three replicates of 10 insects each were used, and a similar number of control insects were injected with ultrapure water and maintained under the same conditions.

[0225] The toxicity of each OAIP 1 to 5 when injected into mealworms at a dose of 3 pmoles/gram is shown in Figure 2.

EXAMPLE 4

Determination of Primary Structure of OAIP 1-5 [0226] Individual OAIPs were reduced and alkylated using 4-vinylpyridine (4VP; protocol adapted from Wang et al., Structure-function studies of omegaatracotoxin, a potent antagonist of insect voltage-gated calcium channels. Eur. J. Biochem. 264:488-494, 1999). Purified OAIPs (20-30 pg) were dissolved in 100 pL of Milli-Q water, before adding an equal volume of 4VP.buffer (0.25 M Tris, 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), pH 8.0). The

25^J solution was incubated at 65°C for 20 min to reduce all disulfide bonds. After 20 min, pL 4VP and 20 pL acetonitrile were added. The alkylation reaction was allowed to proceed in the dark at ambient temperature for 60 min. Alkylated OAIPs (45-450 pmol per sample) were sent to the Australian Proteome Analysis Facility (APAF, Sydney, Australia) and the Adelaide Proteomics Centre (APC, Adelaide Australia) for

N-terminal sequencing. This yielded partial N-terminal sequences for each OAIP.

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PCT/AU2012/001000 [0227] The complete primary structure of each OAIP was then determined by using the N-terminal sequence information in combination with a trancsriptome obtained form the venom gland of S. plumipes. Four venom glands isolated from two S', plumipes spiders and the total RNA was immediately extracted using Trizol (Invitrogen, Carlsbad, CA). The concentration and quality of RNA was measured using a Nanodrop (ND-1000, ThermoScientific, Wilmington, DE, USA) and Bioanalyzer (Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA, USA). An Oligotex Direct mRNA Mini Kit (Qiagen, Hilden, Germany) was used to isolate poly A⁺ mRNA from the total RNA. Elution was performed first in 5 mM Tris-HCl (pH 7.5), and subsequently samples were precipitated with RNAse-free glycogen, sodium acetate, and ethanol. Samples were again resuspended in RNAse-free water, and then the RNA concentration and quality were measured using the Nanodrop and Bioanalyzer. A total of 227 ng of mRNA (23.4 pL, with a concentration of 9.7 ng/pL) was submitted to the Australian Genome Research Facility (Brisbane Node, The University of Queensland,

St Lucia, Queensland, Australia) for pyrosequencing using the Roche 454 GS-FLX platform (Roche, Basel, Switzerland).

[0228] Raw 454 reads were assembled using SeqManNGen (v2, DNAStar, Madison, WI). After assembly, the sequences obtained from N-terminal sequencing of each OAIP were BLASTed against the raw 454 data. Sequence hits were then matched to contigs assembled using SeqManNGen. Complete transcripts were then isolated from the assembled data. This process provided the complete sequence, including the mature peptide sequence (SEQ ID NOs: 4,12,20, 28, 36 57, 58 and 59) signal sequence (SEQ ID NOs: 8, 16, 24, 32 and 40) and propeptide region (SEQ ID NOs: 6,14, 22, 30 and 38), for all five OAIPs isolated from S', plumipes crude venom as shown in Tables 5 and

6.

[0229] Table 5 shows the complete protein sequence obtained for each OAIP, with the signal sequence indicated in black underline and the propeptide sequence in grey. The mature peptide sequence is in bold black with the cysteine residues underlined. C-terminal post-translational amidation is predicted for OAIP-1, -2, and -3 (shown in italics). This process leads to removal of the residues shown in italics and amidation of the remaining C-terminal residue.

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Table 5

OAIP 1	MIFLLPSIISVMLLAEPVLMLGDTEDADLMEMVQLSR PFFNPIIRAVELVELREERQRDCGHLHDPCPNDRPGHR TCCIGLQCRYGKCLVRVGtf*
OAIP 2	MRVLFIIAGLALLSVVCYTSEMKERSSFNEVLSEFFAA DEPQ1 iRDCLGQ WASCEPKNSKCCPNYAC1WKYP WCRYRAGK*
OAIP 3	MKTSVLFAILGLALLFCLSFGVE 1 .EETGRECGGLMTR CDGKTTFCCSGMNCSPTWKWCVYAPGW
OAIP 4	MKASLFAVIFGLVVLCACSFAEDQFASPNELLKSMFVES ΙΊΙΙ'.Ι Ί PI VLGRYCQKWMWTCDAERKCCEDMACELWCKKRLG*
OAIP 5	MLIVILTCALLVIYHAAAAEELEAKDVIESKALATLDEER FECVLKCDIQYNGKNCKGKGENKCSGGWRCRFKLCLKI*

[0230] Table 6 shows a comparison of the OAIP sequences obtained from N-terminal sequencing using either the APC or APAF facilities, and the complete sequences obtained by BLASTing these N-terminal sequences against the transcriptome data. The reduced mass of each OAIP is shown (if each toxin forms three disulfide bonds as expected, the mass would be reduced by 6 Da).

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Table 6

OAIP-1 MR 3724 Da
OAIP-1_454	DCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVR
OAIP-1APAF	DCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVR
OAIP-2, MR 3933 Da
OAIP 2_454	DCLGQWASCEPKNSKCCPNYACTWKYCWPRYRA
OAIP 2_APAF	DCLGQWASCEPKNSKCCPNYACTWKYCWPRYAR
OAIP 2_APC	DCLGQWASCEPKNSKCC
OAIP 3, MR 3540 Da .
OAIP 3_454	ECGGLMTRCDGKTTFCCSGMNCSPTWKWCVYA
OAIP 3 APAF	ECGGLMTRCDGKTTFCCSGMNCSPTWKWCVYA
OAIP 3_APC	ECGGKMGYCDGKTTFCCSGMNCSPTTKW
OAIP 4, MR 3718 Da
OAIP 4 454	YCQKWMWTCDAERKCCEDMACELWCKKRLG
OAIP 4APC	YCQKWMWTCDAERKCCED
OAIP 5, MR 4371 Da
OAIP 5 454	FECVLKCDIQYNGKNCKG-KGENKCSGGWRCRFKLCL KI
OAIP 5_APC	FECVKKCDIQYNGKNCKGGKEENKNKGCW

EXAMPLE 5

Chemical Synthesis of OAIP-1 [0231] Synthetic OAIP-1 (sOAIP-1) was produced via Fmoc solid-phase peptide synthesis using the following Fmoc-protected L-amino acids: Arg(Tos), Asn(Trt), Asp(OtBu), Ala, Cys(Trt), Gln(Trt), Glu, Gly, His(Trt), Ile, Leu, Lys(Boc), Met, Phe, Pro, Ser(tBu), Thr, Tyr(tBu), Trp and Val. sOAIP-1 was synthesized on Wang polystyrene resin preloaded with the first C-terminal amino acid residue at

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0.2 mmol/g scale. Chain assembly was performed following a previously established in situ neutralization protocol (Schnolzer et al., In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences, Int. J. Pept. Protein Res. 40:180-193,1992) The process was carried out using a Symphony

Automatic Peptide Synthesizer (Protein Technologies, Tuscon, AZ, USA). Peptides were de-protected and cleaved from the solid resin with a solution of TFA: triisopropylsilane:water at 90:5:5 ratio for 3 h and evaporated under a stream of N2. The desired product was precipitated in cold diethylether and filtered. The retained crude peptide product was dissolved in a solution of 50% acetonitrile in water acidified with

0.1% TFA. Crude peptide solutions were lyophilized.

[0232] sOAIP-1 was then purified via RP-HPLC using a linear gradient of 15-40% Solvent B over 25 min; the peptide eluted at approximately 28% Solvent B. Mass spectrometry was used to confirm that a peptide of the correct mass had been produced. sOAIP-1, at a concentration of 0.1 mg/mL, was folded overnight at room temperature in ammonium bicarbonate redox buffer (0.1 M NH4HCO3 (pH 8.0), 5 mM GSH/0.5 mM GSSG). A linear 15-20% acetonitrile gradient was used in a final RP-HPLC step to purify the folded sOAIP-1 peptide to homogeneity.

[0233] Figure 3A is a RP-HPLC chromatogram showing fractionation of sOAIP-1 after folding overnight in a redox buffer. Eluted peptides were detected by absorbance at 214 and 276 nm. Correctly-folded sOAIP-1 elutes as a single peak at -25% Solvent B. 500 pg of folding reaction mixture was loaded onto a Cl 8 Vydac RP-HPLC column (5 pm, 4.6 x 250 mm) and eluted using a linear 1% gradient of 1540% Solvent B (dotted line). Peptide masses were determined using an ABI4700 MALDITOF-TOF mass spectrometer using a matrix of 10 mg/mL α-CHCA; the inset shows the m/z profile for the RP-HPLC peak at eluting with a retention time of -21 min, which corresponds to the mass of correctly folded sOAIP-1. The mass obtained,

3718.7 Da, agrees with the predicted mass of fully oxidized sOAIP-1 (3718.3 Da).

[0234] Figure 3B is a RP-HPLC chromatogram showing elution of sOAIP-1 (dashed line) and co-elution of equal amounts of sOAIP-1 and native OAIP-1 purified from venom (black line), with detection at 214 nm. For each chromatogram, -130 pg of peptide was loaded (thus, in the co-elution, -260 pg total of peptide was loaded). The

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PCT/AU2012/001000 linear gradient (dotted line) is the same as in Figure 3A; the peptide eluted at ~25% Solvent B.

EXAMPLE 6

Structure of Synthetic OAIP-1 (0235] The three-dimensional (i.e., tertiary) structure of sOAIP-1 was determined using NMR spectroscopy. Lyophilized sOAIP-1 was resuspended at a final concentration of 700 μΜ in 10 mM H2KPO4 phosphate buffer, pH 5.8, in 95% H2O: 5% D2O. The sample was filtered using a 0.22 μΜ Millipore Ultrafree-MC centrifugal filter, then 300 μΕ was added to a susceptibility-matched 5 mm outer-diameter microtube (Shigemi Inc., Osaka, Japan). A high-resolution ID NMR spectrum and 2D ’H-'H TOCSY, ’H-’H NOESY, ’H-’H ECOSY, 'H-¹⁵N HSQC, and ’H-¹³C HSQC spectra were acquired at 298 K. using a 900 MHz AV ANCE spectrometer (Bruker AXS, Karlsruhe, Germany) equipped with a cryogenically cooled probe. All spectra were recorded with an interscan delay of 1 s.

[0236] The NOESY mixing time was 200 ms, while the isotropic mixing period in the TOCSY experiment was 90 ms. Standard Bruker pulse sequences were used with a WATERGATE pulse sequence for solvent suppression. NMR data were processed using nmrPipe and the Rowland NMR Toolkit. Resonance assignments were made using XEASY. The ’Ha, ¹³Ca, ¹³0β, and ^1SN chemical shifts were used in conjunction with the software program TALOS+ (Shen et al., TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44:213-223,2009) to obtain predictions for the backbone φ and ψ dihedral angles; these were converted to dihedral-angle restraints for use in CYANA using a range corresponding to twice the standard deviation estimated by TALOS+. The

2D NOESY spectrum was automatically assigned and an ensemble of structures calculated without manual intervention using the program CYANA (Guntert,

Automated NMR Structure Calculation With CYANA. In: Protein NMR Techniques, Vol. 278. A.K. Downing, editor, Humana Press, pp. 353-378, 2004). The tolerances used for making automated resonance assignments were 0.025 and 0.020 ppm in the FI and F2 dimensions, respectively. The disulfide-bonding pattern (Cys2-Cys25, Cys9Cys25, and Cysl9-Cys30) was evident from the first round of structure calculations and therefore disulfide bond restraints of 2.0 < d < 2.1 A for Sy(i)—Sy(j), and 3.0 < d< 3.1 A

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PCT/AU2012/001000 for both CP(i)—Sy(j) and Syfi)—C3(j) were used in the final set of structure calculations. Five hydrogen bonds were clearly identified in preliminary rounds of structure calculation, and in subsequent rounds of structure calculations hydrogen bond restraints of 1.7-2.2 A and 2.7-3.2 A were used for the Hn-0 and N-0 distances, respectively.

[0237] In the final round of structure calculations CYANA was used to calculate 100 structures from random starting conformations, then the 20 conformers with the lowest target function, as identified by CYANA, were used to represent the solution structure of sOAIP-1. CYANA assigned 87% (1098 out of 1262) of the NOESY crosspeaks during the automated structure calculations.

[0238] The calculated structure of OAIP-1, shown in Figure 4, comprises three disulfide bonds, in a 1-3,2-4, 3-6 conformation, plus two β-strands towards the C-terminus of the molecule (found from residues 23-26 and 29-32). The disulfide bonds, shown as black tubes, form an inhibitor cystine knot motif in which the Cys2Cys20 and Cys9-Cys25 disulfide bonds and the intervening sections of polypeptide backbone form a 23-residue ring that is pierced by the Cysl 9-Cys30 ring.

EXAMPLE 7

IN VITRO STABILITY ASSESSMENT [0239] The stability of sOAIP-1 was determined ex vivo in insect hemolymph at room temperature (Figure 5A). Fourth-instar H armigera larvae were decapitated and the gut was removed using forceps. The carcasses were spun in a benchtop centrifuge (14,000 g for 10 min) to separate the hemolymph from the exoskeleton. For each time point, 200 pL of undiluted hemolymph was mixed with 30 pg of sOAIP-1. The hemolymph/sOAIP-1 solution was maintained in the dark at room temperature, and immediately before RP-HPLC analysis the hemolymph/sOAIP-1 solution was again spun in a benchtop centrifuge (14,000 g for 10 min). After centrifugation, 30 pg of a control peptide, ω-HXTX-Hvla (Fitches, E. C., Pyati, P., King, G. F., Gatehouse, J. A., 2012, PLoS ONE, 7, e39389), was added and the sample was filtered using a 0.45 pM filter. As shown in Figure 5A, the peptides were separated by RP-HPLC using a linear acetonitrile gradient (5-40% over 40 min), and the identity of each pure, folded toxin was confirmed using mass spectrometry. Figure 5B shows the percentage of intact sOAIP-1 present at each time point, which was determined by comparing the area of the

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PCT/AU2012/001000 sOAIP-1 peak (normalised against that of the ω-HXTX-Hvla peak) to the area of the sOAIP-1 peak at zero time.

[0240] Approximately 40% of sOAIP-1 remained intact after 24 h exposure to hemolymph, while 90% of the peptide was degraded after 72 h and none remained intact after a one-week incubation in undiluted hemolymph. The identity of the sOAIP-1 and ω-HXTX-Hvla rpHPLC peaks was confirmed at each time point based on retention time and mass spectrometric profile.

EXAMPLE 8

Activity of Synthetic OAIP-1 in Mealworms 10 [0241] The insecticidal activity of sOAIP-1 was determined by injection into mealworms or feeding to mealworms. Mealworms (Tenebrio molitor Linnaeus, Coleoptera: Tenebrionidae) of mass ~180 mg/individual were purchased from Pisces Enterprises (Kenmore, Queensland, Australia). For each mealworm, 2.6 μΐ. of sOAIP-1 diluted in ultrapure water was injected into the metathoracic plurite. Injections were performed using a 29.5 gauge insulin syringe (B-D Ultra-Fine, Terumo Medical

Corporation, Elkton, Maryland, USA). At each sOAIP-1 concentration, three replicates of 10 insects each were used, and a similar number of control insects were injected with . ultrapure water and maintained under the same conditions.

[0242] Alternatively, to determine the oral activity of sOAIP-1 mealworms were fed 100 pL of a solid agar diet containing sOAIP-1 at 20 concentrations ranging from 1 pmol to 1 mM. The agar diet allowed a homogenous mixture of sOAIP-land diet to be prepared to ensure an equal dose was delivered to each replicate of 10 individuals (n = 3). Insects were maintained at 24.5°C in the dark at ambient humidity, in sterile Petri dishes.

[0243] LD₅o values (i.e., the dose required to cause 50% mortality) were determined by using mortality at 48 hours post-injection; insects that were dead or moribund were counted as dead. Moribund insects were defined as those that were not feeding or moving independently, and were unable to right themselves when turned on their back. The effects of sOAIP-1 were not reversible once insects reached this level of response; to ensure mortality would occur as a result of toxin exposure within 3-5 days, moribund insects were isolated from the cohort and observed for an additional five days to confirm mortality.

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PCT/AU2012/001000 [0244] The percent mortality in the control group was subtracted from the experimental groups, and the corrected values were then scaled to 100% by multiplying all percent values by a control factor (Equation 1).

Equation 1:

Control factor = 100 /100 - % mortality in control [0245] Equation 2 was then fitted by non-linear regression using GraphPad Prism for Mac v5.0a (software MacKiev, Boston, MA, USA) to the sigmoidal log doseresponse curves to calculate LD50 values.

Equation 2:

Y= 100/[l + 10^A(/ogLDso-X)] [0246] where Y is the percentage mortality, X is the logarithm of the venom dose (in grams crude venom per gram of mealworm), LD50 is the dose required for 50% mortality, and n is the Hill coefficient. Outliers were automatically excluded (Q=1.0%)

I ' (Motulsky & Brown, Detecting Outliers when fitting data with nonlinear regression - a 15 new method based on robust nonlinear regression and the false discovery rate, BMC

Bioinformatics 7:123,2006).

[0247] The LD50 values for mealworms that were either injected with or fed sOAIP-1 are shown in Figures 6A an 6B, respectively. The LD50 value for mealworms injected with sOAIP-1 was 183.8 pmoles/gram and the LD50 value for mealworms fed with sOAIP-1 was 170.5 nmoles/gram.

EXAMPLE 9

ORAL ACTIVITY OF sOAIP-1 IN COTTON BOLLWORM [0248] Cotton bollworms (i.e., larvae of Helicoverpa armigera, Lepidoptera, Noctuidae) were fed 5 pL sOAIP-1 (or water for untreated controls) in 20 pL of agar diet and maintained in 12-well tissue culture dishes.

[0249] The LD50 value was determined as described above using mortality data at 48 hours. The oral LD50 value was 104.5 pmol/g as shown in Figure 7. sOAIP-1 was more potent against cotton bollworms compared to mealworms and termites.

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EXAMPLE 10

SYNERGISTIC ORAL INSECTICIDAL ACTIVITY [0250] Cotton bollworms (Helicoverpa armigera) vjexQ fed 100 pmol of the widely used chemical insecticide imidicloprid (an amount calculated to be the 5 approximate LD50 value for these Lepidoptera at their instar and weight), 100 pmol sOAIP-1, or a 50% mixture of imidicloprid (50 pmol) and sOAIP-1 (50 pmol).

[0251] The 50:50 mixture yielded a mortality of 72% ± 5%, higher than the theoretical additive value of imidicloprid (31% ± 3%) and sOAIP-1 (4% ± 3%) alone (Figure 8). The two insecticides in combination demonstrated synergistic activity.

EXAMPLE 11

FEEDING CHOICE TEST [0252] A choice test was conducted to determine whether sOAIP-1 is repellent. This involved exposing a group of mealworms to both toxin-treated and untreated agar (Figure 9); if both agars were fed on equally, it would suggest that sOAIP-1 is not repellent. Conversely, if the toxin-treated agar was preferentially consumed, it might indicate that sOAIP-1 acts as an attractant.

[0253] According to Dunnett’s Multiple Comparison Test (33), the mortality at 48 h was significantly elevated (P <0.01) above that of the control for all doses except at the lowest two doses (10 and 1 pmol toxin). At 1 pmol toxin, there was no mortality. This indicates that mealworms fed voluntarily on toxin-treated agar, even though there was still untreated agar available to them. The data concur with what was observed when the mealworms were offered only treated agar; the mortality observed in the choice test (where 50% untreated and 50% treated agar was offered) was approximately half that seen in the non-choice test (where only toxin-treated agar was available). This suggests that sOAIP-1 is neither a repellent that repels insects nor an attractant that is preferentially consumed by insects.

EXAMPLE 12

PHENOTYPIC RESPONSE TO OAIP-1 [0254] A scored response test was used to quantify the phenotypic response to sOAIP-1. By comparing the response of the insects injected with sOAIP-lto that of

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PCT/AU2012/001000 . those insects injected only with water, excitatory or depressive effects could be evaluated.

[0255] Phenotypic responses were observed in mealworms 5, 30, and 60 min following injection of sOAIP-1 (Figure 10). A score close to zero represents dead or moribund insects; a score of 2 indicates insects that exhibit an excitatory response but are not paralyzed and can still move independently. Insects scored at 1 exhibited excitatory paralysis, which is categorized as an overstimulation of the nervous system that included constant shaking, rapid leg movements, and uncontrollable spasms resulting in an inability of the insect to move independently (e.g., to right itself when turned upside down).

[0256] Many arachnid toxins inhibit presynaptic voltage-gated ion channels, and this typically induces a depressant response as synaptic transmission is inhibited (King et al., Channels, 2008,2, 100-116). Clearly, sOAIP-1 does not have this mode of action. Rather, the excitatory phenotype induced by sOAIP-1 suggests that it might be an activator of presynaptic voltage-gated ion channels (e.g., it may be an agonist or a gating modifier that slows down channel inactivation) or an agonist of postsynaptic receptors such as nicotinic acetylcholine receptors (the mode of action of neonicotinoid insecticides such as imidacloprid).

EXAMPLE 13

RECOMBINANT EXPRESSION OF OAIP-3 [0257] The gene sequence for OAIP-3 was obtained by reverse translation of the peptide sequence, with optimisation of codons for E. coli expression. Gene construction and subcloning of the synthetic gene into the pLICC expression vector was performed by GeneArt™ (Germany). This vector enables OAIP-3 to be expressed as a (MalE)-His6-MBP-(TEV)-OAIP-3 fusion protein. MalE is a signal sequence used to direct the fusion protein to the E. coli periplasm, the His6 tag is used for affinity purification, and maltose binding protein (MBP) is used to enhance protein solubility. A tobacco etch virus (TEV) protease cleavage recognition site was inserted between the OAlP-3 and MBP coding regions to allow cleavage of the peptide from the fusion protein. Cleavage of the fusion protein with TEV protease results in an additional glycine residue at the N-terminus of OAIP-3, which is a vestige of the TEV cleavage site. This IPTG-inducible construct enables export of the His6-MBP-fusion protein from

- 90 WO 2013/026105

PCT/AU2012/001000 the highly reducing cytoplasm to the E. coli periplasm, where the disulfide-bond folding machinery is located, thus allowing production of correctly folded OAIP-3.

[0258] The plasmid was transformed into E.coli strain BL21 (XDE3) for recombinant OAIP-3 production. Cultures were grown in LB media (LB/Amp) supplemented with 100 pg/mL ampicillin at 37 °C with shaking at 180 rpm. Expression of the toxin gene was induced with 0.2 mM IPTG at an Οϋβοο ~ 1.2 at 20 °C overnight. Cells were harvested the following day by centrifugation at 5000 rpm for 15 min. For the production of ^l3C/¹⁵N-label led toxin, cells were first grown in LB/Amp until OD₆oo reached ~ 0.6, then harvested by centrifugation. The pellet was gently resuspended in

M9 media and further incubated for 2 h at 37°C, 180 rpm before expression with 0.2 mM IPTG, 20°C overnight.

M9 Media (500 mL)

	IX M9 salts	500 mL
	Vitamin solution	1 mL
15	Thiamine solution	1 mL (1 mg/mL)
	Ampicillin	500 pL (100 mg/mL)
	1 M MgSO₄	0.8 mL
	1 M CaCl₂	40 cL
	¹⁵NH4C1	0.5 g
20	^l3C-D-glucose	2g
	5X M9 salts (1 L)
	KH₂PO₄	15g
	Na₂HPO₄	64 g
25	NaCl	5g

[0259] The cell pellet was resuspended in Equilibration Buffer (20 mM Tris, 200 mM NaCl adjusted to pH 8). Whole cell lysate was obtained by lysing cells using a

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Constant Cell Disruptor System (Constant Systems) at 26 KPSI and then the lysate was centrifuged at 18,000 rpm, 4°C for 30 min. The whole cell lysate was then passed through a Ni-NTA Superflow resin (Qiagen) to capture the Hise-tagged fusion protein. Non-specific binding proteins were removed using equilibration buffer containing

15 mM imidazole and then the His6-MBP-OAIP-3 fusion protein was eluted with

500 mM imidazole. The fusion protein was cleaved by adding 500 pg of His6-tagged TEV protease per litre of bacterial culture and the cleavage reaction was allowed to proceed for >12 h at room temperature. The recombinant OAIP-3 liberated by TEV protease cleavage of the fusion protein was further purified using RP-HPLC on a Vydac

Cl8 column (250 x 4.6 mm, 5 pm particle size) using a flow rate of 1 mL/min and a gradient of 25 to 40% Solvent B (0.05% TFA in 90% acetonitrile) in solvent A (0.05% TFA in water) over 15 min.

[0260] Each stage of the expression was analyzed using SDS-PAGE. Significant overexpression of His6-MBP-OAIP-3 fusion protein was observed after

IPTG induction. Almost all of the His6-MBP-OAIP-3 fusion protein was found in the soluble fraction followingcell lysis. None of the His6-MBP-OAIP-3 fusion protein was lost during application of the soluble cell fraction to the Ni-NTA column or during the subsequent washing step with 15 mM imidazole. Most of the His6-MBP-OAIP-3 fusion protein was cleaved successfully with TEV protease to liberate free recombinant

OAIP-3 having the sequence GECGGLMTRCDGKTTFCCSGMNCSPTWKWCVYAP (SEQ ID NO:75).

[0261] The final RP-HPLC purification provided 1.5 mg of recombinant OAIP-3 from 1 L of bacterial culture. The recombinant OAIP-3 was judged to be >95% pure by RP-HPLC, as shown in Figure 11.

[0262] The mass of recombinant OAIP-3 was checked by MALDI-TOF mass spectrometry using a Model 4700 Proteomics Bioanalyser (Applied Biosystems, CA, USA). Samples were spotted (1:1, v:v) with cyano-4-hydroxy-cinnamic acid matrix (5 mg/ mL, 50% acetonitrile/water) matrix. Spectra were collected in positive reflector mode.

[0263] Freeze-dried recombinant OAIP-3 was resuspended at a final concentration of 400 μΜ in 30 mM MES buffer, pH 6, constituted in 5% D2O. The sample was filtered using a Costar® Spin-X® Centrifuge Tube Filters (0.22 pm pore - 92 Η:'·κ/ρ(ι

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Augu:

2012300197 30 Aug 2017 size; Corning, USA), then 300 uL was added to a susceptibility-matched 5 mm outer diameter microtube (Shigemi Inc., Japan). NMR spectra were collected at 298 K on a 900 MHz NMR spectrometer (Bruker BioSpin, Germany ) equipped with a cryogenically-cooled triple resonance probe.

[0264] The structural integrity of OAIP-3 was examined by acquiring a 2D ^!H-¹⁵N

HSQC spectrum on a ¹⁵N-labelled sample. As shown in Figure 12, the HSQC spectrum revealed the expected number of backbone ^!H-¹⁵N connectivities as well as peaks for the sidechain amide groups of the two Trp residues, a pair of sidechain ’H-^N connectivities for the single Asn residue and a single peak for the sidechain amide group of the single Asn residue. The excellent chemical shift dispersion in both the 4i and ^I5N frequency dimensions is characteristic of a protein with a stable tertiary fold.

EXAMPLE 14

INSECTICIDAL ACTIVITY OF rOAIP-3 [0265] The insecticidal activity of recombinant OAIP-3 (rOAIP-3) was confirmed by injection into houseflies (Musca domestica). Injection of rOAIP-3 at a dose of 3.4 micrograms per fly induced paralysis after 30 minutes in all flies and 33% were dead after 24 hours.

[0266] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0267] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0268] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

[0269] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

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Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An isolated or purified proteinaceous molecule which comprises, consists or consists essentially of an amino acid sequence corresponding to a mature peptide or mature peptide together with an amidation signal, wherein the amino acid sequence is

5 selected from the group consisting of:

(a) an amino acid sequence selected from:

DCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVRVGR [SEQ ID NO:4, Orally Active Insecticidal Peptide (OAIP)-l mature peptide and amidation signal]; and

DCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVRV [SEQ ID NO:57,

10 OAIP-1 mature peptide];

(b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in SEQ ID NO: 4 or 57; or (e) an amino acid sequence that is encoded by the nucleotide sequence set forth 15 in SEQ ID NOG (nucleotide sequence encoding OAIP-1 mature peptide and amidation signal), or SEQ ID NO:60 (nucleotide sequence encoding OAIP-1 mature peptide);

(d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 3 or 60, or a

20 complement thereof; or (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in SEQ ID NO: 3 or 60, or a complement thereof, wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more 25 activities selected from the group consisting of: being orally active against insect pests;

increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.
2. A proteinaceous molecule according to claim 1, further comprising an amino acid sequence corresponding to a propeptide region, wherein the amino acid sequence is

30 selected from the group consisting of:

(a) an amino acid sequence:

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DTEDADLMEMVQLSRPFFNPIIRAVELVELREERQR [SEQ ID NO:6,

OAIP-1 propeptide region];

(b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity

5 with the sequence set forth in SEQ ID NO: 6; or (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in SEQ IDNO:5 (nucleotide sequence encoding OAIP-1 propeptide region);

(d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in

10 between) sequence identity with the sequence set forth in SEQ ID NO: 5, or a complement thereof; or (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in SEQ ID NO: 5, or a complement thereof,

15 wherein the proteinaceous molecule further comprising the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.

20
3. A proteinaceous molecule according to claim 1 or claim 2, further comprising an amino acid sequence corresponding to a signal peptide, wherein the amino acid sequence is selected from the group consisting of:

(a) an amino acid sequence:

MIFLLPSIISVMLLAEPVLMLG [SEQ ID NO:8, OAIP-1 signal peptide];

25 (b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in SEQ ID NO: 8; or (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in SEQ ID NO:7 (nucleotide sequence encoding OAIP-1 signal peptide);

-95 Η:'·κφ It

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2012300197 30 Aug 2017 (d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 7, or a complement thereof; or

5 (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in SEQ ID NO: 7, or a complement thereof, wherein the proteinaceous molecule further comprising the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group

10 consisting of: being orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.
4. A proteinaceous molecule according to any one of claims 1 to 3, which comprises, consists or consists essentially of an amino acid sequence corresponding to a

15 precursor peptide having a propeptide region (P) and a mature peptide (M) or mature peptide together with an amidation signal (A), wherein the amino acid sequence is selected from the group consisting of:

(a) an amino acid sequence selected from:

DTEDADLMEMVQLSRPFFNPI1RAVELVELREERQRDCGHLHDPCPND

20 RPGHRTCCIGLQCRYGKCLVRVGR [SEQ ID NO:42, OAIP-1 P + M + A]; and

DTEDADLMEMVQLSRPFFNPIIRAVELVELREERQRDCGHLHDPCPND RPGHRTCCIGLQCRYGKCLVRV [SEQ ID NO:63, OAIP-1 P + M];

(b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity

25 with the sequence set forth in SEQ ID NO: 42 or 63; or (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in SEQ ID NO:41 (nucleotide sequence encoding OAIP-1 P + M + A) or SEQ ID NO:66 (nucleotide sequence encoding OAIP-1 P + M);

(d) an amino acid sequence that is encoded by a nucleotide sequence that 30 shares at least 70% (and at least 71% to at least 99% and all integer percentages in

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2012300197 30 Aug 2017 between) sequence identity with the sequence set forth in SEQ ID NO: 41 or 66, or a complement thereof; or (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth
5 in SEQ ID NO: 41 or 66, or a complement thereof, wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.

10 5. A proteinaceous molecule according to any one of claims 1 to 3, which comprises, consists or consists essentially of an amino acid sequence corresponding to a prepropeptide that includes a signal peptide (S), a propeptide region (P) and a mature peptide (M) or mature peptide together with an amidation signal, wherein the amino acid sequence is selected from the group consisting of:

15 (a) an amino acid sequence selected from:

MIFLLPSIISVMLLAEPVLMLGDTEDADLMEMVQLSRPFFNPIIRAVELV ELREERQRDCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVRVGR [SEQ ID NO: 2, OAIP-1 S + P + M + A]; and

MIFLLPSIISVMLLAEPVLMLGDTEDADLMEMVQLSRPFFNPIIRAVELV 20 ELREERQRDCGHLHDPCPNDRPGHRTCCIGLQCRYGKCLVRV [SEQ ID NO:69,

OAIP-1 S + P + M];

(b) an amino acid sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence similarity or sequence identity with the sequence set forth in SEQ ID NO: 2 or 69; or

25 (c) an amino acid sequence that is encoded by the nucleotide sequence set forth in SEQ ID NO:1 (nucleotide sequence encoding OAIP-1 S + P + M + A) or SEQ ID NO:72 (nucleotide sequence encoding OAIP-1 S + P + M);

(d) an amino acid sequence that is encoded by a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in

30 between) sequence identity with the sequence set forth in SEQ ID NO: 1 or 72, or a complement thereof; or

-972012300197 24 Nov 2017 (e) an amino acid sequence that is encoded by a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in SEQ ID NO: 1 or 72, or a complement thereof, wherein the amino acid sequence of (a), (b), (c), (d) or (e) has any one or more activities selected from the group consisting of: being orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.
6. A construct comprising a nucleic acid molecule operably connected to a heterologous regulatory sequence, wherein the nucleic acid molecule comprises, consists or consists essentially of a nucleotide sequence encoding the amino acid sequence of a proteinaceous molecule as defined in any one of claims 1 to 5.
7. A construct comprising a nucleic acid molecule operably connected to a heterologous regulatory sequence, wherein the nucleic acid molecule comprises, consists or consists essentially of a nucleotide sequence encoding an amino acid sequence corresponding to a mature peptide or mature peptide together with an amidation signal, wherein the nucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence selected from:

gactgtggtcacctgcacgatccatgtcctaatgatcgtcctggacaccgtacgtgctgcataggactccagtgcagat acggtaaatgcctcgtgcgggttggtcgc [SEQ ID

NO:3, nucleotide sequence encoding OAIP-1 mature peptide and amidation signal]; and gactgtggtcacctgcacgatccatgtcctaatgatcgtcctggacaccgtacgtgctgcataggactccagtgcagat acggtaaatgcctcgtgcgggtt [SEQ ID NO:60, nucleotide sequence encoding OAIP-1 mature peptide];

(b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 3 or 60, or a complement thereof;

(c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in SEQ ID NO: 3 or 60, or a complement thereof, wherein the amino acid sequence encoded by the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of: being

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2012300197 30 Aug 2017 orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.
8. A construct according to claim 7, wherein the nucleic acid molecule further 5 comprises a nucleotide sequence encoding an amino acid sequence corresponding to a propeptide region, wherein the nucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence:

gataccgaagatgcagatttgatggagatggttcagttgtctagaccatttttcaatcccattatccgagctgttgaa

10 cttgtggaactacgtgaagaaagacaaaga [SEQ ID NO:5, nucleotide sequence encoding OAIP-1 propeptide region];

(b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 5, or a complement thereof;

15 (c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in SEQ ID NO: 5, or a complement thereof, wherein the amino acid sequence encoded by the nucleic acid molecule further comprising the nucleotide sequence of (a), (b) or (c) has any one or more activities

20 selected from the group consisting of: being orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.
9. A construct according to claim 7 or claim 8, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding an amino acid sequence

25 corresponding to a signal peptide, wherein the nucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence:

atgatatttctactaccttcgatcatttctgttatgcttttggccgagcctgtcctaatgcttgga [SEQ ID NO:7, nucleotide sequence encoding OAIP-1 signal peptide];

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2012300197 30 Aug 2017 (b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 7, or a complement thereof;

(c) a nucleotide sequence that hybridizes under at least medium or high

5 stringency conditions to the sequence set forth in SEQ ID NO: 7, or a complement thereof, wherein the amino acid sequence encoded by the nucleic acid molecule further comprising the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of: being orally active against insect pests; increasing
10 mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.

10. A construct according to any one of claims 7 to 9, wherein the nucleic acid molecule comprises, consists or consists essentially of a nucleotide sequence encoding an amino acid sequence corresponding to a precursor peptide including a propeptide

15 region plus a mature peptide region or mature peptide together with an amidation signal, wherein the nucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence selected from:

gataccgaagatgcagatttgatggagatggttcagttgtctagaccatttttcaatcccattatccgagctgttgaa cttgtggaactacgtgaagaaagacaaagagactgtggtcacctgcacgatccatgtcctaatgatcgtcctggacaccgtac

20 gtgctgcataggactccagtgcagatacggtaaatgcctcgtgcgggttggtcgctag [SEQ ID NO:41, nucleotide sequence encoding OAIP-1 P + M + A]; and gataccgaagatgcagatttgatggagatggttcagttgtctagaccatttttcaatcccattatccgagctgttgaa cttgtggaactacgtgaagaaagacaaagagactgtggtcacctgcacgatccatgtcctaatgatcgtcctggacaccgtac gtgctgcataggactccagtgcagatacggtaaatgcctcgtgcgggtt [SEQ ID NO:66, nucleotide

25 sequence encoding OAIP-1 P +M];

(b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 41 or 66, or a complement thereof;

(c) a nucleotide sequence that hybridizes under at least medium or high 30 stringency conditions to the sequence set forth in SEQ ID NO: 41 or 66, or a complement thereof,

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2012300197 30 Aug 2017 wherein the amino acid sequence encoded by the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of: being orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or

5 preventing insect pests from feeding.
11. A construct according to any one of claims 7 to 9, wherein the nucleic acid molecule comprises, consists or consists essentially of a nucleotide sequence encoding an amino acid sequence corresponding to a prepropeptide including a signal peptide (S), a propeptide region (P) and a mature peptide (M) or mature peptide together with an

10 amidation signal (A), wherein the nucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence selected from: atgatatttctactacettcgatcatttctgttatgcttttg gccgagcctgtcctaatgcttggagataccgaagatgcagatttgatggagatggttcagttgtctagaccatttttcaatcccatt atccgagctgttgaacttgtggaactacgtgaagaaagacaaagagactgtggtcacctgcacgatccatgtcctaatgatcgt

15 cctggacaccgtacgtgctgcataggactccagtgcagatacggtaaatgcctcgtgcgggttggtcgctag [SEQ ID NO:1, nucleotide sequence encoding OAIP-1 S + P + M + A]; and atgatatttctactaccttcgatcatttctgttatgcttttggccgagcctgtcctaatgcttggagataccgaagatgc agatttgatggagatggttcagttgtctagaccatttttcaatcccattatccgagctgttgaacttgtggaactacgtgaagaaag acaaagagactgtggtcacctgcacgatccatgtcctaatgatcgtcctggacaecgtacgtgctgcataggactccagtgca

20 gatacggtaaatgcctcgtgcgggtt [SEQ ID NO:72, nucleotide sequence encoding OAIP-1 S + P + M];

(b) a nucleotide sequence that shares at least 70% (and at least 71% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in SEQ ID NO: 1 or 72, or a complement thereof;

25 (c) a nucleotide sequence that hybridizes under at least medium or high stringency conditions to the sequence set forth in SEQ ID NO: 1 or 72, or a complement thereof, wherein the amino acid sequence encoded by the nucleotide sequence of (a), (b) or (c) has any one or more activities selected from the group consisting of: being

30 orally active against insect pests; increasing mortality of insect pests; stimulating paralysis of insect pests; inhibiting development or growth rate of insect pests; or preventing insect pests from feeding.

- 101 Η:'·κ/ρ(ι

Elenv.?»eii'NRPt)ilW’:DCC\>iZl‘‘-i5607292_i.DOt

30 Augu:

2012300197 30 Aug 2017
12. A microbe comprising the construct of any one of claims 6 to 11.
13. A microbe according to claim 12, wherein the microbe is selected from viruses, bacteria, algae and fungi.
14. A host cell comprising the construct of any one of claims 6 to 11.

5 15. A plant cell comprising the construct of any one of claims 6 to 11.

16. A fusion protein comprising a theraphosid pest-controlling (TPC) agent selected from a proteinaceous molecule according to any one of claims 1 to 5 and a nonTPC peptide or polypeptide.

17. A fusion protein according to claim 16, wherein the non-TPC peptide or 10 polypeptide is Galanthus nivalis agglutinin which improves oral activity of the TPC agent.

18. A composition comprising at least one theraphosid pest-controlling (TPC) agent selected from: a proteinaceous molecule according to any one of claims 1 to 5; or a construct according to any one of claims 6 to 11; or a microbe according to claim 12
15 or claim 13; or a host cell according to claim 14; or a fusion protein according to claim 16 or claim 17; and optionally an agriculturally acceptable carrier, diluent and/or excipient.
19. A composition according to claim 18, which is formulated for oral delivery to insects.
20 20. A composition according to claim 19, wherein the at least one TPC agent is formulated with an attractant for attracting the insects to the composition.
21. A composition according to any one of claims 18 to 20, further comprising another pesticide.
22. A composition according to claim 21, wherein the pesticide is a

25 neonicotinoid pesticide.
23. A method for controlling insect pests, including combating or eradicating infestations of plants, plant products, land and waterways by insect pests, the method comprising administering to a plant or plant part, product or site having or at risk of developing an insect infestation an effective amount of a theraphosid pest-controlling

30 agent selected from: a proteinaceous molecule according to any one of claims 1 to 5; or a construct according to any one of claims 6 to 11; or a microbe according to claim 12 or claim 13; or a host cell according to claim 14; or a fusion protein according to claim 16 or claim 17.

- 102Elenv.?»eii'NRPt)ilW’:DCC\>iZI‘‘-i5607292_i.DOC

30 Augu:

2012300197 30 Aug 2017
24. A method according to claim 23, further comprising co-administration with another pesticide or an agent that enhances the activity of the TPC agent.
25. A method according to claim 24, wherein the pesticide is a neonicotinoid pesticide.

- 103 WO 2013/026105

PCT/AU2012/001000

-1/10FIGURE 1

100ί w 80-

1 2 3 4 5

Toxin

WO 2013/026105

PCT/AU2012/001000

-2/10FIGURE 2

1 2 3 4 5

Toxin

WO 2013/026105

PCT/AU2012/001000

- 3/10 FIGURE 3

A

WO 2013/026105

PCT/AU2012/001000

-4/10FIGURE 4

WO 2013/026105

PCT/AU2012/001000

-5/10FIGURE 5

WO 2013/026105

PCT/AU2012/001000

- 6/10 FIGURE 6

Mortality at 48 hours (%) Mortality at 48 hours (%)

WO 2013/026105

PCT/AU2012/001000

- 7/10 FIGURE 7

FIGURE 8

100η

WO 2013/026105

PCT/AU2012/001000

- 8/10 Response 3 Mortality at 48 h (%) (% of control) g

FIGURE 9

WO 2013/026105

PCT/AU2012/001000

- 9/10 FIGURE 11

WO 2013/026105

PCT/AU2012/001000

-10/10 FIGURE 12

GECGGLMTRCDGKTTFCCSGMNCSPTWKWCVYAP 34 residues,

G=5

P=2

R=1

N/Q=1 ·

W=2 ²³

->

31 BB NH 5SCNH

N-SC • —

R-SC ’24 •22 •l3 —οι
26.

27 ·

0 ·

15.

w-sc o ·

1*6« W

018 0 ₁₉

F2 ppm

- 105

- 106

- 107

- 108

- 109

- 110 - 111 ¹ 112 ¹ 113 ¹ 114

- 115

- 116

- 117

- 118

- 119

- 120 121 ¹ 122 123

- 124

- 125

- 126

127

128

- 129

- 130 131

- 132

- 133

F

P

P m

PCT/AU2012/001000

C;\NRPonbl\DCC\RBR\456323R..I.DOC-24AW/2IH2

SEQUENCE LISTING <110> The University of Queensland KING, Glenn F (US only)

HARDY, Margaret C (US only) <12O> Pest-controlling agents and uses therefor <130> 35112591 <160> 75 <170> Patentln version 3.5 <210> 1 <211> 285 <212> DNA <213> theraphosidae <400> 1 atgatatttc tactaccttc gatcatttct gttatgcttt tggccgagcc tgtcctaatg 60 cttggagata ccgaagatgc agatttgatg gagatggttc agttgtctag accatttttc 120 aatcccatta tccgagctgt tgaacttgtg gaactacgtg aagaaagaca aagagactgt 180 ggtcacctgc acgatccatg tcctaatgat cgtcctggac accgtacgtg ctgcatagga 240 ctccagtgca gatacggtaa atgcctcgtg cgggttggtc gctag 285 <210> 2 , <211> 94· <212> PRT <213> theraphosidae <400> 2

Met Ile Phe Leu Leu Pro Ser Ile lie Ser Val Met Leu Leu Ala Glu 1 5 10 15

Pro Val Leu Met Leu Gly Asp Thr Glu Asp Ala Asp Leu Met Glu Met 20 25 30

Val Gln Leu Ser Arg Pro Phe Phe Asn Pro Ile Ile Arg Ala Val Glu 35 4 0 45

Leu Val Glu Leu Arg Glu Glu Arg Gln Arg Asp Cys Gly His Leu H.i.s 50 55 60

Asp Pro Cys Pro Asn Asp Arg Pro Gly His Arg Thr Cys Cys lie Gly 65 70 75 80

Leu Gin Cys Arg Tyr Gly Lys Cys Leu Val Arg Val Gly Arg 85 90

PCT/AU2012/001000

C:\NRPortbl\DCC\RBR\456.123K_).DOC-24ΛΜ/2ΟΙ2

<210> 3 <211> 108 <212> □NA <213> theraphosidae <400> 3

gactgtggtc acctgcacga tccatgtcct aatgatcgtc ctggacaccg tacgtgctgc ataggactcc agtgcagata cggtaaatgc ctcgtgcggg ttggtcgc

6 0

108 <2l0> 4 <211> 36 <212> PRT <213> theraphosidae <400> 4

Asp Cys Gly His Leu His Asp Pro Cys Pro Asn Asp Arg Pro Gly His 1 5 10 15

Arg Thr Cys Cys Ile Gly Leu Gln Cys Arg Tyr Gly Lys Cys Leu Val 20 25 30

Arg Val Gly Arg 35 <210> 5 <211> 108 <212> DNA <213> theraphosidae <400> 5 gataccgaag atgcagattt gatggagatg gttcagttgt ctagaccatt tttcaatccc attatccgag ctgttgaact tgtggaacta cgtgaagaaa gacaaaga

108 <210> 6 <211> 36 <212> PRT <213> theraphosidae <400> 6

Asp Thr G.lu Asp Ala Asp Leu Met Glu Met Val Gln Leu Ser Arg Pro 15 10 15

Phe Phe Asn Pro Ile lie Arg Ala Val Glu Leu Val Glu Leu Arg Glu 20 25 30

Glu Arg Gln Arg 35

PCT/AU2012/001000

CANRPortbI\DCC\RBR\45632)R I DOC-2-M>K/2»l2

-3 <210>

<211>

<212>

DNA <213 > theraphosidae <400> 7 ' atgatatttc tactaccttc gatcatttct gttatgcttt tggccgag'cc tgtcctaatg cttgga <210> 8 <211> 22 <212> PRT <213> theraphosidae <400> 8

Met Ile Phe Leu Leu Pro Ser Ile lie Ser Val Met Leu Leu Ala Glu 1 5 10 15

Pro Val Leu Met Leu Gly 20 <210> 9 <211> 240 <212> DNA <213> theraphosidae <400> 9

atgagggttc tgttcatcat tgccggatta gccctgcttt ccgttgtttg ctacacttcc 60 gagatgaagg agcgaagctc atttaatgaa gtgctttcgg agttttttgc tgccgacgag 120 cctcaggaaa gagactgtct aggacagtgg gccagttgtg aacctaagaa cagcaagtgc 180 tgccccaact atgcatgtac ttggaaatac ccttggtgca gatatcgcgc tggtaaatag 240

<210> 10 <211> 79 <212> PRT <213> theraphosidae <400> 10 Met Arg r Val Leu Phe Ile lie Ala Gly Leu Ala Leu Leu Ser Val 1 5 10 15

Cys Tyr Thr Ser Glu Met Lys Glu Arg Ser Ser Phe Asn Glu Val Leu 20 25 30

Ser Glu Phe Phe Ala Ala Asp Glu Pro Gln Glu Arg Asp Cys Leu G]y 35 40. 45

PCT/AU2012/001000

C:\NRPonbl\DCC\R BRU563 23K I DOC-2 4AiK/2<) 12

-4Gln Trp Ala Ser Cys Glu 50

Pro Lys Asn Ser Lys 55

Cys Cys Pro Asn Tyr 60

Ala Cys Thr Trp Lys 65

Tyr Pro Trp Cys Arg 70

Tyr Arg Ala Gly Lys 75 <210> 11 <211> 108 <212 > DNA <213> theraphosidae <400> 11 gactgtctag gacagtgggc cagttgtgaa cctaagaaca gcaagtgctg ccccaactat gcatgtactt ggaaataccc ttggtgcaga tatcgcgctg gtaaatag <210> 12 <211> 35 <212> PRT <213> theraphosidae <400> 12

Asp Cys Leu Gly Gin Trp Ala Ser Cys Glu Pro Lys Asn Ser Lys Cys 15 10 15

Cys Pro Asn Tyr Ala Cys Thr Trp Lys Tyr Pro Trp Cys Arg Tyr Arg 20 25 30

Ala Gly Lys 35 <210> 13 <211> 75 <212> DNA <213> theraphosidae <400> 13 tccgagatga aggagcgaag ctcatttaat gaagtgcttt cggagttttt tgctgccgac gagcctcagg aaaga

108 <210> 14 <211> 25 <212> PRT <213> theraphosidae <400> 14

Ser Glu Met Lys Glu Arg Ser Ser Phe Asn Glu Val Leu Ser Glu Phe 1 5 10 15

PCT/AU2012/001000

C:\NRPortbl\DCC\RBR\456323K_l.1X)C-24AW/2IH2

Phe Ala Ala Asp Glu Pro Gln Glu Arg 20 25 <210> 15 <211> 57 <212> DNA <213> theraphosidae <400> 15 atgagggttc tgttcatcat tgccggatta gccctgcttt ccgttgtttg ctacact <210> IS <211> 19 <212> PRT <213 > theraphosidae <400> 16

Met Arg Val Leu Phe Ile Ile Ala Gly Leu Ala Leu Leu Ser Va.I val 15 10 15

Cys Tyr Thr <210> 17 <211> 196 <212> DNA <213> theraphosidae <400> 17

atgaagacat cagttttatt cgccatcttg ggattggctc tgcttttctg cctttca ttt 60 ggagttgaat tggaagagac cggaagggag tgtgggggac taatgacccg ctgtgat.gga .12 0 aagacaacgt tttgctgttc aggtatgaat tgttctccaa cgtggaaatg gtgtgtctat 18 0 gctcctggac gccgttga 198

<210> 18 <211> 65 <212> PRT <213> theraphosidae <400> 18

Met Lys Thr Ser Val Leu Phe Ala lie Leu Gly Leu Ala Leu Leu Phe 1 5 10 15

Cys Leu Ser Phe Gly Val Glu Leu Glu Glu Thr Gly Arg Glu Cys Gly 20 25 30

PCT/AU2012/001000

C:\NRPonbl\DCC\XBRU56123K_l. DOC-24/0H/20I2

-6Gly Leu Met Thr Arg Cys 35

Asp Gly Lys 40

Thr Thr

Phe Cys Cys Ser Gly 45

Met Asn Cys Ser Pro Thr 50

Trp Lys Trp Cys Val 55

Tyr Ala Pro Gly Arg 60

Arg <210> 19 <211> 111 <212> DNA <213> theraphosidae <400> 19 gagtgtgggg gactaatgac ccgctgtgat ggaaagacaa cgttttgctg ttcaggtatg 60 aattgttctc caacgtggaa atggtgtgtc tatgctcctg gacgccgttg a 111 <210> 20 <211> 36 <212> PRT <213> theraphosidae <400> 20

Glu Cys Gly Gly Leu Met Thr Arg Cys Asp Gly Lys Thr Thr Phe Cys 15 10 15

Cys Ser Gly Met Asn Cys Ser Pro Thr Trp Lys Trp Cys Val Tyr Ala 20 25 30

Pro Gly Arg Arg 35 <210> 21 <211> 24 <212> DNA <213> theraphosidae <400> 21 gttgaattgg aagagaccgg aagg 24 <210> 22 <211> 8 <212> PRT <213> theraphosidae <400> 22

Val Glu Leu Glu Glu Thr Gly Arg 1 5

PCT/AU2012/001000

C.\NRPorbl\DCCRBRU5ft.123>1_1 IX)C-24/118/2012

<210> 23 <211> ⁶³ <212> DNA <213> theraphosidae <400> 23 atgaagacat cagttttatt cgccatcttg ggattggctc· tgcttttctg cctttcattt 60 gga 63

<210> 24 <211> 21 <212> PRT <213> theraphosidae <400> 24 Met Lys i Thr Ser Val Leu Phe Ala Ile Leu Gly Leu Ala Leu lieu Phe 1 5 10 15

Cys Leu Ser Phe Gly 20 <210> 25 <211> 243 <212> UNA < 213 > theraphos idae <400> 25

atgaaggctt cactattcgc tgtcatattt ggattggttg tgttgtgcgc ctgctccttt 60 gccgaagatc aatttgcttc gcctaatgaa ctgctgaaat caatgtttgt ggagagtaca 120 catgaactca cacctgaagt ggaaggaaga tattgccaaa aatggatgtg gacctgtgat 18 0 gcagaaagaa aatgctgcga agacatggct tgcgaactgt ggtgcaaaaa gagactcgga 24 0 taa 243

<210> 26 <211> 80 <212> PRT <213> theraphosidae <400> 26

Met Lys Ala Ser Leu Phe Ala Val Ile Phe Gly Leu Val Val Leu Cys 1 5 10 15

Ala Cys Ser Phe Ala Glu Asp Gin Phe Ala Ser Pro Asn Glu Leu Leu 20 25 30

PCT/AU2012/001000

C:\NRPof1bl\DCe\RBR\456WH I.DOC-?.44H</2lH2

-8Lys Ser Met Phe Val Glu Ser Thr His Glu Leu Thr Pro Glu Val Glu 35 40 45

Gly Arg Tyr Cys Gin Lys Trp Met Trp Thr Cys Asp Ala Glu Arg Lys 50 55 60

Cys Cys Glu Asp Met Ala Cys Glu Leu Trp Cys Lys Lys Arg Leu Gly 65 70 75 80 <210> 27 <211> 90 <212> DNA <213> theraphosidae <400> 27 tattgccaaa aatggatgtg gacctgtgat gcagaaagaa aatgctgcga. agacatggct tgcgaactgt ggtgcaaaaa gagactcgga <210> 28 <211> 30 <212 > PRT <213> theraphosidae <400> 28

Tyr Cys Gin Lys Trp Met Trp Thr Cys Asp Ala Glu Arg Lys Cys Cys 1 5 10 15

Glu Asp Met Ala Cys Glu Leu Trp Cys Lys Lys Arg Leu Gly 20 25 30 <210> 29 <211> 87 <212> DNA <213> theraphosidae <400> 29 gaagatcaat ttgcttcgcc taatgaactg ctgaaatcaa tgtttgtgga gagtacacat gaactcacac ctgaagtgga aggaaga <210> 30· <211> 29 <212> PRT <213> theraphosidae <400> 30

Glu Asp Gin Phe Ala Ser Pro Asn Glu Leu Leu Lys Ser Met Phe Val 15 10 15

PCT/AU2012/001000

C:\NRPonbl\DCC\R8R\456i21K_ I DOC-2-MiH/2(H 2

-9Glu Ser Thr His Glu Leu Thr Pro Glu Val Glu Gly Arg 20 25 <210> 31 <2ll> 63 <212> DNA <213> theraphosidae <400> 31 atgaaggctt cactattcgc tgtcatattt ggattggttg tgttgtgcgc ctgctccttt gcc <210> 32 <2ll> 21 <212 > PRT <213> theraphosidae <400> 32

Met Lys Ala Ser Leu Phe Ala Val lie Phe Gly Leu Val Val Leu Cys

1 5 10 . 15

Ala Cys Ser Phe Ala 20 <210> 33 <211> 237 <212> DNA <213 > theraphosidae <400> 33

atgttgattg tcattctgac atgtgctctg ttggttattt atcacgccgc agcagcggag 60 gaacttgaag caaaagatgt gatagaatct aaagcactag caactctgga tgaagaaa.ga 120 ttcgaatgtg ttttgaaatg cgacattcaa tacaatggga aaaattgtaa gggcaaagga 18 0 gagaacaaat gttcaggagg atggagatgc cgttttaaat tgtgtctgaa aataLaa 23 7

<210> 34 <211> 78 <212> PRT <213> theraphosidae <400> 34

Met Leu Ile Val lie Leu Thr Cys Ala Leu Leu Val Tie Tyr His Ala 1 5 10 15

Ala Ala Ala Glu Glu Leu Glu Ala Lys Asp Val Ile Glu Ser Lys Ala 20 25 30

PCT/AU2012/001000

ClNRPortbhDCC\RBR\456.12.1H. I.DOC-?.4A>K/2I)I2

Leu Ala Thr Leu Asp Glu Glu Arg Phe Glu Cys Val Leu Lys Cys Asp 35 40 45 lie Gln Tyr Asn Gly Lys Asn Cys Lys Gly Lys Gly Glu Asn Lys Cys 50 55 60

Ser Gly Gly Trp Arg Cys Arg Phe Lys Leu Cys Leu Lys lie 65 70 75 <210> 35 <211> 117 <212> DNA <213> theraphosidae <400> 35 ttcgaatgtg ttttgaaatg cgacattcaa tacaatggga aaaattgtaa gggcaaagga gagaacaaat gttcaggagg atggagatgc cgttttaaat tgtgtctgaa aatataa

1 1 7

<210> 36 <211> 38 <212> PRT <213> theraphosidae <400> 36 Phe Glu Cys Val Leu :

Lys Gly Lys Gly Glu Asn Lys Cys Ser Gly Gly Trp Arg Cys Arg Phe 20 25 30

Lys Leu Cys Leu Lys Ile 35 <210> 37 <211> 63 .

<212> DNA <213> theraphosidae <400> 37 gaggaacttg aagcaaaaga tgtgatagaa tctaaagcac tagcaactct ggatgaagaa aga <210> 38 <211> 21 <212> PRT <213> theraphosidae <400> 38

PCT/AU2012/001000

C:\NRPortbl\DCC\RBRM$6'2iK_ I.DOC-24ANU20I2

- 11 Glu Glu Leu Glu Ala Lys Asp Val Ile Glu Ser Lys Ala Leu Ala Thr

1 5 10 15 Leu Asp Glu Glu Arg 20 <210> 39 <211> 57 <212> DNA <213> theraphosidae <400> 39 atgttgattg tcattctgac atgtgctctg ttggttattt atcacgccgo agcagcg <210> 40 <211> 19 <212> PRT <213> theraphosidae <400> 40 Met Leu lie Val Ile Leu Thr Cys Ala Leu Leu Val lie Tyr His Ala 1 5 10 15

Ala Ala Ala <210> 41 <211> 219 <212> DNA <213> theraphosidae <400> 41

gataccgaag atgcagattt gatggagatg gttcagttgt ctagacoatt tttcaatccc 60 attatccgag ctgttgaact tgtggaacta cgtgaagaaa gacaaagaga ctgtggtcac 120 ctgcacgatc catgtcctaa tgatcgtcct ggacaccgta cgtgctgcat aggactccag 180 tgcagatacg gtaaatgcct cgtgcgggtt ggtcgctag 219

<210> 42 <211> 72 <212> PRT <213> theraphosidae <400> 42

Asp Thr Glu Asp Ala Asp Leu Met Glu Met Val Gln Leu Ser Arg Pro 1 5 10 15

Phe Phe Asn Pro Ile Ile Arg Ala Val Glu Leu Val Glu Leu Arg Glu 20 25 30

PCT/AU2012/001000

C \NRPonbl\DCC\RBR\45f>37.3KJ DQC-2W)K/2IH2

12Glu Arg Gln Arg Asp Cys Gly His Leu His Asp Pro Cys Pro Asn Asp 35 40 45

Arg Pro Gly His Arg Thr Cys Cys Ile Gly Leu Gln Cys Arg Tyr Gly 50 55 60

Lys Cys Leu Val Arg, Val Gly Arg 65 70 <210> 43 <211> 183 <212> DNA <213 > theraphosidae <400> 43

tccgagatga aggagcgaag ctcatttaat gaagtgcttt cggagttttt tgCtgccgac 60 gagcctcagg aaagagactg tctaggacag tgggccagtt gtgaacctaa gaacagcaag 120 tgctgcccca actatgcatg tacttggaaa tacccttggt gcagatatcg cgctggtaaa 180 tag 183

<210> . 44 <211> 60 <212> PRT. <213> theraphosidae <400> 44 Ser Glu ί Met Lys Glu A

Phe Ala Ala Asp Glu Pro Gln Glu Arg Asp Cys Leu Gly Gln Trp Ala 20 25 30

Ser Cys Glu Pro Lys Asn Ser Lys Cys Cys Pro Asn Tyr Ala Cys Thr 35 40 45

Trp Lys Tyr Pro Trp Cys Arg Tyr Arg Ala Gly Lys 50 55 60 <210> 45 <211> 135 <212> DNA <213> theraphosidae <400> 45 gttgaattgg aagagaccgg aagggagtgt gggggactaa tgacccgctg tgatggaaag

PCT/AU2012/001000

C:\NRJ>ortbl\DCCRBRU56323«_l OOC-2WK/2III2

- 13 acaacgtttt gctgttcagg tatgaattgt tctccaacgt ggaaatggtg tgtctatgct 120 cctggacgcc gttga 135

<210> 46 <211> 44 <212> PRT <213> theraphosidae <400> 46 Val Glu Leu Glu Glu '

Cys Asp Gly Lys Thr Thr Phe Cys Cys Ser Gly Met Asn Cys Ser Pro 20 25 30

Thr Trp Lys Trp Cys Val Tyr Ala Pro Gly Arg Arg 35 40 <210> 47 <211> 180 <212> DNA <213> theraphosidae <400> 47

gaagatcaat ttgcttcgcc taatgaactg ctgaaatcaa tgtttgtgga gagtacacat. 60 gaactcacac ctgaagtgga aggaagatat tgccaaaaat ggatgtggac ctgtgataca 12 C gaaagaaaat gctgcgaaga catggcttgc gaactgtggt gcaaaaagag actcagataa 18 0

<210> 48 <211> 59 <212> PRT <213> theraphos idae <400> 48 Glu Asp i Gln Phe Ala ;

Glu Ser Thr His Glu Leu Thr Pro Glu Val Glu Gly Arg Tyr Cys Gln 20 25 30 .

Lys Trp Met Trp Thr Cys Asp Ala Glu Arg Lys Cys Cys Glu Asp Met 35 40 45

Ala Cys Glu Leu Trp Cys Lys Lys Arg Leu Gly 50 55

PCT/AU2012/001000

CANRPonhl\DCC\RBR\4%)?H 1.ΙΧΧ?-24Λ)Κ/2ι»Ι2

- 14<210> 49 <211> 180 <212> DNA <213> theraphosidae <400> 49

gaggaacttg aagcaaaaga tgtgatagaa tctaaagcac tagcaactct ggatgaagaa 60 agattcgaat gtgttttgaa atgcgacatt caatacaatg ggaaaaattg taagggcaaa 12.0 ggagagaaca aatgttcagg aggatggaga tgccgtttta aattgtgtct gaaaatataa 180

<210> 50 <211> 59 <212> PRT <213> theraphos idae <400> 50 Glu Glu i Leu Glu Ala :

Leu Asp Glu Glu Arg Phe Glu Cys Val Leu Lys Cys Asp Ue Gln Tyr 20 25 30

Asn Gly Lys Asn Cys Lys Gly Lys Gly Glu Asn Lys Cys Ser Gly Gly 35 40 45

Trp Arg Cys Arg Phe Lys Leu Cys Leu Lys lie 50 55 <210> 51 <211> 234 <212> DNA <213> theraphosidae <400> 51

atgttgattg tcattctgac atgtgctctg ttggttattt atcacgccgc agcagcggag 60 gaacttgaag caaaagatgt gatagaatct aaagcactag caactctgga tgaagaaaga 12 0 ttcgaatgtg ttttgaaatg cgacattaaa tacgatggga aaaattgtaa gggcaaagga 180 gagaagaaat gttcaggagg atggagatgc cgttttaaat tgtgtctgaa aata 234

<210> 52 <211> 78 <212> PRT <213> theraphosidae <400> 52

Met Leu Ile Val Ile Leu Thr Cys Ala Leu Leu Val I.le Tyr His Ala 15 10 ' 15

PCT/AU2012/001000

C:\MRPonbl\DCCVU3RUSW2.1H_ 1 DOC-24fllH/2lll2

Ala Ala Ala Glu Glu Leu Glu Ala 20

Leu Ala Thr Leu Asp Glu Glu Arg 35 40

Ile Lys Tyr Asp Gly Lys Asn Cys 50 55

Ser Gly Gly Trp Arg Cys Arg Phe 65 70 <210> 53 <211> 114 <212> DNA <213> theraphosidae <400> 53 ttcgaatgtg ttttgaaatg cgacattaaa gagaagaaat gttcaggagg atggagatgc

<210> 54 <211> 38 <212> PRT <213> theraphosidae <400> 54

Phe Glu Cys Val Leu Lys Cys Asp ^{1 5}

Lys Gly Lys Gly Glu Lys Lys Cys 20

-15Lys Asp Val lie Glu Ser Lys Ala 25 30

Phe Glu Cys Val Leu Lys Cys Asp 45

Lys Gly Lys Gly Glu Lys Lys Cys 60

Lys Leu.Cys Leu Lys Ile 75 tacgatggga aaaattgtaa gggcaaagga cgttttaaat tgtgtctgaa aata lie Lys Tyr Asp Gly Lys Asn Cys .10 15

Ser Gly Gly Trp Arg Cys Arg Phe 25 30

114

Lys Leu Cys Leu Lys Ile

35 <210> 55 <211> 177 <212> DNA <213> theraphosidae <400> 55

gaggaacttg aagcaaaaga tgtgatagaa agattcgaat gtgttttgaa atgcgacatt tctaaagcac tagcaactcl ggatgaagaa aaatacgatg ggaaaaattg taagggcaaa

1 20 ggagagaaga aatgttcagg aggatggaga tgccgtttta aattgtgtct gaaaata

177

PCT/AU2012/001000

C:\NRPonbl\DCC\RBR\456323H_l,DOC-244W/2(>l2

- 16<210> 56 <211> 59 <212> PRT <213 > theraphosidae <400> 56

Glu Glu Leu Glu Ala Lys Asp Val Ile Glu Ser Lys Ala Leu Ala Thr 1 5 10 15

Leu Asp Glu Glu Arg Phe Glu Cys Val Leu Lys Cys Asp Ile Lys Tyr 20 25 30

Asp Gly Lys Asn Cys Lys Gly Lys Gly Glu Lys Lys Cys Ser Gly Gly 35 40 ’ 45

Trp Arg Cys Arg Phe Lys Leu Cys Leu Lys Ile 50 55 <210> 5 7 <211> 34 <212> PRT <213> theraphosidae <400> 57

Asp Cys Gly His Leu His Asp Pro Cys Pro Asn Asp Arg Pro Gly His 1 5 10 15 .

Arg Thr Cys

Cys Ile Gly Leu Gin 20

Cys

Arg

Tyr

Gly

Lys

Cys Leu Va ! 30

Arg Val <210> 58 <211> 33 <212> PRT <213> theraphosidae <400> 58

Asp Cys Leu Gly Gin Trp Ala Ser Cys Glu Pro Lys Asn Ser Lys Cys 1 5 10 15

Cys Pro Asn Tyr Ala Cys Thr Trp Lys Tyr Pro Trp Cys Arg Tyr Arg 20 ’ 25 30

Ala

PCT/AU2012/001000

C:\NRPonbl\DCC\R13R\456 UMJ.DOC··;Ζ4ΛΜ/2Ο12

- 17<210> 59 <211> 33 <212> PRT <213> theraphosidae <400> 59

Glu Cys Gly Gly Leu Met Thr Arg Cys Asp Gly Lys Thr Thr Phe Cys . 1 5 10 .15

Cys Ser Gly Met Asn Cys Ser Pro Thr Trp Lys Trp Cys Val Tyr Ala 20 25 30.

Pro <210> 60 <211> 102 <212> DNA <213> theraphosidae <400> 60 gactgtggtc acctgcacga tccatgtcct aatgatcgtc ctggacaccg tacgtgctgc ataggactcc agtgcagata cggtaaatgc ctcgtgcggg tt

102 <210> 61 <211> 99 <212> DNA <213> theraphosidae <400> 61 gactgtctag gacagtgggc cagttgtgaa cctaagaaca gcaagtgctg ccccaactat gcatgtactt ggaaataccc ttggtgcaga tatcgcgct <210> 62 <211> 99 <212> DNA <213> theraphosidae <400> 62 gagtgtgggg gactaatgac ccgctgtgat ggaaagacaa cgttttgctg ttcaggtatg aattgttctc caacgtggaa atggtgtgtc tatgctcct <210> 63 <211> 70 <212 > PRT <213> theraphosidae <400 > 63

PCT/AU2012/001000

C.\NRPortW\l>CC\RBRU56523« J.DOC-2WJU2DI2

Asp Thr Glu Asp Ala Asp Leu Met Glu Met Val Gln Leu Ser Arg 15 Pro 1 5 10 Phe Phe : Asn Pro Ile Ile Arg Ala Val Glu Leu Val GLu Leu Arg G1 u 20 25 30 Glu Arg Gln Arg Asp Cys Gly His Leu His Asp Pro Cys Pro Asn Asp 35 40 45 Arg Pro Gly His Arg Thr Cys Cys Ile Gly Leu Gin Cys Arg Tyr Gly 50 55 60 Lys Cys Leu Val Arg Val 65 70 <210> 64 <211> 58 <212> PRT <213> theraphos idae <400> 64 Ser Glu Met Lys Glu Arg Ser Ser Phe Asn Glu Val Leu Ser Glu Phe 1 5 10 1.5 Phe Ala Ala Asp Glu Pro Gln Glu Arg Asp Cys Leu Gly Gln Trp Ala 20 25 30 Ser Cys Glu Pro Lys Asn Ser Lys Cys Cys Pro Asn Tyr Ala Cys Thr 35 40 45 Trp Lys Tyr Pro Trp Cys Arg Tyr Arg Ala 50 55 <210> 65 <211> 41 <212> PRT <213> theraphosidae <400> 65 Val Glu Leu Glu Glu Thr Gly Arg Glu Cys Gly Gly Leu Met Thr Arg 1 5 10 15 Cys Asp Gly Lys Thr Thr Phe Cys Cys Ser Gly Met Asn Cys Ser Pro 20 25 30

Thr Trp Lys Trp Cys Val Tyr Ala Pro

PCT/AU2012/001000

CANRPonbf\DCC\RBRU56l21«,.l DOC-JWiH/2(il2

- 19 - 35 4 0 <210> 66 <211> 210 <212> DNA <213> theraphos idae <400> 66 gataccgaag atgcagattt gatggagatg gttcagttgt ctagaccatt tttcaatccc 60 attatccgag ctgttgaact tgtggaacta cgtgaagaaa gacaaagaga ctgtggtcac 12 0 ctgcacgatc catgtcctaa tgatcgtcct ggacaccgta cgtgctgcat aggactccag 180 tgcagatacg gtaaatgcct cgtgcgggtt 21 0 <210> 67 <211> 174 <212> DNA <213> theraphosidae <400> 67 tccgagatga aggagcgaag ctcatttaat gaagtgcttt cggagttttt tgctgccgac 60 gagcctcagg aaagagactg tctaggacag tgggccagtt gtgaacctaa gaacagcaag 120 tgctgcccca actatgcatg tacttggaaa tacccttggt gcagatatcg cgct .174 <210> 68 <211> 123 <212> DNA <213> theraphosidae <400> 68 gttgaattgg aagagaccgg aagggagtgt gggggactaa tgacccgctg tgatggaaag 60 acaacgtttt gctgttcagg tatgaattgt tctccaacgt ggaaatggtg tgtctatgct 120 cct 123 <210> 69 <211> 92 <212> PRT <213> theraphosidae ) <400> 69 Met Ile Phe Leu Leu Pro Ser Ile Ile Ser Val Met Leu Leu Ala GJ u 1 5 10 15 Pro Val Leu Met Leu Gly Asp Thr Glu Asp Ala Asp Leu Met Glu Met 2 0 25 30 Val Gln Leu Ser Arg Pro Phe Phe Asn Pro He Ile Arg Ala Val Glu

PCT/AU2012/001000

C:\NRPortbl\LX.C\RBR\4W2'»K I ΕΧΧ2.4ΛΙΧ/2ΟΙ2

35 40 45 Leu Val Glu Leu Arg Glu Glu Arg Gin Arg Asp Cys Gly Hi.s Leu H.is ⁵⁰ 55 60 Asp Pro Cys Pro Asn Asp Arg Pro Gly His Arg Thr Cys Cys lie Gly 65 70 75 80 Leu Gin Cys Arg Tyr Gly Lys Cys Leu Val Arg Val 85 90 <210> 70 <211> 77 <212> PRT <213> theraphosidae <400> 70 Met Arg Val Leu Phe Ile Ile Ala Gly Leu Ala Leu Leu Ser Val Val 1 5 10 15 Cys Tyr Thr Ser Glu Met. Lys Glu Arg Ser Ser Phe Asn Glu Val lieu 20 25 30 Ser Glu Phe Phe Ala Ala Asp Glu Pro Gin Glu Arg Asp Cys Leu G.1 y 35 40 45 Gin Trp Ala Ser Cys Glu Pro Lys Asn Ser Lys Cys Cys Pro Asn Tyr 50 55 60 . Ala Cys Thr Trp Lys Tyr Pro Trp Cys Arg Tyr Arg Ala 65 70 75 <210> 71 <211> 62 <212> PRT <213> theraphosidae <400> 71 Met Lys Thr Ser Val Leu Phe Ala Ile lieu Gly Leu Ala Leu Leu Phe 1 5 10 15 Cys Leu Ser Phe Gly Val Glu Leu Glu Glu Thr Gly Arg Glu Cys G.1 y 20 25 30 Gly Leu Met Thr Arg Cys Asp Gly Lys Thr Thr Phe Cys Cys Ser Gly 35 40 45

PCT/AU2012/001000

C:\NRPoitbl\DCC\RBRUyi32.‘W,I.OOC-24A)R/2012

-21 Met Asn Cys Ser Pro Thr Trp Lys Trp Cys Val Tyr Ala Pro 50 55 60 <210> 72 <211> 276 <212> DNA <213> theraphosidae <400> 72

atgatatttc tactaccttc gatcatttct gttatgcttt tggccgagcc tgtcctaatg 60 cttggagata ccgaagatgc agatttgatg gagatggttc agttgtctag accatttttc 120 aatcccatta tccgagctgt tgaacttgtg gaactacgtg aagaaagaca aagagactgt. 180 ggtcacctgc acgatccatg tcctaatgat cgtcctggac accgtacgtg ctgcatagga 240 ctccagtgca gatacggtaa atgcctcgtg cgggtt 276

<210> 73 <211> 231 <212> DNA <213> theraphosidae <400> 73

atgagggttc tgttcatcat tgccggatta gccctgcttt ccgttgtttg ctacacttcc 60 gagatgaagg agcgaagctc atttaatgaa gtgctttcgg agttttttgc tgccgacgag 120 cctcaggaaa gagactgtct aggacagtgg gccagttgtg aacctaagaa cagcaagtgc 18 0 tgccccaact atgcatgtac ttggaaatac ccttggtgca gatatcgcgc t 231

<210> 74 <211> 186 <212> DNA <213> theraphosidae <400> 74 gctcct

cagttttatt cgccatcttg ggattggctc tgcttttctg cctttcattt 60 tggaagagac cggaagggag tgtgggggac taatgacccg ctgtgatgga 12.0 tttgctgttc aggtatgaat tgttctccaa cgtggaaatg gtgtgtctat 180 186

<210> 75 <211> 34 <212> PRT <213> theraphosidae <400> 75

Gly Glu Cys Gly Gly Leu Met Thr Arg Cys Asp Gly Lys Thr Thr Phe

PCT/AU2012/001000

C:\NRPortbhDCC\RBRU5612m J.DOf-24W2M2

-221 5 10 15

Cys Cys Ser Gly Met Asn Cys Ser Pro Thr Trp Lys Trp Cys Val Tyr 20 25 30

Ala Pro