US 20060178292A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0178292 A1 Ruvkun et al. (43) Pub. Date: Aug. 10, 2006

(54) METHODS AND COMPOSITIONS 0F Related Us, Application Data ECDYSOZOAN MOLT INHIBITION (60) Provisional application No. 60/437,235, ?led on Dec. (75) Inventors: Gary Ruvkun, Newton, MA (US); 31, 2002. Alison Frand, Cambridge, MA (US) Publication Classi?cation Correspondence Address: CLARK & ELBING LLP (51) IIlt- Cl 101 FEDERAL STREET A01N 37/18 (2006.01) BOSTON, MA 02110 (US) C12Q 1/68 (2006.01) G01N 33/567 (2006.01) G01N 33/53 (2006.01) (73) Assignee: The General Hospital Corporation, (52) US. Cl...... 514/2; 435/6; 435/7.2 Boston, MA (US) (57) ABSTRACT (21) Appl_ No; 10/540,445 In general, this invention relates to nucleic acid and amino acid sequences involved in molting and the use of these (22) PCT Filed; Dec 31, 2003 sequences as targets for the development of compounds that disrupt EcdysoZoan molting, and are useful as insecticides, (86) PCT No.1 PCT/US03/41788 nematicides, and anti-parasitic agents. Patent Application Publication Aug. 10, 2006 Sheet 1 0f 5 US 2006/0178292 A1 Patent Application Publication Aug. 10, 2006 Sheet 2 0f 5 US 2006/0178292 A1

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METHODS AND COMPOSITIONS OF compound, Where an alteration in expression identi?es the ECDYSOZOAN MOLT INHIBITION candidate compound as a candidate compound that modu lates molting. STATEMENT AS TO FEDERALLY SPONSORED [0008] In various embodiments of the previous aspects, RESEARCH the method identi?es a compound that increases or decreases [0001] This Work Was supported in part by the National transcription of a mlt nucleic acid molecule. In other Institutes of Health (NIH GM 44619). The government may embodiments of the previous aspects, the method identi?es have certain rights in this invention. a compound that increases or decreases translation of an mRNA transcribed from the mlt nucleic acid molecule. In BACKGROUND OF THE INVENTION still other embodiments of the identi?cation methods described herein, the compound is a member of a chemical [0002] In general, the invention features methods and library. In preferred embodiments, the cell is in a nematode. compositions that disrupt molting and are therefore useful targets for pesticides. [0009] Typically, a compound that decreases transcription or translation of a mlt nucleic acid molecule is useful in the [0003] Nematodes represent one out of every ?ve animals invention. For some applications, hoWever, a compound that on the planet, and virtually all plant and animal species are increases transcription or translation of a mlt nucleic acid targeted by at least one parasitic nematode. Plant-parasitic molecule is useful, for example, a mlt nucleic acid (e.g., nematodes reduce the yield of the World’s 40 major food W08F4.6, F09B12.1, or W01F3.3) that When overexpressed staples resulting in losses of approximately 12.3% annually. leads to larval arrest or death, or a mlt nucleic acid (e.g., Parasitic nematodes also damage human and domestic ani C17G1.6, CD4.6, C42D8.5, F08C6.1) that encodes a mal health. Lymphatic ?lariasis and elephantiasis are among secreted protease, Which degrades EcdysoZoan cuticle and the most devastating human tropical diseases. The World leads to larval arrest or death. Health Organization estimated that these diseases affected 120 million people WorldWide in 1992. [0010] In a related aspect, the invention provides yet another method for identifying a candidate compound that [0004] The impact of nematodes on human, animal, and disrupts molting in an EcdysoZoan. The method involves (a) plant health has resulted in the search for effective nemati providing a cell expressing a MLT polypeptide; (b) contact cides. BenZimidaZoles and avermectins are tWo common ing the cell With a candidate compound; and (c) comparing nematicides, Which target microtubule assembly and muscle the biological activity of the MLT polypeptide in the cell activity, respectively. Unfortunately, resistance to these contacted With the candidate compound to a control cell not compounds is increasingly common. In addition, these com contacted With said candidate compound, Where an alter pounds can have toxic effects on humans and other animals. ation in the biological activity of the MLT polypeptide Moreover, these nematicides are not effective against all identi?es the candidate compound as a candidate compound nematodes. Thus more effective and speci?c nematicides are that disrupts molting. required. [0011] In various embodiments, the cell is a nematode cell SUMMARY OF THE INVENTION or a mammalian cell. In other embodiments, the MLT polypeptide is a protease. In still other embodiments, the [0005] The present invention features improved methods biological activity of MLT polypeptide is monitored With an and compositions for inhibiting molting in EcdysoZoans, enZymatic assay or an immunological assay. In other pre including nematodes, parasitic nematodes, and insects. ferred embodiments, the cell is in a nematode and the biological activity is monitored by detecting molting. [0006] In one aspect, the invention provides a method for identifying a candidate compound that disrupts molting in an [0012] In another related aspect, the invention provides EcdysoZoan (e.g., an insect or nematode). The method yet another method for identifying a candidate compound includes the steps of: (a) providing a cell expressing a mlt that disrupts molting. The method includes the steps of: (a) nucleic acid molecule or an ortholog of a mlt nucleic acid contacting a nematode With a candidate compound; and (b) molecule; (b) contacting the cell With a candidate com comparing molting in the nematode contacted With the pound; and (c) comparing the expression of the mlt nucleic candidate compound to a control nematode not contacted acid molecule in the cell contacted With the candidate With said candidate compound, Where an alteration in molt compound With the expression of the nucleic acid molecule ing identi?es the candidate compound as a candidate com in a control cell not contacted With said candidate com pound that disrupts molting. pound, Where an alteration in expression identi?es the candidate compound as a candidate compound that disrupts [0013] In yet another related aspect, the invention pro molting. vides a yet further method of identifying a candidate com pound that disrupts EcdysoZoan molting. The method [0007] In a related aspect, the invention provides another includes the steps of: (a) contacting a cell containing a mlt method for identifying a candidate compound that disrupts nucleic acid regulatory region fused to a detectable reporter molting in a nematode. The method includes the steps of: (a) gene With a candidate compound; (b) detecting the expres providing a nematode cell expressing a mlt nucleic acid sion of the reporter gene; and (c) comparing the reporter molecule; (b) contacting the nematode cell With a candidate gene expression in the cell contacted With the candidate compound; and (c) comparing the expression of the mlt compound With a control cell not contacted With the candi nucleic acid molecule in the cell contacted With the candi date compound, Where an alteration in the expression of the date compound With the expression of the nucleic acid reporter gene identi?es the candidate compound as a can molecule in a control cell not contacted With said candidate didate compound that disrupts molting. US 2006/0178292 A1 Aug. 10, 2006

[0014] In various embodiments of the previous aspect, the that inhibits molting (e.g., C01H6.5, C17G1.6, C45B2.7, alteration is an alteration in the timing of reporter gene F11C1.6, F18C12.2, F29D11.1, F53G12.3, F56C11.1, expression of at least 10%, 20%, 30%, 40%, 50%, 60%, or K04F10.4, T05C12.10, T27F2.1, Y23H5A.7, and ZK270.1). even 70%, 80%, 90%, 95%, or 99% relative to the timing of In other embodiments, the naturally occurring mlt nucleic expression in a control nematode not contacted With the acid encodes a component of a secretory pathWay (e.g., candidate compound. In another embodiment, the alteration ZK1014.1, H15N14.1, F26H9.6, Y63D3A.5, C56C10.3, is an alteration in the level of expression of the reporter gene ZK180.4, F57H12.1, C39F7.4, Y113G7A.3, R160.1, of at least 10%, 20%, 30%, 40%, 50%, 60%, or even 70%, C02C6.1, E03H4.8, F59E10.3, K12H4.4, D1014.3, 80%, 90%, 95%, or 99% relative to the level of expression C13B9.3, F43D9.3). In other embodiments, the naturally in a control nematode not contacted With the candidate occurring mlt nucleic acid encodes a protein that functions compound. In another embodiment, the alteration is an in protein synthesis (e.g., B0336.10, B0393.1, C04F12.4, alteration in the cellular expression pattern of the reporter C23G10.3, D1007.6, F28D1.7, F35H10.4, F37C12.11, gene relative to the cellular expression pattern in a control F37C12.9, F40F11.1, F53A3.3, T01C3.6, T05F1.3, nematode not contacted With the candidate compound. Y45F10D.12). In still other embodiments, the inactivation [0015] In another related aspect, the invention provides a or inhibition of a naturally occurring mlt nucleic acid method for identifying a candidate compound that disrupts produces mlt defects in less than 5% of larvae (e.g., EcdysoZoan molting. The method includes the steps of: (a) C09F12.1, C09H10.2, C17H12.14, C37C3.2, C37C3.3, contacting a MLT polypeptide With a candidate compound; D2085.1, EEED8.5, F10E9.7, F19F10.9, F28F8.5, F32D1.2, and (b) detecting binding of said candidate compound to F35H10.4, F41E7.1, F42A8.1, F54B3.3, F55A3.3, F56F3.5, said MLT polypeptide, Wherein said binding identi?es said H06I04.4a, K06A4.6, K10D6.1, R06A10.1, T07D10.1, candidate compound as a candidate compound that disrupts Y17G7A.2, Y23H5A.7, Y38F2AL.3, Y41D4B.21, molting. Y41D4B.5, Y41D4B.5, Y45F10B.5, Y55H10A.1, [0016] In other aspects, the invention generally features an ZK1236.3, ZK265.5, ZK265.6, ZK652.1). isolated RNA mlt nucleic acid inhibitor comprising at least [0017] In preferred embodiments, the naturally occurring a portion of a naturally occurring mlt nucleic acid molecule mlt nucleic acid molecule is an ortholog of a mlt nucleic acid of an organism, or its complement, Where the mlt nucleic molecule. The ortholog is selected from the group consisting acid is selected from the group consisting of any or all of the of any one or all of the folloWing M90806, NMi134578, following B0024.14, C09G5.6, C11H1.3, C23F12.1, AY075331, BG310588, BE758466, BG227161, BM346811, B02725, C34G6.6, C37C3.3, C42D8.5, CD4.4, CD4.6, BG226227, BF169279, BE580288, BG893621, BQ625515, D1054.15, F08C6.1, F09B12.1, F16H9.2, F18A1.3, BI746672, AA471404, BE579677, BI500192, BI782938, F20G4.1, F25B4.6, F33A8.1, F33C8.3, F38H4.9, F40G9.1, BI073876, BF060055, AI723670, BI746256, BM882137, F41C3.4, F41H10.7, F45G2.5, F49C12.12, F52B11.3, BM277122, BM880769, BI501765, BE581131, AI539970, F53B8.1, F54A5.1, F54C9.2, F57B9.2, H04M03.4, BE580231, BE238916, AY060635, NMi143476, H19M22.1, K05C4.1, K06B4.5, K07C5.6, K07D8.1, AC008339, L02793, NMi079167, 102727, NMi139674, K08B4.1, K09H9.6, M03F4.7, M03F8.3, M162.6, M6.1, NMi079763, NMi057268, NMi137449, NMi079419, M88.6, R05D11.3, R07E4.6, R11G11.1, T01C3.1, T01H3.1, NMi080092, AAF51201, NMi057698, NMi080132, T14F9.1, T19B10.2, T23F2.1, T24H7.2, W01F3.3, NMi132335, AJ487018, NMi080072, AY094832, W08F4.6, W09B6.1, W10G6.3, Y111B2A.14, Y37D8A.10, NMi057520, NMi136653, NMi078644, AY075331, Y38F2AL.3, Y48B6A.3, ZC101.2, ZK1073.1, ZK1151.1, M90806, NMi079419, NMi080092, AAF51201, ZK262.8, ZK430.8, ZK686.3, ZK783.1, ZK970.4, NMi057698, NMi134578, AY071265, AY060235, C09F12.1, C09H10.2, C17H12.14, C37C3.2, D2085.1, NMi078577, NMi057621, AY089504, NMi135238, EEED8.5, F10E9.7, F19F10.9, F28F8.5, F32D1.2, X78577, AY118647, NMi140652, AY113364, F35H10.4, F41E7.1, F42A8.1, F54B3.3, F55A3.3, F56F3.5, NMi079972, X58374, NMi132550, AY052122 H06I04.4a, K06A4.6, K10D6.1, R06A10.1, T07D10.1, AY060893, AY058709 AA161577, CAAC01000031, Y17G7A.2, Y38F2AL.3, Y41D4B.21, Y41D4B.5, CAAC01000016, BI744615, BG224680, AW114337, Y45F10B.5, Y55H10A.1, ZK1236.3, ZK265.5, ZK265.6, BM281377, BU585500, BG577863, BQ091075, ZK652.1, Y54E10BR.5, B0513.1, R06A4.9, Y105E8B.1, AW257707, BF014893, BQ613344, CAAC01000088, Y47D3B.1, Y54F10AL.2, T17H7.3, H27M09.5, F45E10.2, BG735742, CAAC01000028, AA110597, BI863834, F25H8.6, K04A8.6, ZC13.3, T19A5.3, F32D8.6, F53F4.3, AI987143, BI782814, BI744849, and BG735807. F56C9.12, T25B9.10, ZK154.3, Y37D8A.19, Y37D8A.21, Y71F9AL.7, Y51H1A.3, W03F9.10, ZK945.2, ZK637.4, [0018] In other preferred embodiments, the naturally C30F8.2, F32H2.9, Y87G2A.5, Y53F4B.22, Y77E11A.13, occurring mlt nucleic acid molecule is a Drosophila ortholog C15H11.7, Y113G7B.23, C53H9.1, W09C5.6, T24B8.1, of a mlt nucleic acid molecule. The Drosophila ortholog is Y71A12B.1, C26C6.3, C42D8.5, F53G12.3, Y41D4B.10, selected from the group consisting of any one or all of the and F10C1.5, or an ortholog ofany or all ofthese mlt nucleic folloWing reflNMf079167, gblM90806, reflNMf079419, acid molecules, Where the RNA mlt nucleic acid inhibitor reflNMi080092, gblAY075331, ref NMi057698, reflNMi comprises at least a portion of a naturally occurring mlt 132335, reflNMf134871, gblAAF51201, reflNMf136653, nucleic acid inhibitor, or is capable of hybridiZing to a reflNMi057520, reflNMi080132, gblAY094832, naturally occurring mlt nucleic acid molecule, and decreases emblAJ487018, reflNMi080072, emblAJ011925, reflNMi expression from a naturally occurring mlt nucleic acid 078644, reflNMf132550, reflNMi079972, gblAY089504, molecule in the organism. In some embodiments, the natu emblX78577, gblAY118647, gblAY071265, reflNMi rally occurring mlt nucleic acid had been previously iden 140652, reflNMi078577, emblX58374, reflNMf134578, ti?ed as functioning in molting, but had not been identi?ed gblAY058709, gblAY060235, gblAY052122, AY060893, as the target for a nematicide, insecticide, or other compound gblAY113364, reflNMf135238, reflNMf057621, reflNMi US 2006/0178292 A1 Aug. 10, 2006

136498, reflNMf143476, reflNMf137449, gblM16152, in a control sample not having a parasitic infection, thereby reflNMi057268, reflNMf139674, gblL02793, diagnosing the organism as having a parasitic infection. gblAY060635, gblAC008339. [0026] In another aspect, the invention provides a method [0019] In other preferred embodiments of the previous for diagnosing an organism having a parasitic infection. The aspects, the RNA mlt nucleic acid inhibitor is a double method involves detecting an increased level of a MLT stranded RNA molecule that decreases expression in the polypeptide in a sample from the organism relative to the organism by at least 10%, 20%, 30%, 40%, 50%, 60%, or level in a control sample not having a parasitic infection, even 70%, 80%, 90%, 95%, or 99% from a naturally thereby diagnosing the organism as having a parasite infec occurring mlt nucleic acid molecule. In other preferred tion. In one embodiment, this method of detection is an embodiments, the RNA mlt nucleic acid inhibitor is an immunological method involving an antibody against a antisense RNA molecule that is complementary to at least MLT polypeptide. six, seven, eight, nine, ten, ?fteen, tWenty, tWenty-?ve, thirty, forty, ?fty, seventy-?ve, or one hundred nucleotides [0027] In other related aspects, the invention provides a biocide including a biocide excipient and a mlt nucleic acid, of the mlt nucleic acid molecule and decreases expression in or portion thereof, that disrupts EcdysoZoan molting by at the organism by at least 10%, 20%, 30%, 40%, 50%, 60%, least 10%, 20%, 30%, 40%, 50%, 60%, or even 70%, 80%, or even 70%, 80%, 90%, 95%, or 99% from a nucleic acid molecule to Which it is complementary. In other preferred 90%, 95%, or 99%. embodiments, the RNA mlt nucleic acid inhibitor is an [0028] In other related aspects, the invention provides a siRNA molecule that comprises at least ?fteen, sixteen, biocide including a biocide excipient and an RNA mlt seventeen, eighteen, nineteen, tWenty, tWenty-one, tWenty nucleic acid inhibitor (e.g., double-stranded RNA, antisense tWo, tWenty-three, tWenty-four, tWenty-?ve, or tWenty-six RNA, or siRNA), or portion thereof, that disrupts Ecdyso nucleic acids of a mlt nucleic acid molecule and decreases Zoan molting by at least 10%, 20%, 30%, 40%, 50%, 60%, expression in said organism by at least 10%, 20%, 30%, or even 70%, 80%, 90%, 95%, or 99%. 40%, 50%, 60%, or even 70%, 80%, 90%, 95%, or 99%. [0029] In other related aspects, the invention provides a [0020] In related aspects, the invention features a vector biocide including a biocide excipient and a MLT polypep comprising a mlt nucleic acid that encodes a MLT polypep tide, or portion thereof, or an ortholog of a MLT polypeptide tide or a nucleic acid encoding an RNA mlt nucleic acid that disrupts EcdysoZoan molting by at least 10%, 20%, inhibitor (e.g., double-stranded RNA, antisense RNA, or 30%, 40%, 50%, 60%, or even 70%, 80%, 90%, 95%, or siRNA), positioned for expression, and a host cell (e.g., 99%. plant, animal, or bacterial cell) containing the vector. For [0030] In other aspects, the invention provides an insec some applications, the vector used is a vector described in ticide including an insecticide excipient and a MLT polypep Fraser et al. (Nature, 408:325-30, 2000), hereby incorpo tide or portion thereof, encoded by a MLT nucleic acid, or rated by reference. ortholog, that disrupts insect molting by at least 10%, 20%, [0021] In another aspect, the invention provides a method 30%, 40%, 50%, 60%, or even 70%, 80%, 90%, 95%, or for reducing or ameliorating a parasitic nematode infection 99%. in an organism (e.g., a human or domestic mammal, such as [0031] In other related aspects, the invention provides an a coW, sheep, goat, pig, horse, dog, or cat). The method insecticide including an insecticide excipient and a mlt includes contacting the organism With a mlt nucleic acid or nucleic acid, or portion thereof, or ortholog, and disrupts an RNA mlt nucleic acid inhibitor (e.g., double-stranded insect molting by at least 10%, 20%, 30%, 40%, 50%, 60%, RNA, antisense RNA, or siRNA). or even 70%, 80%, 90%, 95%, or 99%. [0022] In a related aspect, the invention provides a method [0032] In other related aspects, the invention provides an for reducing or ameliorating a parasitic nematode infection insecticide including an insecticide excipient and an RNA in an organism (e.g., a human or domestic mammal, such as mlt nucleic acid inhibitor (e.g., double-stranded RNA, anti a coW, sheep, goat, pig, horse, dog, or cat). The method sense RNA, or siRNA) that disrupts insect molting by at includes contacting the organism With a MLT polypeptide. least 10%, 20%, 30%, 40%, 50%, 60%, or even 70%, 80%, [0023] In other related aspects, the invention provides a 90%, 95%, or 99%. pharmaceutical composition including a MLT polypeptide [0033] In other aspects, the invention provides a nemati or portion thereof, encoded by a mlt nucleic acid or an cide including a nematicide excipient and an MLT polypep ortholog of the nucleic acid molecule, and a pharmaceutical tide, or portion thereof, encoded by a mlt nucleic acid excipient, that ameliorates a parasite infection in an animal. molecule, or ortholog. [0024] In other related aspects, the invention provides a [0034] In other related aspects, the invention provides a pharmaceutical composition including a mlt nucleic acid or nematicide including a nematicide excipient and a mlt an RNA mlt nucleic acid inhibitor (e.g., double-stranded nucleic acid, or portion thereof, or ortholog, that disrupts RNA, antisense RNA, or siRNA), or portion thereof, and a nematode molting by at least 10%, 20%, 30%, 40%, 50%, pharmaceutical excipient, Which ameliorates a parasite 60%, or even 70%, 80%, 90%, 95%, or 99%. infection in an animal. [0035] In other related aspects, the invention provides a [0025] In another aspect, the invention provides a method nematicide including a nematicide excipient and an RNA of diagnosing an organism having a parasitic infection. The mlt nucleic acid inhibitor (e.g., double-stranded RNA, anti method involves contacting a sample from the organism sense RNA, or siRNA), that disrupts nematode molting by With a mlt nucleic acid probe and detecting an increased at least 10%, 20%, 30%, 40%, 50%, 60%, or even 70%, level of a mlt nucleic acid in the sample relative to the level 80%, 90%, 95%, or 99%. US 2006/0178292 A1 Aug. 10, 2006

[0036] In another related aspect, the invention provides a R11G11.1, T01c31, T011131, T05c1210, T14F9.1, transgenic organism (e.g., EcdysoZoan) expressing a mlt T19B10.2, T23P21, T24H7.2, T27F2.1, W01P3.3, nucleic acid molecule or an RNA mlt nucleic acid inhibitor W08P46, W09B6.1, W10G63, Y111B2A.14, Y37D8A.10, (e.g., double-stranded RNA, antisense RNA, or siRNA) at a Y38P2AL3, Y48B6A.3, Zc1012, ZK1073.1, ZK1151.1, level suf?cient to disrupt molting in the progeny of an ZK62.8, ZK270.1, ZK430.8, ZK686.3, ZK783.1, ZK970.4, EcdysoZoan (e.g., a nematode, a parasitic nematode, or an c09P121, c0911102, c17111214, c37c32, 1320851, insect) breeding With the transgenic organism relative to a 1313131385, P101397, P19P10.9, P28P8.5, P321312, control nematode, parasitic nematode, or insect not bred P3511104, P411371, F42A8.1, P541333, P55A33, P56P3.5, With the organism. In various embodiments, the mlt nucleic acid molecule or RNA mlt nucleic acid inhibitor is expressed H06I04.4a, K06A4.6, K10D6.1, R06A10.1, T07D10.1, under the control of a conditional promoter. In some appli Y17G7A.2, Y23H5A.7, Y38P2AL3, Y41D4B.21, cations, for the control of a population of EcdysoZoan pests, Y41D4B5, Y41D4B5, Y45F10B5, Y55H10A.1, a transgenic organism expressing a mat nucleic acid mol ZK12363, ZK2655, ZK265.6, ZK652.1, ZK1014.1, ecule or an RNA mlt nucleic acid inhibitor, or portion H15N14.1, F26H9.6, Y63D3A5, c56c103, ZK180.4, thereof, under the control of a conditional promoter, for F57H12.1, c39P7.4, Y113G7A.3, R1601, c02c61, example, may be released into an area infested With an 13031148, P5913103, K12H4.4, 1310143, c131393, EcdysoZoan pest (e.g., a nematode or insect pest). The P431393, 130336.10, 1303931, c04P12.4, c23G103, transgenic organism transmits the mlt nucleic acid transgene Dl007.6, F28D1.7, F35H10.4, F37C12.11, F37C12.9, during mating With Wild-type EcdysoZoan pests to disrupt F40F11.1, F53A3.3, T01C3.6, T05F1.3, Y45F10D.12, or molting in the progeny, and controls a population of Ecdyso Y54E10BR5, B0513.1, R06A4.9, Y105E8B.1, Y47D3B.1, Zoan pests. Y54F10AL.2, T17H7.3, H27M095, F45E10.2, F25H8.6, K04A8.6, ZC13.3, T19A5.3, F32D8.6, F53F4.3, F56C9.12, [0037] In other related aspects, the invention provides a T25B9.10, ZK154.3, Y37D8A.19, Y37D8A.21, transgenic plant expressing a mlt nucleic acid or an RNA mlt Y71F9AL.7, Y51H1A.3, W03F9.10, ZK945.2, ZK637.4, nucleic acid inhibitor (e.g., double-stranded RNA, antisense C30F8.2, F32H2.9, Y87G2A5, Y53F4B.22, Y77E11A.13, RNA, or siRNA), or portion thereof, Where a cell of the plant C15H11.7, Y113G7B.23, C53H9.1, W09C5.6, T24B8.1, expresses the mlt nucleic acid or RNA mlt nucleic acid inhibitor at a level suf?cient to disrupt molting in an Ecdyso Y71A12B.1, C26C6.3, C42D85, F53G12.3, Y41D4B.10, F10C15, or a portion thereof, or an ortholog of any or all of Zoan (e.g., a nematode, a parasitic nematode, or an insect) these nucleic acids. In other embodiments, the mlt nucleic that contacts (e.g., feeds on) the plant relative to a control acid is a component ofa secretory pathWay (e.g. ZK1014.1, nematode, parasitic nematode, or insect not contacted With H15N14.1, F26H9.6, Y63D3A5, C56C10.3, ZK180.4, the plant. F57H12.1, C39F7.4, Y113G7A.3, R160.1, C02C6.1, [0038] In other aspects, the invention provides a trans E03H4.8, F59E10.3, K12H4.4, D1014.3, C13B9.3, and genic organism (e.g., insect or domestic mammal, such as a F43D9.3). In other embodiments, the mlt nucleic acid is a coW, sheep, goat, pig, or horse) expressing a mlt nucleic acid protein that functions in protein synthesis and produces mlt or an RNA mlt nucleic acid inhibitor (e.g., double-stranded defects in less than 5% of larvae (e.g. B0336.10, B0393.1, RNA, antisense RNA, or siRNA), or portion thereof, at a C04F12.4, C23G10.3, Dl007.6, F28D1.7, F35H10.4, level suf?cient to disrupt molting in a nematode, a parasitic F37C12.11, F37C12.9, F40F11.1, F53A3.3, T01C3.6, nematode, or an insect that contacts, (e.g., parasitiZes or T05F1.3, Y45F10D.12). feeds on) the transgenic organism relative to a control [0040] In preferred embodiments of any of the above nematode, parasitic nematode, or insect not contacted With the organism. Such transgenic organisms Would be expected aspects, a mlt ortholog is any or all of the folloWing mlt to be more resistant to parasitic nematode infection than nucleic acids: M90806, NMi134578, AY075331, control organisms not expressing a transgene. In preferred BG310588, BE758466, BG227161, BM346811, embodiments, the transgenic organism is an insect host BG226227, BF169279, BE580288, BG893621, BQ625515, organism (e.g., black?y) capable of being infected With an BI746672, AA471404, BE579677, BI500192, BI782938, EcdysoZoan parasite (e.g., nematode) that spends part of its BI073876, BF060055, AI723670, BI746256, BM882137, life cycle as an insect parasite and part of its life cycle as a BM277122, BM880769, BI501765, BE581131, AI539970, human parasite. Expression of the transgene in the trans BE580231, BE238916, AY060635, NMi143476, genic host organism inhibits molting in the EcdysoZoan AC008339, L02793, NMi079167, 102727, NMi139674, parasite, and is useful in controlling a human parasitic NMi079763, NMi057268, NMi137449, NMi079419, infection. NMi080092, AAF51201, NMi057698, NMi080132, NMi132335, AJ487018, NMi080072, AY094832, [0039] In preferred embodiments of the above aspects, a NMi057520, NMi136653, NMi078644, AY075331, mlt nucleic acid is any one or all of the folloWing B0024.14, M90806, NMi079419, NMi080092, AAF51201, C0lH6.5, C09G5.6, C11H1.3, C17G1.6, C23F12.1, NMi057698, NMi134578, AY071265, AY060235, B02725, C34G6.6, C37C3.3, C42D85, C45B2.7, CD4.4, NMi078577, NMi057621, AY089504, NMi135238, CD4.6, D1054.15, F08C6.1, F09B12.1, F11C1.6, F16H9.2, X78577, AY118647, NMi140652, AY113364, P18A13, P18c122, P20G41, P251346, P2913111, NMi079972, X58374, NMi132550, AY052122, F33A8.1, P33c83, P381149, F40G9.1, P41c3.4, AY060893, AY058709, AA161577, CAAC01000031, F41H10.7, F45G25, P49c1212, P5213113, P531381, CAAC01000016, BI744615, BG224680, AW114337, P53G123, F54A5.1, P54c92, P56c111, P571392, BM281377, BU585500, BG577863, BQ091075, H04M03.4, H19M22.1, K04F10.4, K05C4.1, K06B45, AW257707, BF014893, BQ613344, CAAC01000088, K07C5.6, K07D8.1, K08B4.1, K09H9.6, M03P47, BG735742, CAAC01000028, AA110597, BI863834, M03P83, M162.6, M6.1, M88.6, R0513113, R07E4.6, AI987143, BI782814, 11744849, BG735807. US 2006/0178292 A1 Aug. 10, 2006

[0041] In other preferred embodiments of any of the above [0051] By “parasitic nematode” is meant any nematode aspects, a Drosophila ortholog includes any or all of the that lives on or Within the cells, tissues, or organs of a following mlt nucleic acids: reflNMf079167, gblM90806, genetically distinct host organism (e.g., plant or animal). For reflNMi079419, reflNMi080092, gblAY075331, reflNMi example, parasitic nematodes include, but are not limited to, 057698, reflNMi132335, reflNMi134871, gblAAF51201, any ascarid, ?larid, or rhabditid (e.g., Onchocerca volvulus, reflNMi136653, reflNMi057520, reflNMi080132, Ancylosloma, Ascaris, Ascaris lumbricoides, Ascaris suum, gblAY094832, emblAJ487018, reflNMi080072, Baylisascaris, Baylisascaris procyonis, Brugia malayi, emblAJ011925, reflNMi078644, reflNMf132550, Diro?aria, Diro?aria immilis, Dracunculus, Haemonchus reflNMi079972, gblAY089504, emblX78577, conlorlus, Helerorhabdilis bacleriophora, Loa [0a, root gblAY118647, gblAY071265, reflNMi140652, reflNMi knot nematodes, such as Meloidogyne, M arenaria, M 078577, emblX58374, reflNMi134578, gblAY058709, chitwoodi, M graminocola, M graminis, M hapla, M gblAY060235, gblAY052122, AY060893, gblAY113364, incognila, Necalor, M microzyla, and M naasi, cyst nema reflNMi135238, reflNMi057621, reflNMi136498, todes (for example, Helerodera sp. such as H. schachlii, H. reflNMi143476, reflNMi137449, gblM16152, reflNMi glycines, H. sacchari, H. oryzae, H. avenue, H. cajani, H. 057268, reflNMil39674, gblL02793, gb‘AY060635, elachisla, H. goellingiana, H. graminis, H. medilerranea, H. gb‘AC008339. molhi, H sorghi, and H zeae, or, for example, Globodera sp. such as G. roslochiensis and G. pallida) root-attacking [0042] In other preferred embodiments of any of the nematodes (for example, Rolylenchulus reniformis, Elen previous aspect, the nucleic acid sequence is selected from chuylus semipenelrans, Pralylenchus brachyurus, Radopho those listed in Tables 1A, 1B, 4A-4D, or 7. lus cilrophilus, Radopholus similis, Xiphinema americanum, [0043] By “biocide” is meant any agent, compound, or Xiphinema rivesi, Paralrichodorus minor, Helerorhabdilis molecule that sloWs, delays, inhibits, or arrests the growth, heliolhidis, and Bursaphelenchus xylophilus), and above viability, molting, or reproduction of any EcdysoZoan by at ground nematodes (for example, Anguina funesla, Anguina least 5%, 10%, 20%, 30%, 40%, 50%, 60%, or even by as Zrilici, Dilylenchus dipsaci, Dilylenchus myceliphagus, and much as 70%, 80%, 90%, 95%, or 99%. Aphenlenchoides besseyi), Paraslrongyloides Zrichosuri, [0044] By “EcdysoZoan” is meant the clade of organisms Prislionchuspacl?cus, Sleinernema, Slrongyloides slercora that molt. EcdysoZoans include arthropods, tardigrades, ony lis, Slrongyloides ralli, Toxocara canis, Trichinella spiralis, chophorans, nematodes, nematomorphs, kinorhynchs, loric Trichuris muris or Wuchereria bancro?i). iferans, and priapulids. [0052] By “nematicide” is meant any agent, compound, or molecule that sloWs, delays, inhibits, or arrests the groWth, [0045] By “molting” is meant the shedding and synthesis viability, molting, or reproduction of any nematode by at of cuticle that occurs during the life cycle of an EcdysoZoan, least 5%, 10%, 20%, 30%, 40%, 50%, 60%, or even by as such as a nematode or insect. much as 70%, 80%, 90%, 95%, or 99%. [0046] By “disrupts molting” is meant that the process of [0053] By “insecticide” is meant any agent, compound, or cuticle shedding is delayed, inhibited, sloWed, or arrested. In some applications, the molting process is disrupted by larval molecule that sloWs, delays, inhibits, or arrests the groWth, viability, molting, or reproduction of any insect by at least arrest. 5%, 10%, 20%, 30%, 40%, 50%, 60%, or even by as much [0047] By “mlt nucleic acid” is meant a nucleic acid as 70%, 80%, 90%, 95%, or 99%. molecule, or an ortholog thereof, Whose inactivation (e.g., [0054] By “anti-parasitic” is meant any agent, compound, by RNAi) results in a molting defect or larval arrest phe or molecule that ameliorates a parasitic infection in a host notype in an EcdysoZoan. RNAi of a mlt gene results in a organism. In some applications, an anti-parasitic agent Mlt phenotype or larval arrest phenotype in at least 1%, 3%, sloWs, delays, inhibits, or arrests the groWth, viability, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or even in 70%, 80%, molting, or reproduction of a parasite in a host organism. 90%, 95%, or 99% of the larvae exposed to dsRNA expressing bacteria. [0055] By “ortholog” is meant any polypeptide or nucleic acid molecule of an organism that is highly related to a [0048] By “RNA mlt nucleic acid inhibitor” is meant a double-stranded RNA, antisense RNA, or siRNA, or portion reference protein or nucleic acid sequence from another organism. The degree of relatedness may be expressed as the thereof, that When administered to an EcdysoZoan results in probability that a reference protein Would identify a a molting defect or larval arrest phenotype. Typically, an RNA mlt nucleic acid inhibitor comprises at least a portion sequence, for example, in a blast search. The probability that a reference sequence Would identify a random sequence as of a mlt nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of an ortholog is extremely loW, less than e_lo, e_2O, e_3o, e_4o, a mlt nucleic acid molecule. For example, a mlt nucleic acid e_5O, e_75, e_loo. The skilled artisan understands that an molecule includes any or all of the nucleic acids listed in ortholog is likely to be functionally related to the reference Tables 1A, 1B, 4A-4D, and 7. protein or nucleic acid sequence. In other Words, the ortholog and its reference molecule Would be expected to [0049] By “MLT polypeptide” is meant any amino acid ful?ll similar, if not equivalent, functional roles in their molecule encoded by a mlt nucleic acid. Typically, a MLT respective organisms. polypeptide functions in molting in an EcdysoZoan (e.g., [0056] Drosophila melanogasler orthologs of C. elegans nematode or insect). mlt genes include, but are not limited to, reflNMf079167, [0050] By “parasite” is meant any multicellular organism gb‘M90806, reflNMf079419, reflNMi080092, that lives on or Within the cells, tissues, or organs of a gb‘AY075331, ret NMi057698, reflNMf132335, reflNMi genetically distinct host organism. 134871, gblAAF51201, reflNMf136653, reflNMi057520, US 2006/0178292 A1 Aug. 10, 2006

reflNMi080132, gblAY094832, emb]AJ4870l8, reflNMi 080072, emblAJ01925, reflNMi078644, reflNMi132550, reflNMi079972, gblAY089504, emblX78577, gblAY118647, gblAY071265, reflNMi140652, reflNMi 078577, emblX58374, reflNMi134578, gblAY058709, gblAY060235, gblAY052122, AY060893, gblAY113364, reflNMi135238, reflNMi057621, reflNMi136498, reflNMi143476, reflNMi137449, gblM16152, reflNMi 057268, reflNMi139674, gblL02793, gblAY060635, and gblAC008339. [0057] Nematode orthologs of C. elegans mlt genes include, but are not limited to, BG310588 in Onchocerca volvulus (e_l2l); BE758466 in Brugia malayi (e_lo4); BG227l6l2 in Slrongyloides slercoralis (e_84); BM3468ll in Paraslrongyloides Zrichosuri (e_89); BG226227 in Slrongyloides slercoralis (9e 24); BFl69279 in Trichuris [0059] By “portion” is meant a fragment of a protein or muris (4e_ll); BG893621 in Slrongyloides ralli (2e_2o); nucleic acid that is substantially identical to a reference BQ625515 in Meloidogyne incognila (3e_25); Bl746672 in protein or nucleic acid, and retains at least 50% or 75%, Meloidogyne arenaria (6e_3l); AA47l404 in Brugia malayi more preferably 80%, 90%, or 95%, or even 99% of the (2568); BE579677 in Slrongyloides slercoralis (2553); biological activity of the reference protein or nucleic acid Bl500l92 in Prislionchus paci?cus (2569); Bl782938 in using a molting assay as described herein. Ascaris suum (9e_52); Bl073876 in Slrongyloides ralli (le' [0060] By “isolated polynucleotide” is meant a nucleic 41); BF060055 in Haemonchus conlorlus (4e_l8); Al723670 acid (e.g., a DNA) that is free of the genes, Which, in the in Brugia malayi (8e_4o); Bl746256 in Meloidogyne naturally occurring genome of the organism from Which the arenaria (3.00515); BM882137 in Paraslrongyloides Zricho nucleic acid molecule of the invention is derived, ?ank the suri (6e_33); BM277122 in Trichuris muris (6e_l5); gene. The term therefore includes, for example, a recombi BM880769 in Meloidogyne incognila (3e_4l); Bl50l765 in nant DNA that is incorporated into a vector; into an autono Meloidogyne arenaria; BE58ll3l in Slrongyloides ster mously replicating plasmid or virus; or into the genomic coralis (le_34); Al5399702 in Onchocerca volvulus (e_38); DNA of a prokaryote or eukaryote; or that exists as a BE5802318 in Slrongyloides slercoralis (e_35); BE2389166 separate molecule (for example, a cDNA or a genomic or in Meloidogyne incognila (e_l7); BE580288 in Slrongy cDNA fragment produced by PCR or restriction endonu loides slercoralis, AAl6l577 in Brugia malayi (e_39); clease digestion) independent of other sequences. In addi CAAC0l0000l6 in C. briggsae; Bl7446l5 in Meloidogyne tion, the term includes an RNA molecule that is transcribed javanica (4e-44); BG224680 Slrongyloides slercoralis (4e from a DNA molecule, as Well as a recombinant DNA that 44); AWll4337 Prislionchus pacl?cus (e_4l), BM281377 in is part of a hybrid gene encoding additional polypeptide Ascaris suum (2e_4l); BU585500 in Ascaris lumbricoides, sequence. BG577863 in Trichuris muris (e_24); BQ091075 in Slrongy loides ralli (6e_l4); AW257707 in Onchocerca volvulus; [0061] By “polypeptide” is meant any chain of amino BF0l4893 in Slrongyloides slercoralis (7e-35); BQ6l3344 acids, regardless of length or post-translational modi?cation in Meloidogyne incognila (5e_47); CAAC0l000088 in C. (for example, glycosylation or phosphorylation). Briggsae, BG735742 in Meloidogyne javanica (4e_l4); [0062] By an “isolated polypeptide” is meant a polypep CAAC0l000028; AAll0597 in Brugia malayi (3556); tide of the invention that has been separated from compo Bl863834 in Paraslrongyloides Zrichosuri (3e_69); nents that naturally accompany it. Typically, the polypeptide Al987l43 in Prislionchus paci?cus (3560); Bl7828l4 in is isolated When it is at least 60%, by Weight, free from the Ascaris suum; Bl744849 in Meloidogyne javanica; and proteins and naturally occurring organic molecules With BG735807 in Meloidogyne javanica (6e_38). Which it is naturally associated. Preferably, the preparation [0058] Of particular interest are orthologs of the following is at least 75%, more preferably at least 90%, and most genes: B002414, C01H6.5, C09G5.6, C11H1.3, C17G1.6, preferably at least 99%, by Weight, a polypeptide of the C23F12.1, B02725, C34G6.6, c37c33, c42138.5, invention. An isolated polypeptide of the invention may be c45B2.7, c1344, CD4.6, 131054.15, P08c61, F09B12.1, obtained, for example, by extraction from a natural source, F11C1.6,F16H9.2,F18A1.3,F18C12.2,F20G4.1,F25B4.6, by expression of a recombinant nucleic acid encoding such P2913111, F33A8.1, P33c83, F38H4.9, P40G91, a polypeptide; or by chemically synthesizing the protein. F4lC3.4, F4lHl0.7, F45G2.5, F49Cl2.l2, F52Bll3, Purity can be measured by any appropriate method, for F53B8.l, F53Gl2.3, F54A5.l, F54C9.2, F56Cll.l, example, column chromatography, polyacrylamide gel elec F57B9.2, H04M03.4, Hl9M22.l, K04Fl0.4, K05C4.l, trophoresis, or by HPLC analysis. K06B4.5, K07C5.6, K07D8.1, K08B4.1, K09H9.6, [0063] By “substantially identical” is meant a polypeptide M03F4.7, M03P8.3, M162.6, M61, M88.6, R05D11.3, or nucleic acid molecule exhibiting at least 50% identity to R07E4.6, RllGll.l, T0lC3.l, T0lH3.l, T05Cl2.l0, a reference amino acid sequence (for example, any one of T14P91, T19B10.2, T23F2.1, T24H7.2, T27P21, the amino acid sequences described herein) or nucleic acid W0lF3.3, W08F4.6, W09B6.l, Wl0G6.3, YlllB2A.l4, sequence (for example, any one of the nucleic acid Y37D8A.l0, Y38F2AL3, Y48B6A.3, ZCl0l.2, ZKl073.l, sequences described herein). Preferably, such a sequence is ZK1151.1, ZK262.8, ZK270.1, ZK430.8, ZK6863, at least 60%, more preferably 80%, and most preferably ZK783.1, ZK970.4, C09Fl2.l, C09Hl0.2, Cl7Hl2.l4, 90% or even 95% identical at the amino acid level or nucleic c37c32, 1320851, EEED8.5, F10E9.7, P19P10.9, P28P8.5, acid to the sequence used for comparison. US 2006/0178292 A1 Aug. 10, 2006

[0064] Sequence identity is typically measured using acid or amino acid sequence. In one embodiment, the sequence analysis software (for example, Sequence Analysis decrease in expression or biological activity is at least 10%, Software Package of the Genetics Computer Group, Uni relative to a control, more desirably 25%, and most desirably versity of Wisconsin Biotechnology Center, 1710 University 50%, 60%, 70%, 80%, 90%, or more. The anti-sense nucleic Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or acid may contain a modi?ed backbone, for example, phos PILEUP/PRETTYBOX programs). Such softWare matches phorothioate, phosphorodithioate, or other modi?ed back identical or similar sequences by assigning degrees of bones knoWn in the art, or may contain non-natural inter homology to various substitutions, deletions, and/or other nucleoside linkages. modi?cations. Conservative substitutions typically include substitutions Within the folloWing groups: glycine, alanine; [0072] By “siRN ” is meant a double stranded RNA that valine, isoleucine, leucine; aspartic acid, glutamic acid, complements a region of an mRNA. Optimally, an siRNA is asparagine, glutamine; serine, threonine; lysine, arginine; 21, 22, 23, or 24 nucleotides in length and has a 2 base and phenylalanine, tyrosine. In an exemplary approach to overhang at its 3' end. siRNAs can be introduced to an individual cell, tissue, organ, or to a Whole animals. For determining the degree of identity, a BLAST program may example, they may be introduced systemically via the blood be used, With a probability score betWeen e'3 and e'100 stream. Such siRNAs are used to doWnregulate mRNA indicating a closely related sequence. levels or promoter activity. Desirably, the siRNA is capable [0065] By “transformed cell” is meant a cell into Which (or of decreasing the expression or biological activity of a into an ancestor of Which) has been introduced, by means of nucleic acid or amino acid sequence. In one embodiment, recombinant DNA techniques, a polynucleotide molecule the decrease in expression or biological activity is at least encoding (as used herein) a polypeptide of the invention. 10%, relative to a control, more desirably 25%, and most [0066] By “positioned for expression” is meant that the desirably 50%, 60%, 70%, 80%, 90%, or more. The siRNA may contain a modi?ed backbone, for example, phospho polynucleotide of the invention (e.g., a DNA molecule) is rothioate, phosphorodithioate, or other modi?ed backbones positioned adjacent to a DNA sequence that directs tran scription and translation of the sequence (i.e., facilitates the knoWn in the art, or may contain non-natural intemucleo side production of, for example, a recombinant polypeptide of linkages. the invention, or an RNA molecule). [0073] By “hybridize” is meant pair to form a double stranded molecule betWeen complementary polynucleotide [0067] By “speci?cally binds” is meant a compound or antibody Which recognizes and binds a polypeptide of the sequences (e.g., genes listed in Tables 1A, 1B, 4A-4D, and invention, but Which does not substantially recognize and 7), or portions thereof, under various conditions of strin bind other molecules in a sample, for example, a biological gency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) sample, Which naturally includes a polypeptide of the inven Methods Enzymol. 1521399; Kimmel, A. R. (1987) Methods tion. Enzymol. 152:507) For example, stringent salt concentration Will ordinarily be less than about 750 mM NaCl and 75 mM [0068] By “derived from” is meant isolated from or having trisodium citrate, preferably less than about 500 mM NaCl the sequence of a naturally occurring sequence (e.g., a and 50 mM trisodium citrate, and most preferably less than cDNA, genomic DNA, synthetic, or combination thereof). about 250 mM NaCl and 25 mM trisodium citrate. LoW [0069] By “immunological assay” is meant an assay that stringency hybridization can be obtained in the absence of relies on an immunological reaction, for example, antibody organic solvent, e.g., formamide, While high stringency binding to an antigen. Examples of immunological assays hybridization can be obtained in the presence of at least include ELISAs, Western blots, immunoprecipitations, and about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions Will ordi other assays knoWn to the skilled artisan. narily include temperatures of at least about 30° C., more [0070] By “anti-sense” is meant a nucleic acid sequence, preferably of at least about 37° C., and most preferably of at regardless of length, that is complementary to the coding least about 42° C. Varying additional parameters, such as strand or mRNA of a nucleic acid sequence. In one embodi hybridization time, the concentration of detergent, e.g., ment, an antisense RNA is introduced to an individual cell, sodium dodecyl sulfate (SDS), and the inclusion or exclu tissue, organ, or to a Whole animals. Desirably the anti-sense sion of carrier DNA, are Well knoWn to those skilled in the nucleic acid is capable of decreasing the expression or art. Various levels of stringency are accomplished by com biological activity of a nucleic acid or amino acid sequence. bining these various conditions as needed. In a preferred In one embodiment, the decrease in expression or biological embodiment, hybridization Will occur at 30° C. in 750 mM activity is at least 10%, relative to a control, more desirably NaCl, 75 mM trisodium citrate, and 1% SDS. In a more 25%, and most desirably 50%, 60%, 70%, 80%, 90%, or preferred embodiment, hybridization Will occur at 37° C. in more. The anti-sense nucleic acid may contain a modi?ed 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% backbone, for example, phosphorothioate, phosphorodithio formamide, and 100 ug/ml denatured salmon sperm DNA ate, or other modi?ed backbones knoWn in the art, or may (ssDNA). In a most preferred embodiment, hybridization contain non-natural intemucleoside linkages. Will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ug/ml ssDNA. [0071] By “double stranded RNA” is meant a complemen Useful variations on these conditions Will be readily appar tary pair of sense and antisense RNAs regardless of length. ent to those skilled in the art. In one embodiment, these dsRNAs are introduced to an individual cell, tissue, organ, or to a Whole animals. For [0074] For most applications, Washing steps that folloW example, they may be introduced systemically via the blood hybridization Will also vary in stringency. Wash stringency stream. Desirably, the double stranded RNA is capable of conditions can be de?ned by salt concentration and by decreasing the expression or biological activity of a nucleic temperature. As above, Wash stringency can be increased by US 2006/0178292 A1 Aug. 10, 2006

decreasing salt concentration or by increasing temperature. advantages of the invention Will be apparent from the For example, stringent salt concentration for the Wash steps detailed description, and from the claims. Will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and mo st preferably less than about 15 mM BRIEF DESCRIPTION OF THE DRAWINGS NaCl and 1.5 mM trisodium citrate. Stringent temperature [0081] FIGS. 1A-1E are micrographs shoWing Mlt phe conditions for the Wash steps Will ordinarily include a notypes associated With RNAi of mlt-24, mlt-18, mlt-12, temperature of at least about 25° C., more preferably of at and mlt-13 in nematodes visualized using Nomarski optics. least about 42° C., and most preferably of at least about 68° FIGS. 1A and 1B are micrographs shoWing the Mlt phe C. In a preferred embodiment, Wash steps Will occur at 25° notype of a mlt-24(RNAi) nematode. FIG. 1C is a micro C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. graph shoWing the Mlt phenotype of a mlt-18(RNAi) nema In a more preferred embodiment, Wash steps Will occur at tode. FIG. 1D is a micrograph shoWing the Mlt phenotype 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% of a mlt-12(RNAi) nematode. FIG. IE is a micrograph SDS. In a most preferred embodiment, Wash steps Will occur shoWing the Mlt phenotype of a mlt-13(RNAi) nematode. at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and Black arroWs indicate Where excess cuticle remains attached 0.1% SDS. Additional variations on these conditions Will be to the larvae. readily apparent to those skilled in the art. Hybridization techniques are Well knoWn to those skilled in the art and are [0082] FIGS. 2A-2D shoW that molting genes are described, for example, in Benton and Davis (Science expressed in a pulse before each molt. FIG. 2A is a series 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. of micrographs shoWing ?uorescence from mlt-12::gfp-pest Sci, USA 72:3961, 1975); Ausubel et al. (Current Protocols early in L1, at the L1/ L2 molt, and early in L2. The L2 larvae in Molecular Biology, Wiley Interscience, NeW York, 2001); Was ?uorescent before molting. Black arroWs indicate Berger and Kimmel (Guide to Molecular Cloning Tech cuticle separated from the body. FIGS. 2B and 2C are niques, 1987, Academic Press, NeW York); and Sambrook et graphs shoWing the percentage of Worms that Were ?uores al., Molecular Cloning: A Laboratory Manual, Cold Spring cent overtime, on a scale normalized to the period betWeen Harbor Laboratory Press, NeW York. molts for each Worm under observation. The bar at the top of the graph indicates the Worm’s developmental stage. FIG. [0075] By “transgene” is meant any piece of DNA Which 2B shoWs results for Ex[mlt-12::gfp-pest] (dashed line) or is inserted by arti?ce into a cell and typically becomes part Ex[mlt-10::gfp-pest] (solid line) larvae scored for detectable of the genome of the organism Which develops from that ?uorescence and for molting once per hour from late in the cell. Such a transgene may include a gene that is partly or L1 stage until early adulthood. FIG. 2C shoWs cycling entirely heterologous (i.e., foreign) to the transgenic organ ?uorescence in Worms expressing mlt-13::gfp-pest (dashed ism, or may represent a gene homologous to an endogenous line) or mlt-18::gfp-pest (solid line), observed in the hypo gene of the organism. A transgene of the invention may dermis and seam cells. FIG. 2C shoWs Northern analysis of encode a MLT polypeptide or an RNA mlt nucleic acid mlt-10 messenger RNA levels. Ribosomal RNA stained With inhibitor. ethidium-bromide provides a loading control. [0076] By “transgenic” is meant any cell Which includes a [0083] FIGS. 3A-3H are micrographs shoWing GFP ?uo DNA sequence Which is inserted by arti?ce into a cell and rescence associated With Pmlt-18::GFP-PEST and Pmlt becomes part of the genome of the organism Which develops 13::GFP-PEST expression in transgenic nematodes. FIGS. from that cell, or part of a heritable extra chromosomal array. 3A, 3C, and 3E are micrographs shoWing GFP ?uorescence As used herein, transgenic organisms may be either trans in transgenic Pmlt-18::GFP-PEST expressing nematodes genic vertebrates, such as domestic mammals (e.g., sheep, during early L1, L1/L2 molt, and early L2. FIGS. 3B, 3D, coW, goat, or horse), mice, or rats, transgenic invertebrates, and 3F are micrographs of nematodes visualized using such as insects or nematodes, or transgenic plants. Nomarski optics. The black arroW in FIG. 2D indicates shedding of the cuticle at the L1/L2 molt. Worms Were [0077] By “cell” is meant a single-cellular organism, cell synchronized after hatching and monitored through larval from a multi-cellular organism, or it may be a cell contained development. FIGS. 3G and 3H are micrographs of nema in a multi-cellular organism. todes shoWing GFP ?uorescence in transgenic Pmlt [0078] By “differentially expressed” is meant a difference 13::GFP-PEST expressing nematodes during early L2 and in the expression level of a nucleic acid. This difference may L1/L2 molt. The inset in FIGS. 3G and 3H is a micrograph be either an increase or a decrease in expression, When of the transgenic nematode visualized using Nomarski compared to control conditions. optics. [0079] By “therapeutic compound” is meant a substance [0084] FIG. 4A is a graph shoWing the percentage of that affects the function of an organism. Such a compound animals that Were ?uorescent before a defective molt, nor may be, for example, an isolated naturally occurring, semi malized to the percentage of control larvae that Were ?uo synthetic, or synthetic agent. For example, a therapeutic rescent before molting from the same stage. Ex[mlt-12: :gfp compound may be a drug that targets a parasite infecting a pest], indicated With black bars, or Ex[mlt-10::gfp-pest] host organism. A therapeutic compound may decrease, sup larvae, indicated With gray bars, Were fed bacteria express ing dsRNA for each gene indicated. “n” indicates the num press, attenuate, diminish, arrest, or stabilize the develop ber of larvae observed. PairWise chi-square tests indicated ment or progression of disease, disorder, or infection in a eukaryotic host organism. that the decreased fraction of ?uorescent Ex[mlt-12::gfp pest] larvae after RNAi of nhr-23 or acn-1, and of ?uores [0080] The invention provides for compositions and meth cent Ex[mlt-10::gfp-pest] larvae after RNAi of nhr-23, acn ods useful for inhibiting molting in an Ecdysozoan (e.g., a 1, or mlt-12, relative to control animals, is signi?cant, With parasitic nematode, nematode or insect). Other features and p<0.001 in all 5 tests. US 2006/0178292 A1 Aug. 10, 2006

[0085] FIG. 4B is a graph that shows the percentage of speci?cally disrupt molting processes common to Ecdyso late L4 larvae with detectable ?uorescence, for selected gene Zoans, and given this speci?city are unlikely to adversely inactivations. Ex[mlt-10::gfp-pest] larvae were fed bacteria e?‘ect human health. The third class includes mlt genes expressing dsRNA for each gene indicated. Values represent whose inactivation by RNA results in highly penetrant molt the weighted average of two independent trials. defects (e.g., those molt genes listed in Tables 1A and Table 1B). Tables 1A and 1B include genes not previously iden [0086] FIGS. 5A-5G are a series of micrographs showing ti?ed as being involved in molting (e.g., B0024.14, expression of molting gene gfp fusion genes in worms. FIGS. 5A-C show expression from mlt-24::gfp-pest. FIG. C09G5.6, C11H1.3, C23F12.1, B02725, C34G6.6, 5A shows ?uorescence in the hypodermis (arrow) and seam c37c35, C42D8.5, c1344, CD4.6, 131054.15, F08C6.1, cells (arrowhead) of an L4 larvae. FIG. 5B shows ?uores P09B12.1, F16H9.2, F18A13, P20G4.1, F25B4.6, cence in the rectal gland. The solid line traces the tail of the F33A8.1, F33C83, F38H4.9, F40G9.1, F41C3.4, worm, the dashed line outlines the intestine. FIG. 5C is a F41H10.7, F45G2.5, F49C12.12, F52B11.3, F53B8.1, pair of micrographs showing ?uorescence and Nomarski F54A5.1, F54C9.2, F57B9.2, H04M03.4, H19M22.1, images of the vulva of a young adult. FIG. 5D-5F are K05C4.1, K06B4.5, K07C5.6, K07D8.1, K08B4.1, micrographs showing expression of acn-1::gfp-pest in a K09H9.6, M03F4.7, M03F8.3, M162.6, M6.l, M88.6, worm. FIG. 5D shows ?uorescence in the excretory gland, R05D11.3, R07E4.6, R11G11.1, T01C3.1, T01H3.1, duct, and pore cells (Exc), and in the glial cells (G) of T14F9.1, T19B10.2, T23F2.1, T24H7.2, W01F3.3, interlabial neurons of larvae (lateral view). FIG. 5E shows W08F4.6, W09B6.1, W10G6.3, Y111B2A.14, Y37D8A.10, ?uorescence in the excretory gland (GN) and duct cells. A Y38F2AL.3, Y48B6A.3, ZC101.2, ZK1073.1, ZK1151.1, solid line traces the worm, and a dashed line outlines the ZK262.8, ZK430.8, ZK686.3, ZK783.1, ZK970.4, posterior bulb of the pharynx. FIG. 5F shows ?uorescence Y54E10BR.5, B0513.1, R06A4.9, Y105E8B.1, Y47D3B.1, in the hypodermis and seam cells of a late L1 larvae. FIG. Y54F10AL.2, T17H7.3, H27M09.5, F45E10.2, F25H8.6, 5G shows ?uorescence from mlt-18::gfp-pest in the hypo K04A8.6, ZC13.3, T19A5.3, F32D8.6, F53F4.3, F56C9.12, dermis (arrow) and seam cells (arrowhead) of a late L1 T25B9.10, ZK154.3, Y37D8A.19, Y37D8A.21, larvae. FIG. 5H shows ?uorescence from mlt-13::gfp in the Y71F9AL.7, Y51H1A.3, W03F9.10, ZK945.2, ZK637.4, hypodermis and seam cells of a late L3 larvae. The seam cell C30F8.2, F32H2.9, Y87G2A.5, Y53F4B.22, Y77E11A.13, ?uorescence from mlt-24::gfp-pest was observed only near C15H11.7, Y113G7B.23, C53H9.1, W09C5.6, T24B8.1, the L4/Adult molt, when the cells terminally differentiate Y71A12B.1, C26C6.3, C42D8.5, F53G12.3, Y41D4B.10, and fuse, whereas seam-cell ?uorescence from mlt-13::gfp and F10C1.5) as well as genes not previously suggested as targets for insecticides or nematicides (e.g., C0lH6.5, pest and milt-18::gfp-pest was observed most often near larval-to-larval molts, when the cells divide. The anterior of C17G1.6, C45B2.7, F11C1.6, F18C12.2, F29D11.1, the worm is at the right in all panels. F53G12.3, F56C11.1, K04F10.4, T05C12.10, T27F2.1, Y23H5A.7, and ZK270.1). A fourth class includes mlt genes involved in the neuroendocrine control of molting. Such DESCRIPTION OF THE INVENTION genes are expected to be conserved between nematodes and [0087] The post-embryonic development of C. elegans insects (e.g., Drosophila). C. elegans neuronal control genes proceeds through four larval stages that are separated by are often refractory to RNAi; thus, RNAi against neuroen periodic molts when the collagen-like cuticle that encases docrine control genes is likely to effect molting in only a the worm’s body is shed and synthesiZed anew. As reported small percentage of larvae. Neuroendocrine control genes in more detail below, genes important for molting in C. will likely be identi?ed among mlt genes whose inactivation elegans were identi?ed by the present inventors through a by RNA interference results in molting defects in less than genome-wide screen using bacterial-mediated RNA-inter 5% of larvae (e.g., C09F12.1, C09H10.2, C17H12.14, ference (RNAi) to reduce gene function. Molting (mlt) gene C37C3.2, D2085.1, EEED8.5, F10E9.7, F19F10.9, F28F8.5, inactivation by RNAi caused larvae to become trapped in F32D1.2, F35H10.4, F41E7.1, F42A8.1, F54B3.3, F55A3.3, old cuticle while attempting to molt. Inactivation of these genes, their orthologs in EcdysoZoans, or their encoded proteins by genetic or chemical means is expected to block molting and larval development in virtually any EcdysoZoan (e.g., nematodes and insects). F53F4.3, F56C9.12, T25B9.10, ZK154.3, Y37D8A.19, Y37D8A.21, Y71F9AL.7, Y51H1A.3, W03F9.10, ZK945.2, [0088] Four classes of genes central to molting function ZK637.4, C30F8.2, F32H2.9, Y87G2A.5, Y53F4B.22, have been identi?ed. The ?rst class includes mlt genes that Y77E11A.13, C15H11.7, Y113G7B.23, C53H9.1, function speci?cally in nematodes (e.g., C09G5.6, C17G1.6, W09C5.6, T24B8.1, and Y71A12B.1. Additional mlt genes C23F12.1, C34G6.6, F08C6.1, F09B12.1, F16B4.3, may be identi?ed using a nematode strain having enhanced F18A1.3, F45G2.5, F49C12.2, F53B8.1, H04M03.4, susceptibility to RNAi. H19M22.2,K07D8.1,M6.1,M88.6,T05C12.10,W01F3.3, W08F4.6, Y111B2A.14, ZK262.8, ZK270. 1, and ZK430.8). [0089] These compositions and methods are described The protein products of such genes are likely to function in further below. the execution phase of nematode molting and represent RNAi Library Screen attractive targets for the development of highly speci?c nematicides. The second class includes mlt genes conserved [0090] To systematically identify genes required for molt in insects and nematodes, but not present in humans or yeast ing in C. elegans, a library of 16,757 bacterial clones was (e.g., C0lH6.5, F11C1.6, F52B11.3, and ZK686.3). Nem used. Each HT115(DE3) E. coli clone (Timmons et al., Gene aticides and insecticides targeting such mlt genes, or their 263:103-112, 2001) expressed a double-stranded RNA cor orthologs in insects or parasitic nematodes, are likely to responding to a single open reading frame (ORF) predicted US 2006/0178292 A1 Aug. 10, 2006

in the C. elegans genome (Fraser et al., Nature, 408:325-30, and daf-7(el372) Were fed bacterial clones expressing 2000). Approximately 85% of all ORFs predicted to be dsRNA for each molting gene and cultivated at restrictive present in the genome of C. elegans Were represented in this temperature (250 C.) for 3 days, such that control animals all library. Approximately 2,000 additional clones, Which are became dauers. Animals Were then shifted to permissive publicly available through the Vidal lab ORFeome project at temperature (150 C.) for 2 days, alloWing control animals to Harvard University (Orfeome project, Harvard University molt to the L4 stage. Observation of L2d or dauer larvae Website) Were also screened. The genes listed in Table 1B With the Mlt phenotype, in either genetic background, indi Were identi?ed in this screen. cated that a given gene inactivation disrupted the L2d/dauer [0091] Brie?y, the bacterial colonies from each plate of the or dauer/L4molt. library Were inoculated into 96-Well microtiter dishes con taining 300 ul of LB With 50 ug/ml of ampicillin. The Nomenclature bacteria Were then cultured for approximately sixteen hours [0095] C. elegans genes Whose inactivation by RNAi at 30° C. 30 ul of each overnight culture Was plated onto a single Well of a 24-Well plate containing Nematode GroWth caused a molting defect, or Mlt phenotype, are shoWn in Medium (NGM)-agar, IPTG (8 mM ?nal concentration), Tables 1A, 1B, 4A-4D, 7 and 8. These genes are identi?ed and carbenicillin (25 ug/ml). by a C. elegans gene name and by an open reading frame number. Genes not previously assigned a C. elegans gene [0092] Early Ll larvae from Wild-type (N2) Worms Were name are identi?ed herein as mlt-l2 to mlt-93. Eleven genes isolated using standard techniques, and approximately identi?ed in our screen had been previously identi?ed as tWenty larvae Were added to each Well. The Worms Were then functioning in molting, but had not been previously identi incubated in individual Wells at 200 C. for tWo and a half ?ed as targets for a nematicide, insecticide, or other com days With one of the 16,757 bacterial clones serving as a pound that inhibits molting. These genes include C0lH6.5 food source. Nematodes in each Well Were examined for molting defects by visual inspection using a standard light (nhr-23), C45B2.7 (ptr-4), FllCl.6 (nhr-25), F18Cl2.2 microscope. These assays Were carried out “blind” (i.e., the (rme-8), F29Dll.l (lrp-l), F53G123, F56Cll.l, K04F10.4 researcher examining the nematode’s molting phenotype (bli-4), T05Cl2.l0 (qhg-l), T27F2.l (C. elegans Skip), and Was unaWare of the identity of the bacterial clone present in ZK270.1 (ptr-23). Orthologs of these genes Were not previ the Well at the time the phenotype Was scored). A molting ously identi?ed. Some genes not previously identi?ed as defect Was identi?ed by the presence of larvae With unshed functioning in molting had been previously assigned a C. cuticle attached to their bodies (the Mlt phenotype). Molting elegans gene name. In keeping With C. elegans nomencla defects Were never observed in control larvae fed on bacteria ture practices, genes previously assigned a C. elegans gene transformed With an empty vector. The majority of control name have not been renamed. larvae greW into gravid adult nematodes and sired progeny during the time of observation. As a positive internal control Mlt Phenotypes for the ef?cacy of post-embryonic RNAi, Wild-type N2 [0096] Post-embryonic RNAi against milt genes listed in larvae Were concurrently fed HTl 15(DE3) bacteria express Tables 1A and 1B produced molting-speci?c defects in ing dsRNA corresponding to a knoWn mlt gene, lrp-l. 5-100% of larvae (Table 1A and Table 1B). The majority of [0093] C. elegans genes required for molting are listed in these Worms also exhibited a larval arrest phenotype. This Tables 1A, 1B, 4A-4D, 7, and 8. Open reading frames list identi?es target genes by C. elegans cosmid name and initially identi?ed as causing a Mlt phenotype Were veri?ed open reading frame number. Homology searches using the by re-screening tWo additional times. The identity of the blast algorithm and information available at Wormbase gene represented by each bacterial colony Was veri?ed by (WWW.Wormbase.org), a central repository of data on C. sequencing. This Was accomplished by sequencing the insert elegans, Were used to identify the function of encoded in the plasmid DNA isolated from the bacterial clone using proteins. At least three mlt genes, mlt-24, mlt-25, and primers complementary to ?anking sequence present in the mlt-27, encode proteins predicted to function as secreted vector L440 (Timmons et al., Nature 3911806-811, 1998). proteases. These proteases are likely to function in the [0094] To evaluate the dauer molt, hatchlings of the tem process of cuticle release, or, possibly, in the processing of perature-sensitive, dauer constitutive mutants daf-2(el370) peptide molting hormones.

TABLE lA

RNAi Produced Molt Defects in 5il00% of Exposed Larvae

Gene ORF Accession No. Function Reference for Mlt phenotypee mlt-l9 B0024.l4 reflNMi073255 Pro-collagen nhr-23 C0lH6.5 reflNMi059638 nuclear hormone receptor Kostrouchova et al., 19981 transcription factor reflNMfO 6391 0 cuticle collagen CllHl.3 reflNMi077984 mlt-24 C17Gl .6 reflNMi077268 Metalloprotease, secreted Morita et al., 20022 mlt-20 C23Fl2.l re?NMi077180 endothelial actin-binding protein repeats mlt-21 B02725 same as above endothelial actin-bindin g protein repeats US 2006/0178292 A1 Aug. 10, 2006 11

TABLE lA-continued

RNAi Produced Molt Defects in 5*100% of Exposed Larvae

Gene ORF Accession No. Function Reference for Mlt phenotypee mlt-14 C34G6.6 reflNMi059305 repetitive Cys motifs; 4 PAN domains mlt-22 C37C3.3 mlt-27 C42D8.5 reflNMi076466 Angiotension converting enzyme, metalloprotease ptr-4 C45B2.7 reflNMi076612 sterol-sensing domain Zugasti et al., 20023 mlt-23 CD4.4 reflNMi072073 coiled coil mlt-28 CD4.6 pirlT32525 protease mlt-29 D1054.15 reflNMi073362 G-protein beta WD-40 repeats beta-transducin-like mlt-21 C26C6.3 NMiO59708. Astacin metalloprotease acn-l C42D8.5 NMi076466 Angiotension converting enzyme mlt-2O F08C6.1 reflNMi076885 ADAM/reprolysin metalloprotease, 12 of Thrombospondin type I domain mlt-13 F09B12.1 reflNMi078111 MAM domains, secreted nhr-25 F11C1.6 reflNMi077761 nuclear hormone recptor Gissendanner and Sluder, 20004 mlt-3O F16H9.2 reflNMi077722 nuclear hormone receptor lir-1 F18A1.3 emblAJ130959 Transcription factor like lin-26 rme-8 F18C12.2 gblAF372457 endocytosis DNA] domain Zhang et al., 20015 mlt-31 F20G4.1 reflNMi059784 mlt-32 F25B4.6 reflNMi072095 hydroxymethlglutaryl-CoA synthase lrp-l F29D11.1 reflNMi059726 LDL-receptor related Yochem et al., 19996 let-858 F33A8.1 reflNMi063962 mlt-33 F33C8.3 reflNMi078044 tetraspanin mlt-34 F38H4.9 reflNMi069846 Hs PZAB serine/threonine phosphatase mlt-35 F40G9.1 reflNM_064775 Ankryin repeats mlt-36 F41C3 .4 reflNMi062446 elo-5 F41H10.7 reflNPi500793 GNS1/SUR4 family mlt-17 F45G2.5 reflNMi067371 SS pancreatic trypsin inhibitor mlt-38 F49C12.12 reflNMi069234 transmembrane protein mlt-15 F52B11.3 reflNPi502699 4 PAN domains. Secretory protein mlt-39 F53B8.1 pirHT22551 Hu PLECl orthologue; plectin, kakapo homolog mlt-4O F53G12.3 reflNMi058283 NADPH oXidase Fraser, 20007 mlt-41 F54A5.1 reflNMi058402 stc-1 F54C9.2 reflNMi063407 Heat shock 70 Kd protein (HSP70) F53G12.3 animal haem peroxidase; gp91/phox1 DuOX F56C11.1 reflNMi058285 NADPH oXidase; animal haem Fraser, 20007 peroxidase; gp91/phox1 mlt-42 F57B9.2 reflNMi066115 TX human 1 Proline Rich, 1 Glycosylytransferase family 5 mlt-43 H04M03 .4 reflNPiS 00 8 84 let-805 H19M22.1 reflNMi065198 myotactin form A bli-4 KO4F10.4 reflNMi059427 subtilase protease Thacker et al., 19958 mlt-44 KO5C4.1 pirlT23336 proteasome subunit mlt-45 KO6B4.5 reflNMi074499 nuclear hormone receptor mlt-46 KO7C5.6 reflNMi073260 Zinc ?nger mup-4 KO7D8.1 reflNMi066244 mup-4 ion-channel SEA domains, Ca-binding EGF domains lag-1 KO8B4.1 reflNMi068515 DNA-binding protein, IPT/TIG domain mlt-47 KO9H9.6 reflNMi058707 homologueof Dm Peter Pan, Which is required for larval growth mlt-48 M03F4.7 reflNMi076443 calcium binding protein, EF-hand family 13X mlt-49 M03F8.3 reflNMi072146 crn HAT (Half-A-TPR) repeat 10X, TPR repeat 3X mlt-50 M1626 reflNMi075434 ifc-2 M6.1 reflNMi075732 intermediate ?lament protein A pan-1 M88.6 reflNMi065523 lecuine-rich repeats ran-4 R05D11.3 reflNMi059921 Nuclear import; Nuclear Transport Factor 2 (NTFZ) homologue US 2006/0178292 A1 Aug. 10, 2006 12

TABLE lA-continued

RNAi Produced Molt Defects in 5*100% of Exposed Larvae

Gene ORF Accession No. Function Reference for Mlt phenotypee kin-2 R07E4.6 reflNMi076598 mlt-52 R11G11.1 reflNMi070836 nuclear homrone receptor mlt-53 T01C3.1 reflNMi074284 WD domain, G-beta repeats X13 mlt-54 T01H3.1 reflNMi063258 proteolipid protein PPAl like protein Y41D4B.10 NMi067707 Delta-serrate ligand precursor qhg-l T05C12.10 reflNMi063324 hedgehog-like, hint module Wang et al., 19999 mlt-55 T14F9.1 reflNMi076011 ATPase subunit mlt-56 T19B10.2 reflNMi073447 secretory protein mlt-57 T23F2.1 reflNMi076531 glycosyltransferase mlt-58 T24H7.2 reflNMi062848 Heat shock protein hsp70, Cytochrome b/b6 Ce Skip T27F2.1 reflNMi073549 Drosophila puff speci?c protein Kostrouchova et al., 200210 BX42 like F10C1.5 NMi062737 DSX DNA binding domain mlt-18 W01F3.3 reflNMi075592 multiple BPTI-like domains, secretory protein mlt-12 W08F4.6 reflNMi061358 novel secretory protein mlt-59 W09B6.1 reflNMi061521 acetyl-CoA carboxylase ifa-2 W10G6.3 reflNMi078247 intermediate ?lament protein pqn-8O Y111B2A.14 reflNMi067244 prion-like mlt-60 Y37D8A.10 reflNMi067275 transmembrane protein mlt-61 Y38F2AL.3 reflNMi067786 ATPase mlt-62 Y48B6A.3 reflNMi067371 5'—3' eXonuclease domain; eggshell protein unc-52 ZC101.2 reflNMfO 64645 basement membrane proteoglycan mlt-63 ZK1073.1 reflNMi078233 mlt- 64 ZK1151.1 reflNM_0 60597 plectrin mlt-65 ZK262.8 reflNMi075208 Myosin head (motor domain) ptr-23 ZK270.1 reflNMi061202 sterol-sensing domain SchulZe et al., 200211 mlt-11 ZK430.8 reflNMi062376 animal haem peroxidase; ShTk domain mlt-67 ZK686.3 reflNMi066290 Ankryin repeat mlt-16 ZK783.1 reflNMi066269 ECM micro?bril component (Hs FBN-l homolog) mlt-68 ZK970.4 reflNMfO 63 816 H+—transporting ATPase lKostrouchova et al., Proc. Natl. Acad. Sci. 99: 955449559, 2002 2Morita et al, EMBO 23: 106341073. 3Zugasti et al., 2002 European Worm Meeting 4Gissendanner et al, Dev. Biol, 221: 259472, 2000 5Zhang et al., Mol. Biol. Cell, 12: 2011421, 2001 6Yochem et al., Development, 126: 5974606 7Fraser et al., Nature, 408: 325430, 2000 8Thacker et al., Genes Dev. 9: 956471, 1995 9Wang et al., 1999, International Worm Meeting l0Kostrouchova et al., Proc. Natl. Acad. Sci. 98: 736045, 2001 llSchulZe et al., 2002 European Worm Meeting

[0097]

TABLE 1B

Genes identi?ed in RNAi screen of clones from Vidal Orfeome Pro'ect

Predicted Gene Brief Molecular I.D./ Gene name Accession # Domains High frequency of Mlt phenotype

Y54E10BR.5 reflNMi058691 Signal Peptidase B0513.1 gei-5 reflNMi070273 GEX-3 interacting protein R06A4.9 reflNMi064584 WD domain, G beta repeats, HMGl/Y DNA binding domain Y105E8B.1 lev-11 reflNMi06113 8 tropomyosin Y47D3B.1 re?NMi067064 DUF23 Y54F10AL.2 est-1 reflNMi065164 telomerase subunit T17H7.3 re?NMi064848 H27M09.5 reflNMi059558 novel US 2006/0178292 A1 Aug. 10, 2006

TABLE lB-continued

Genes identi?ed in RNAi screen of clones from Vidal Orfeome Proiect

Predicted Gene Brief Molecular I.D./ Gene name Accession # Domains

F45E10.2 refNMi063970 solute carrier family 22 member F25H8.6 refNMi069384 DNA binding, BED Zinc ?nger K04A8.6 refNMi072260 F-box ZC13.3 re?NMi075772 MAM domain T19A5.3 re?NMi072907 novel lOW frequency of Mlt phenotype

F32D8.6 emo-1 refNMi073377 Protein translocation — Sec61 ortholog F53F4.3 refNMi073966 novel F5 6C9.12 refNRiOO 1470 novel T25B9.10 ref NMi069598 endo/exonuclease phosphatase family ZK154.3 mec-7 refNMi076912 beta-tubulin Y37D8A.19 ref NMi067286 novel secreted protein Y37D8A.21 ref NMi067285 RNA binding, RNP domain Y71F9AL.7 ref NMi058666 novel transmembrane protein Y51H1A.3 ref NMi064506 NADH dehydrogenase 1 beta subcomplex 8 19 kDa like W03F9.10 ref NMi070740 DUF382, Proline rich, PSP, HMG-l DNA binding ZK945.2 pas-7 ref NMi063776 proteosome alpha subunit ZK637.4 ref NMi066563 novel putative nuclear protein C30F8.2 refNMi059114 H+ transporting ATPase C subunit F3 2H2.9 tba-6 ref NMiO 6001 8 tubulin alpha Y87G2A.5 vrs-2 ref NMi060976 cytoplasmic valyl tRNA syhtethase Y53F4B.22 arp-1 ref NMi064707 actin like Y77E11A.13 npp-20 refNMi067686 nuclear core protein, related to essential transport protein SEC1 C15H11.7 pas-1 refNMi074170 26s proteosome subunit Y113G7B.23 psa-1 ref NMi075505 SWUSNF complex chromatin remodeling C53H9.1 rpl-27 refNMi058504 large ribosomal subunit 27 W09C5.6 rpl-31 refNM_060990 large ribosomal subunit 31 T24B8.1 rpl-32 refNMi063533 large ribosomal subunit 32 Y71A12B.1 rps-6 refNMi061034 small ribosomal subunit S6

Cuticle Retention Phenotypes The majority of gene inactivations also disrupted molts into, or out of, dauer, an alternative developmental stage that is [0098] All Mlt larvae failed to fully shed their cuticles. For adapted for survival in unfavorable conditions and example, RNAi against mlt-12, mlt-13, mlt-18, and mlt-24 resembles the infective form of parasitic nematodes. Gen resulted in larvae partially encased in a sheath of unshed erally, animals that failed to complete a molt also ceased to cuticle (FIGS. 1A-1E). The Mlt phenotype observed in these animals resembled the phenotype of lrp-1 (RNAi) develop, but they Would occasionally escape old cuticle after nematodes. lrp-1 Was previously shoWn to be required for several hours, only to become trapped again at the next molt, molting (Yochem et al., Development, 126: 597-606, 1999). as observed in qhg-1(RNAi) larvae. [0099] Interestingly, speci?c differences Were observed in [0100] Reproductive Phenotype cuticle retention among Mlt larvae. The tissue of mlt [0101] While the majority of Mlt larvae arrest develop 13(RNAi) animals remained tethered to old cuticle expelled ment and die, possibly as a consequence of starvation, Mlt from the buccal cavity, suggesting a defect early in the animals trapped in cuticle during the L4-to-adult transition execution of molting (FIG. 1E). In contrast, unc-52(RNAi) occasionally produced a limited number of progeny. This nematodes arrested With sheaths of cuticle extending from Was observed in qhg-l (RNAi), nhr-23(RNAi), and mlt their posteriors, and appeared paralyzed except for small 13(RNAi) animals. head movements. The phenotype of unc-52(RNAi) nema todes suggested a defect in the ?nal stages of ecdysis. [0102] Phenotype Associated With Secretory PathWay Undetached cuticle Was observed around the most anterior Defects portion of mlt-12(RNAi) animals (FIG. 1D). This anterior region corresponds to the location of the cells hyp2 through [0103] RNAi against many genes knoWn to function in the hyp6. Approximately 20% of mlt-24(RNAi) animals had secretory pathWay, such as the Worm orthologs of the vesicle cuticular sheaths Wrapped around their mid-sections (FIGS. coat proteins SEC-23 and B-COP, disrupted molting (Table 1A and 1B). The discovery of phenotypic classes among Mlt 2). Those secretory pathWay components that gave a Mlt larvae indicated that sets of mlt genes likely act together at phenotype When inactivated by RNAi are listed in Table 2. speci?c steps of ecdysis, or that some mlt genes are required The genes are listed by C. elegans cosmid name and open for apolysis of cuticle covering only one or tWo regions of reading frame number. Homology searches using the blast the body. Further, most, if not all, genes uncovered appear algorithm and information available at Wormbase (WWW essential for all four molts, since their inactivation produces .Wormnbaseorg), a central repository of data on C. elegans, molting-defective larvae at several developmental stages. Were used to identify the function of encoded proteins. US 2006/0178292 A1 Aug. 10, 2006

TABLE 2 TABLE 3A

RNAi against Secretory Pathway Components Produced Molt Defects RNAi Produc?d Molt D?f?cts in L655 than 5% of Expos?d Larva‘?

G616 ORF Mol?cular Function or Idmtity Gene ORF Molecular Function/Protein Domains rpl-23 B0336.10 ribosomal protein nsf-1 H15N14.1 vesicular fusion; like human NSF rpS_0 B03931 ribosomal protein fab-5 F26H9-6 ms sup?rfamily GTPase rpl-14 C04F12.4 ribosomal protein L14 tfg-l Y63D3A.5 part of COPII complex; vesicle trafficking rps-3 C23G10.3 ribosomal protein snf-7 C5 6C10.3 vacuolar sorting rps-lO D1007.6 ribosomal protein sar-l ZK180.4 GTP-binding protein fps-23 P281317 ribosomal Protein arf-3 F57H12.1 lGTP-binding protein rpm-7 P3511104 Qbosomal Prom? rab-1 C39F7.4 ras-family rpsiIlI ri?osomai protein sec-23 Y113G7A.3 COPII complex vesicular transport rps_ ' IT Osoma pro T11 I I I rps-ll F4OF11.1 ribosomal protein dpy-23 R160.1 Clathrin adaptor complexes medium chain 7x IPSQZ F53A3 3 ribosomal protein dyn'l C02C6-1 dynamin family 8X rps-16 T01C3.6 ribosomal protein mlt-69 E03H4.8 beta coatomer-like $5.19 T05F1_3 ribosomal protein $19 mlt-70 F59E10.3 Clathrin adaptor complex small chain rpl-18 Y45F10D.12 ribosomal protein m1t_71 K12H4_4 Signal peptidase mlt-75 CO9F12.1 secretory protein mlt_72 B10143 alpha_SNAPI NSF attachm?nt protein mlt-76 C09H10.2 Forkhead-associated (FHA) domain mlt-73 C13B9.3 clathrin adaptor mg]; gTPaSIe f d I HUB/IFS 2 . m - . omain oun in x Ink-74 113112392 181601 liamllyf 1 G B k A N mlt'79 B20851 ' Om‘) 0g 0 got‘ ( en 3“ °°' 0' mog-5 EEED8.5 RNA helicase DEAD/DEAH box helicase NMJ62446) mig-lO F10E9.7 PH domain F38A1.8 SRP-54 (GenBank Acc. No. NMi171254) mlpgo F19F10I9 mlt-8l F28F8.5 mlt-82 F32D1.2 ATP synthase epsilion chain I I I I vha-5 F35H10.4 H+ ion transport V-type ATPase 116 kDa subunit [0104] Interestmgly, the bod1es of an1mals undergolng family RNAi against secretory pathway genes tended to disinte- Ink-83 F41E7-1 TM G-prot?in beta WD-40 repeats grate over time, distinguishing them from other Mlt larvae. mlt'84 1:42AM TGFB path mlt-85 F54B3.3 AAAATPase The 1solat1on of s1xteen secretory pathway genesI 1n a screen mlt_86 F55A3I3 general chromatin factor for larvae unable to molt 1nd1cated that a functlonal secre- m1t_87 1:561:35 Ribosomal protein 53A tory pathway is needed either to generate new cuticle or to mlt-88 H06I04.4a 4 ubiquitin domains, CH2 Zinc ?nger export enzymes that allow larvae to break free of the old m1t'89 K06A4-6 Cuticle mlt-90 K10D6.1 GABA receptor beta subunit ' mlt-91 R06Al0.l [0105] Larval Arrest Phenoty es mlt-92 T07D10.1 transmembrane protein p lin-29 Y17G7A.2 lin-29 [0106] RNAi against genes shown in Table 3A produced mlt'93 Y23H5A'7 ammoacyl'tRNA Synthetas? vha-11 Y38F2AL.3 ATPase molting defects in less than ?ve percent of larvae, and also Y41D4BI21 produced an early larval arrest phenotype (i.e., arrest in the Y41D4B.5 ion channel protein L1 or L2 stage) in the majority of animals. RNAi against Y45F10B-5 genes shown in Table 3B produced molting defects in 10% Y55H10A'1 Cadh?rin elegansor less ofcosmid larvae. nameThis listand identi?es open reading the target frame genes number. by C. ZK265I6 G_pmt6in coupled Tempter Homology searches using the blast algorithm and informa- ZK652.1 small nuclear ribonucleoprotein tion available at wormbase (www.wormbase.org), a central repository of data on C. elegans, were used to identify the function of encoded proteins. [0107]

TABLE 3B

Gene inactivations that cause molting defects in 10% or less of larvae

Gene ORF Accession # Molecular Identity B03481 reflNMi070727 nematode-speci?c protein family clc-1 CO9F12.1 reflNMi077446 claudin-like C23F12.1 reflNMi077l79 endothelial actin-binding protein repeats C37C3.2 gblU64857 domain found in IFZB/IFS CD4.4 reflNMi072073 coiled 4-coil domain pas-6 CD4.6 reflNMi07207l proteosome subunit cdc-5 D1081.8 reflNMi059902 myb-like DNA binding domain pyr-1 D2085.1 reflNMi063437 glutamine-dependent carbamoyl-phosphate synthase