AU3349101A - 5HT3 receptors of nematodes, polynucleotide molecules encoding same, and antagonists thereof - Google Patents

Description

WO 01/61000 PCT/AU01/00150 1 5HTg RECEPTORS OF NEMATODES. POLYNUCLEOTIDE MOLECULES ENCODING SAME. AND ANTAGONISTS THEREOF Field of the Invention: 5 The present invention relates to the identification of compounds for the control of nematodes, insects and other invertebrate pests. More particularly, the present invention relates to the isolation and cloning of polynucleotide molecules encoding nematode 5-HT, receptors. The receptors may be used in assays for the identification and/or assessment of candidate nematicidal, 10 insecticidal and other pesticidal compounds. Background to the Invention: Many species of nematodes are parasites of considerable medical, veterinary and agricultural significance. For example. nematodes of the 15 Orders Strongylida, Strongyloides, Ascaradida, Oxvurida and Trichocephalida include many species that cause disease in humans, sheep, cattle, pigs and other species. Further, nematodes of the Orders Tylenchida and Aphelenchida. and others, include species which are parasitic of important crop plants and fungi. 20 It has been conservatively estimated that plant parasitic nematodes cause US $77 billion worth of damage to major food crops annually [Evans, K. and Haydock, P. 1999. Control of plant parasitic nematodes. Pesticide Outlook. 107-111]. There are, unfortunately. very few control options for plant parasitic nematodes. Fumigants such as methyl bromide are generally 25 being withdrawn from sale and use, because of their detrimental effects on the ozone laver, whilst the remaining available agents are among the most toxic and undesirable pesticides in current use. Animal parasitic nematodes infect humans, companion animals and livestock and cause serious morbidity and economic loss worldwide. This 30 group of parasites includes hookworms and roundworms. They can cause anaemia. loss of weight, hyperimmune reactions and other complications, including death of livestock. There are currently a small number of human and veterinary drugs available for their treatment but, particularly in the veterinary field, the efficacy of a number of existing drugs is dropping 35 because of resistance development.

WO 01/61000 PCT/AUO1/00150 2 For the reasons described, there is an ongoing need for the identification of new nematizidal compounds. Pharyngeal pumping is the basis of nematode feeding and the ability of nematodes to maintain their "hydrostatic skeleton" [Brownlee, D.J.A. et al. 5 1997. Actions of the anthelmintic ivermectin on the pharngeal muscle of the parasitic nematode. Ascaris suum. Parasitology, 115: 553-5611. The pharyngeal pump of nematodes is already a well-established target organ for anthelminthics and nematicidal agents. In particular, inhibition of pharyngeal pumping is a major mode of action of ivermectin, an extremely 10 successful modern nematicide and insecticide. Ivermectin acts on inhibitory glutamate receptors present in the pharynx and other tissues of nematodes and insect [Brownlee, D.J.A. et al. 1997, supra]. Identification of new molecular targets in the nematode pharynx would greatly facilitate the discovery process for nematicidal compounds and insecticidal compounds 15 since knowledge of a molecular target can assist in lead compound choice and/or design. Further, for targets representing molecular receptors, the possibility of isolating the receptor gene offers the prospect of using cloned receptors to screen natural product collections and synthetic compound libraries. Active molecules recovered from these screens may have utility in 20 controlling nematodes, insects and other pests. Examples of this include the macrocyclic lactone nematicides (avermectins) which were originally registered as anthelminthics but are now being used increasingly as insecticides. The damage caused by insect pests is better known and characterised 25 than that caused by nematodes. For example, the worldwide market for chemical insecticides is about US $12 billion, mostly in crop protection, but also in animal and public health. The market is growing at about 5% p.a. These costs of control are only a fraction of the economic costs of losses to crops and livestock worldwide. The role of insects in vectoring major 30 diseases to humans is also well-known. In particular, sucking plant pests such as aphids and plant-hoppers are second only to caterpillars in their econormc importance and their value as a market for insecticides. They are particularly important in Europe and Asia. Whilst there are some existing pesticides active against these pests, a number of them are highly toxic and 35 development of resistance is also causing problems. Thus, there is a great need for new classes of insecticides active on these pests.

WO 01/61000 PCT/AU01/00150 3 Also, insects with piercing and sucking mouthparts are the main vectors of diseases to humans and livestock. These vectors include mosquitoes (e.g. malaria, Japanese encephalitis, dengue fever etc.), higher flies (e.g. onchocerciasis) and true bugs (e.g. trypanosomiosis). Existing control 5 measures are increasingly reliant on pesticides (e.g. permethrin-treated mosquito nets), because of the absence or failure of drug treatments. Therefore, there is also a need for new classes of insecticides active against these pests. Serotonin

(

5 -Hvdroxytrvptamine, 5-HT) is known to have a number of 10 profound effects on the behaviour of Caenorhabditis elegans and other nematodes. In C. elegans, exogenously applied 5-HT results in reduced locomotion, increased pharyngeal pumping, increased egg-laying and decreased defaecation. It is also involved in male mating behaviour. These effects of exogenous 5-HT are believed to occur because 5-HT is a natural 15 nematode neurotransmitter that serves these behaviours. For example, two serotonergic neurones (NSM) are located over the pharynx whilst the HSNL and HSNR serotonergic neurones connect with the vulva. However, it is quite likely, based on the known biology of 5-I-IT in vertebrates that each of these behaviours is controlled by action on different receptors present in 20 different cells. Vertebrate serotonin receptors are known to fall into two distinct multigene superfamilies. One of these, the rhodopsin/-adrenergic receptor superfamily, includes 7 -transmemberane G-protein-linked receptors of the 5 HTI. 5-HT2, 5-HT 4 , 5-HT 5 , 5-HT, and 5-HT 7 classes. Receptors of the 5-HT 3 25 class belong to the nicotinic-acetylcholine receptor (nAChR), GABA-, glycine and glutamate-gated ion channel superfamily and are pentameric, 4 membrane-spanning ligand-gated ion channels. It is generally observed that physiologically expressed pentameric receptors of this family comprise two or more types of subunits, with each type being the product of a distinct gene. 30 While functioning ion channels may be obtained experimentally with a pentamer composed of identical subunits, these do not behave identically with respect to their channel conductance properties, to the heteropentameric ion channel that is present in vivo. In the case of the mammalian 5-HT 3 gated ion-channel, faithful electrophysiology has only been obtained with a 35 heteromeric ion channel containing both 5-HTA and 5-HT 3 a subunits [Davies, P.A. et al. 1999. The 5-HT 3 B subunit is a major determinant of serotonin WO 01/61000 PCT/AU01/00150 4 receptor function. Nature. 397, 359-363]. The mammalian 5-HT 3 receptors are known to form channels that gate the passage of cations across the'cell membrane and when activated they tend to excite the cell. In this respect, as in many others, their closest relatives are the nicotinic acetylcholine 5 receptors. GABAA-gated, glycine-gated, and the invertebrate-specific glutamate-gated ion channels all gate the passage of anions and their activation generally hyperpolarises the cell membrane. It should be noted that, although ligand-gated ion-channels are often heteropentamers (i.e. composed of five receptor subunits with the subunits being the products of 10 two or more genes, as was unequivocally demonstrated for the mammalian 5 HT, receptor [Davies, P.A. et al., 1999, supra], it is possible to form functional channels with only a single subunit and, furthermore, expressed single subunits bind serotonergic ligands with expected authentic pharmacology [(e.g. Maricq, A.V. et c., 1991. Primary structure and functional expression of 15 the 5-HT, receptor. a serotonin-gated ion channel. Science, 254)]. This means that it is possible with known 5-H1T, receptors to perform functional and radioligand binding screens for agonists and antagonists using expressed single subunits. In the case of the 7 -transmembrane 5-HT receptors, a number of 20 invertebrate homologues have now been cloned. These have significant sequence differences from the vertebrate examples but are nevertheless interpretable within the molecular phylogeny generated for the vertebrate receptors. To the extent that they have been studied there are also some pharmacological differences between vertebrate and invertebrate receptors. 25 Until now, there has been no report of the cloning of a cationic 5-HT 3 receptor subunit from an invertebrate species. One electrophysiological study of the snail Helix aspersa, has been reported which measured a channel with conductance properties and serotonergic pharmacology indicative of a 5

HT

3 receptor [Green. K.A. et al. 1996. Ligand gated ion channels opened by 5 30 HT in molluscan neurones. Brit. J. Pharmacol. 119: 602-608]. However. one of the most eminent researchers in the field of C. elegans neurobiology has stated that "[t]here are no sequences in the C. elegans database with significant similarity to the ionotropic serotonin receptor subclass 5-HT 3 " [Niacaris, T.. and Avery L., Expression patterns of candidate serotonin 35 receptors. The Worm Breeder's Gazette. 15:20, 1998]. The present inventors have now isolated from the invertebrate species, C. elegans, three WO 01/61000 PCT/AU01/00150 5 polynucleotide molecules encoding 5-HT receptor subunits which, based on sequence motif analysis and overall homology, show them to be 5-HTI receptor subunits. The present inventors have also obtained pharmacological evidence indicating that nematode pharyngeal pumping (which is known to 5 be involved in nematode feeding and maintenance of internal hydrostatic pressure) is under the control of a receptor with 5-HT 3 characteristics, and furthermore, that double stranded RNA-mediated gene suppression of one of these genes reduces the pharyngeal response to exogenous serotonin. The cloning of the disclosed C. elegans 5-HT 3 receptor subunits is therefore of 10 considerable importance for the identification of novel nematicidal compounds. Very recently, Ranganathan, R. et al. (2000. MOD-1 is a serotonin-gated chloride channel that modulates locomotarv behaviour in C. elegans. Nature. 408: 470-475) have described a novel serotonin-gated ion channel from the 15 nematode C. elegans. This seems to control nematode locomotion in response to food, gates anions, unlike all other known 5-HT receptors, and has novel pharmacology in that it does not respond to 5-HT 3 -specific inhibitors but does respond to some other serotonergic agents. These results indicate the existence of a second class of serotonin-gated ion-channels 20 distinct from the class that includes 5-IIT 3 A and 5-HTB . The results of Ranganathan et al. do not indicate whether there is a 5-HT 3 receptor in C. elegans or other invertebrates. Disclosure of the Invention: 25 Thus, in a first aspect, the present invention provides an isolated polynucleotide molecule encoding an invertebrate 5-HT 3 receptor subunit consisting of a nucleotide sequence which substantially corresponds to any one of those shown as SEQ ID NO:1-6, or which shows greater than 75% (more preferably, greater than 85%, most preferably greater than 95%) 30 homology to any or all of the nucleotide sequences shown as SEQ ID NO:1-6. The polynucleotide molecule of the first aspect, may be operably linked to nucleotide sequence elements necessary for, or to enhance. expression. For instance, the polynucleotide molecule may be operably linked to any suitable promoter sequence (e.g. constitutive or inducible promoters), 35 enhancer sequence or other element which regulates expression. Conveniently, the polynucleotide molecule of the first aspect may be WO 01/61000 PCT/AUO1/00150 6 introduced into an expr ssion cassette or vector for expression of the encoded 5-HT 3 receptor subunit. Thus, in a second aspect, the present invention provides an expression cassette or an expression vector comprising a polynucleotide molecule of the 5 first aspect. In a third aspect, the present invention provides a mammalian, insect, plant, yeast or bacterial host cell transformed with the expression cassette or vector of the second aspect. The host cell of the third aspect may be used to express one or more 10 types of 5-HT, receptor subunits (e.g. 5-HTA and 5-HT receptor subunits) to allow assembly within said cell of homomeric or heteromeric 5-HT 3 receptors. For the production of heteromeric 5-HT, receptors, the host cell is transformed with two or more expression cassettes or vectors, wherein each comprises a polynucleotide molecule encoding a different 5-HT 3 receptor 15 subunit. Alternatively, a single expression vector might be used which comprises two or more polynucleotide molecules each encoding a different 5 HT, receptor subunit. Preferably, said host cell expresses the polynucleotide molecule(s) such that the 5-HT 3 receptors are expressed onto the surface of a host cell. 20 In a fourth aspect. the present invention provides a method of producing 5-HT 3 receptors, comprising culturing the host cell of the third aspect under conditions enabling the expression of the polynucleotide molecule(s) and, optionally, recovering the expressed receptors. Preferably, the host cell is mammalian or of insect origin. Where the 25 cell is mammalian, it is presently preferred that it be a COS cell. Chinese hamster-ovary (CHO) cell or human embryonic kidney 293 cell. Where the cell is of insect origin, it is presently preferred that it be an insect Sf9 cell. In a preferred embodiment, the 5-HT3 receptors are expressed onto the surface of the host cell. 30 In a fifth aspect, the present invention provides an invertebrate 5-HT3 receptor comprising at least one subunit which is characterised by an N terminal amino acid sequence selected from the following: MIICYSCLTV (SEQ ID NO: 7), MLLPILLHFL (SEQ ID NO: 8) or MRRRFEIGIA (SEQ ID NO: 9), 35 or a functionally equivalent fragment of said receptor, in a substantially pure form.

WO 01/61000 PCT/AU01/00150 7 Preferably, the at least one subunit has an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 10, 11 or 12. In a sixth aspect, the present invention provides an assay for identifying and/or assessing nematicidal compounds, said assay comprising 5 contacting a 5-HT 3 receptor or a functionally equivalent fragment thereof according to the fifth aspect, or a cell transfected with and expressing 5-HT3 receptors from one or more expression cassette(s) or vector(s) of the third aspect, with a candidate nematicidal compound under conditions enabling the activation of 5-HT receptors, and detecting an increase or decrease in 10 activity of said 5-HT 3 receptor(s) or functionally equivalent fragment thereof. An increase or decrease in activity of the 5-HT 3 receptor(s) or functionally equivalent fragment thereof may be detected by measuring changes in cell membrane potential or Ca> levels. The step of contacting may involve contacting the 5-HT, receptor(s) or 15 functionally equivalent fragment thereof simultaneously with the candidate nematicidal compound and a serotonergic ligand. In a seventh aspect, the present invention provides an assay for identifying and/or assessing nematicidal compounds, said assay comprising contacting a 5-HT 3 receptor or a functionally equivalent fragment thereof 20 according to the fifth aspect, or a cell transfected with and expressing 5-HT3 receptors from one or more expression cassette(s) or vector(s) of the third aspect. with a predetermined amount of a suitably labelled serotonergic ligand together with a predetermined amount of a candidate nematicidal compound under conditions wherein said serotonergic ligand and said 25 candidate nematicidal compound competitively bind to the 5-HT 3 receptor(s) or functionally equivalent fragment thereof. and determining the amount of bound and/or unbound labelled serotonergic ligand. The determined amount of bound and/or unbound labelled serotonergic ligand enables a calculation of the relative binding affinity of the 30 candidate nematicidal compound to the 5-HT3 receptor(s) or functionally equivalent fragment thereof. The suitably labelled serotonergic ligand is preferably 5-hydroxytryptamine (5-HT). The serotonergic ligand may be labelled with, for example, any of radioisotopes (e.g. H 3 ), enzymes, biotin/avidin, a 35 fluorescent molecule or a chemiluminescent molecule.

WO 01/61000 PCT/AU01/00150 8 In the assay of the seventh aspect, the 5-HT 3 receptor, functionally equivalent fragment, or cell expressing 5-HT 3 receptors are preferably anchored to a support (e.g. the well surfaces of a 96-well plate). This allows unbound serotonergic ligand to be readily washed away by methods routine 5 in the art. In addition, the polynucleotide molecule of the first aspect, especially those comprising all or, at least, a 10 nucleotide part of any of the nucleotide sequences shown as SEQ ID NO: 1-6, may be used to probe for polynucleotide sequences encoding homologous 5-HT 3 receptor subunits from other species. 10 Such probes may be prepared using routine molecular cloning techniques [as described in, for example. Sambrook, J. et al. 1989. Molecular cloning: a laboratory manual, 2nd edition. Vol. 1-3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor] of a polynucleotide molecule according to the first aspect or a part thereof into a multiple cloning site of a suitable 15 bacterial plasmid (e.g. a multicopy bacterial plasmid such as those of the pUC series including pBlueScript (Stratagene, La Jolla, California), followed by transformation of a bacterial cell line (e.g. DH10B; Life Technologies, Grand island, New York) by well known electroporation techniques. Growth of the host bacterial cell line to high density in small scale liquid culture and 20 isolation of plasmid DNA by alkaline lysis followed by phenol chloroform purification may be conducted as described in Sambrook, J. et al. 1989, supra] (so-called mini-plasmid preparation) or by any one of a number of commercially available kits based on solid-phase adsorption of plasmid DNA followed by selective elution (e.g. "ultraclean MiniPlasmid Prep Kit" Mo Biol 25 Laboratories Inc.. Solana Beach, California). Subsequently, the cloned polynucleotide molecule or a part thereof for use as probes. may be excised from the plasmids by restriction endonuclease digestion as is well known in the art. Alternatively, the probes may be generated by polymerase chain reaction (PCR) amplification using a polynucleotide molecule according to 30 the first aspect or a part thereof as template DNA and two PCR primers of suitable length (e.g. 15-30 nucleotides), one of which is homologous to the 5' terminus of the polynucleotide molecule or part thereof and the other being homologous to the 3' terminus of the polynucleotide molecule or part thereof. PCR may conveniently be performed according to any of the methods well 35 known in the art (e.g. as described in Innis, M.A. et al. 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, California).

WO 01/61000 PCT/AU01/00150 9 Purification of the probes may be achieved by size fractionation on a low-melting point agarose gel, followed by detection of the band by et&lidium bromide staining or staining with any other fluorescent nucleic acid dye under ultraviolet illumination, excision of the agarose region containing the 5 probes, and recovery of the probes from the agarose matrix by melting and selective precipitation of DNA [e.g. Sambrook, J. et al. 1989, supra) or by chemical solubilisation of the agarose followed by adsorption of the probes using glass milk or any one of a number of commercially available kits based on solid-phase recovery of DNA from agarose gel matrices (e.g. QIAquick PCR 10 purification kit QIAGEN GmbH, Hilden. Germany). Labelling of the probes can be achieved by any of the methods well known in the art. including nick translation using a P" label. random-prime labelling with P 32 using. for example, the Giga Prime DNA-Labelling Kit (GeneWorks. Adelaide) or labelling with biotin or digoxigenin, or by tagging 15 the probes with a hapten which is detectable using an enzymatic label such as peroxidase, HRP or luciferase to generate a coloured product or chemiluminescence or other luminescent signal. Target DNA (e.g. cDNA and genomic libraries) for probing with the probes may be prepared from those species from which it is desired to obtain 20 polynucleotide sequences homologous to a polynucleotide molecule of the present invention starting with approximately 100mg (or more) of tissue which is derived from an animal or plant parasitic nematode species (e.g. Haemonchus contortus. Ascaris suum. Ascaridia galli. Pratylenchus sp, Globodera sp., Meloidogyne incognita, or from any species of insect including 25 those that have pest status in the orders Lepidoptera (e.g. Helicoverpa sp, Heliothis sp), Diptera (e.g. Lucilia sp, Simulium sp, Anopheles sp. Culex sp. Aedes sp.), Hemiptera (e.g. Mvzus sp, Aphis sp), or tissue from any other invertebrate, particularly those with piercing and/or sucking mouthparts and a mode of feeding or attachment to the host that involves sucking or pumping 30 of the alimentary canal). The starting tissue may comprise the whole organism or it may comprise the pharynx with its associated neural structures, or the entire alimentary canal with its associated neural structures. Ideally, the starting tissue will be obtained from a life-stage known to express a 5-HT, receptor. This information may be obtained by RNA blotting analysis 35 of a range of life-stages using the probes, but if this information is unavailable WO 01/61000 PCT/AUO1/00150 10 or unobtainable then it is convenient and possible to use tissue from mixed life-stages as the starting material. mRNA for the production of a cDNA library is conveniently prepared from the source tissue using a range of commercially available kits such as 5 the QuickPrep Micro RNA Purification Kit (Amersham Pharmacia Biotech Inc., Piscataway, N.J.). Alternatively, traditional methods such as that of Chomczvnski P. and Sacchi N. (1987. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162: 156-159) may be used for preparation of total RNA followed by 10 purification of mRNA by oligo-dT cellulose chromatography (Sambrook J. et al., 1989. supra). cDNA may also conveniently be prepared from mRNA using many commercially available kits such as the TimeSaver cDNA Synthesis Kit (Ainersham Pharmacia Biotech Inc.. Piscataway, N.J.) and the cDNA pool that can be generated is inserted into commercially available 15 lambda arms by conventional ligation techniques. A number of precut variants of lambda phage are available commercially. One suitable variant is lambda gt1o - Nod, phosphatased from Promega Corporation (Madison, Wisconsin). Others include lambda-Zap (Stratagene, La Jolla, California) or lambda-Excel (Amersham Pharmacia Biotech Inc., Piscataway, N.J.). The 20 pool of lambda phage DNA with the the cDNA inserts can then be assembled into a library of infectious phage particles by mixing with commercially available lambda packaging extracts such as MaxPlax packaging extracts (Epicentre Technologies, Corporation. Madison, Wisconsin). The cDNA library is then available for screening. It is also possible to produce cDNA 25 expression libraries by minor variations of these procedures (Sambrook, J. et c. 1989; supra). Recovery of homologous polynucleotide sequences may be achieved as follows. Library screenin g 30 Once libraries have been constructed they may be used to infect Escherichia coli cells of a line supporting lytic infection (e.g. in the case of lambda strain lambda gt10 the strain used would be C600hfl Promega, Corporation. Madison, Wisconsin) which are plated out on a suitable agar (Sambrook, J. et al. 1989, supra). Plaque lifts may be taken onto 35 nitrocellulose or nylon (e.g. Hybond N+, Amersham Pharmacia Biotech Inc., Piscataway, N.J.) or other suitable filters. Assemblages of the order of 50,000- WO 01/61000 PCT/AU01/00150 11 100,000 or more independent phage from such a cDNA may be readily. screened at a single time. Tc do this, the filters are probed with the labelled probes. Then to recover polynucleotide sequences encoding homologous receptor subunits from species other than C. elegans it is necessary to 5 perform the probing and washing procedures at range of stringencies including low stringency. Therefore, probing and washing is conducted in the range of 0.1-5 times SSC (Sambrook, J. et al. 1989. supra) and in the temperature range 65-45'C. To ensure that homologous polvnucleotide sequences are recovered from distantly related nematode species a method of 10 phylogenetic walking may be employed. This involves using the relevant sequence from C. elegans as a probe to recover the homologous sequence from a phylogenetically, relatively-closely related species. for example by preparing and screening a library from another rhabditid nematode such as Steinernema carpocapsae, then by repeating the steps described above to 15 prepare a library from a more distantly related nematode for example a strongylid nematode such as Haemonchus contortus and use the C. elegans probes and probes made from the homologous sequence recovered from S. carpocapsae to screen the H. contortus library and recover the homologous receptor polynucleotide sequence from the latter species. This process may 20 be repeated to isolate the homologous polynucleotide sequence from any nematode species by reiterating the steps of probe generation. cDNA library construction and screening of the library as described above. Polvmerase chain reaction methods An alternative approach to isolating, from a second species, 25 polynucleotide sequences encoding homologous receptor subunits is to use the polymerase chain reaction (PCR) to amplify homologous probes from the second species and then to label and use these probes to isolate clones from the appropriate cDNA library, as described above. In such a process, degenerate PCR primers are designed to include all possible nucleotide 30 combinations encoding the known amino acids at highly or moderately well conserved positions within the sequence. Identification of such highly conserved or moderately conserved positions may be achieved by comparing each of the amino acid sequences shown as SEQ ID NO: 10, 11 and 12 with the amino acid sequences of homologous 5-HT, receptors. such as those 35 shown in Figure 3. In addition, any other homologous invertebrate sequences may be included in such a comparison, such as published expressed WO 01/61000 PCT/AU01/00150 12 sequence tags for homologous cDNAs identifiable by BLAST-searching of the publicly available nucleic acid databases such as the GenBank and EMBL databases and homologous sequences recovered by BLAST searching of the other invertebrate genome databases, in particular the Drosophila 5 melanogaster genome database. Such comparisons are made using multiple alignment algorithms, for example the "pileup" "clustalw" and "eclustalw" algorithms available within the GCG sequence analysis package, written by the Genetics Computing Group at the University of Wisconsin. Similar algorithms are available for most major computing platforms. 10 Homologous polynucleotide molecules may be used to express 5-HT, receptor subunits from other invertebrate species, which can be employed in assays akin to those of the seventh and eighth aspects. Thus, in an eighth aspect, the present invention provides an assay for identifying and/or assessing nematicidal. insecticidal and/or other pesticidal 15 compounds. said assay comprising; (i) isolating a polynucleotide molecule from an invertebrate other than C. elegans wherein said polynucleotide molecule comprises a nucleotide sequence which encodes a 5-HT, receptor subunit and represents a homologue of any or all of the nucleotide sequences shown as SEQ ID NO: 1-6, (ii) expressing said polvnucleotide molecule to 20 produce 5-HT, receptors or functionally equivalent fragments thereof, (iii) contacting at least one of said produced 5-HT 3 receptors or functionally equivalent fragments thereof to a candidate nematicidal. insecticidal and/or other pesticidal compound under conditions enabling the activation of 5-HT receptors. and (iv) detecting an increase or decrease in activity of said 25 produced 5-HT, receptor(s) or functionally equivalent fragment(s) thereof. The 5-HT, receptor(s) or functionally equivalent fragment(s) thereof mentioned in step (iii) may be present on the surface of a cell (e.g. a host cell expressing the polynucleotide molecule mentioned in step (i)), or a cell lysate (e.g. a lysate of a host cell expressing the polynucleotide molecule mentioned 30 is step (i)), or may be in a substantially purified form. In a ninth aspect, the present invention provides an assay for identifying and/or assessing nematicidal. insecticidal and/or other pesticidal compounds, said assay comprising; (i) isolating a polynucleotide molecule from an invertebrate other than C. elegans wherein said polynucleotide 35 molecule comprises a nucleotide sequence which encodes a 5-HT, receptor subunit and represents a homologue of any or all of the nucleotide sequences WO 01/61000 PCT/AU01/00150 13 shown as SEQ ID NO: 1-6, (ii) expressing said polynucleotide molecule .to produce 5-HT, receptors or functionally equivalent fragments thereof, (iii) contacting at least one of said produced 5-HT, receptors or functionally equivalent fragments thereof with a predetermined amount of a suitably 5 labelled serotonergic ligand together with a predetermined amount of a candidate nematicidal, insecticidal and/or other pesticidal compound under conditions wherein said serotonergic ligand and said candidate compound competitively bind to the 5-HT, receptor(s) or functionally equivalent fragment(s) thereof, and (iv) determining the amount of bound and/or labelled 10 serotonergic ligand. In a tenth aspect, the present invention provides a nematicidal compound identified by the assay of the seventh or eighth aspects. In an eleventh aspect, the present invention provides a nematicidal, insecticidal and/or other pesticidal compound identified by the assay of the 15 ninth or tenth aspects. In a further aspect, the present invention provides a method of killing a helminth (e.g. a nematode, cestode or other flatworms), said method comprising exposing said helminth to an effective amount of a compound which alters the activity of a 5-HT 3 receptor of said helminth. 20 Preferably, said compound inhibits stimulation of the 5-HT 3 receptor by a serotonergic ligand. An effective amount of said compound may be in the range of 100 iM or less, preferably 104M or less, more preferably 1ijM or less at the whole organism level. The compound may be provided in combination with a veterinary- or pharmaceutically-acceptable carrier. or any 25 other carrier as may be appropriate. In a yet further aspect, the present invention provides a composition for killing a helminth comprising an effective amount of a compound which alters the activity of a 5-HT, receptor of a helminth. It has also been found that effective insecticides against certain insects 30 (e.g. sucking insects such as aphids or other insects with muscular feeding mechanisms) are provided by compounds which alter the activity of a 5-HT, receptor of such insects. Thus, in still further aspect, the present invention provides a method of killing an insect, said method comprising exposing said insect to an effective 35 amount of a compound which alters the activity of a 5-HT 3 receptor of said insect.

WO 01/61000 PCT/AU01/00150 14 Preferably, said ccmpound inhibits stimulation of the 5-HT 3 receptors by a serotonergic ligand. An effective amount of said compound may be in the range of 1004I or less preferably 10 .M or less, more preferably 1pI or less at the whole organism level. The compound may be provided in 5 combination with an agriculturally-acceptable carrier. In yet a still further aspect, the present invention also provides an insecticidal composition comprising an effective amount of a compound which alters the activity of a 5-HT, receptor of an insect. Reference to percent homology made in this specification have been 10 calculated using the BLAST program blast as described by Altschul, S.F. et al. 1997 Capped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Research. Vol. 25, No. 17. pp. 3389 3402 (1997). The term "5-HT, receptor" as used herein refers to a receptor having one 15 or more of the following features (1) - (3): (1) Is a serotonin-gated molecular ion-channel which gates the conductance of cations and is composed of five receptor subunits each of which has a nicotinicoid transmembrane topology (N-terminus, large extracellular domain, 3 transmembrane helices. large intracellular domain, 1 20 transmembrane helix, C-terminus). (2) If it is a mammalian 5-HT, receptor. is composed of subunits with a high degree of homology to other known mammalian 5-HT 3 A or 5-HT 3 B subunits. If it is an invertebrate 5-HT 3 receptor. is composed of subunits with a higher level of amino acid homology to mammalian 5-HT, receptor subunits 25 than to known mammalian nicotinic acetylcholine receptor subunits. (3) Has a characteristic pharmacological profile in that it binds to a subset of known 5-HT 3 -specific agonists and antagonists with greater selectivity than it binds to agonists and antagonists of other 5-HT receptor classes. The term "serotonergic ligand" as used herein refers to any compound 30 that selectively binds to one or more subclasses of serotonin receptor. It may activate the receptor (agonists) or prevent other ligands binding without itself activating the receptor (antagonist) or it may have a mixed agonist/antagonist character. The term "substantially corresponds" as used herein in relation to 35 nucleotide sequences is intended to encompass minor variations in the nucleotide sequence which due to degeneracy in the DNA code do not result WO 01/61000 PCT/AU01/00150 15 in a change in the encoled invertebrate 5-HT 3 receptor. Further, this term is intended to encompass other minor variations in the sequence which'may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded 5 protein. The term "substantially corresponding" as used herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in biological activity of the invertebrate 5-HT 3 receptor. These variations may include 10 conservative amino acid substitutions. The substitutions envisaged are: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P. Na-alklamino acids. The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated step, 15 component or feature or group of steps. components or features with or without the inclusion of a further step, component or feature or group of steps. components or features. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the 20 purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. 25 The invention will hereinafter be described with reference to the following non-limiting examples and accompanying figures. Brief description of the accompanying figures: Figure 1 provides dose-response curves for serotonin action on 30 pharvngeal pumping. Figure la is a dose response curve that was determined for serotonin by videotaping C. elegans under Nomarski optics and counting the rate of pharyngeal pumping over the range 325 pi - 6.3 mM serotonin. Figure lb shows a dose response curve for the effect of serotonin on pharyngeal pumping rate obtained using a plate-based assay recently 35 described in International Patent Application No. PCT/AUOO/01476. Any minor differences between the curves of Figures la and lb are accounted for WO 01/61000 PCT/AU01/00150 16 by the differences in experimental detail and the fact that in the microscopical assay, the rate o; pumping is counted over a period of 1-3 minutes. whilst in the plate-based assavs the fluorescence intensity represents the accumulation of ingested material over 60 minutes. 5 Figure 2 a typical dose-response curve for the inhibitory effect of MDL72222 on the serotonin-stimulated pharyngeal pumping of C. elegans in the presence 0.325mM serotonin. The IC,, of MDL 72222 in this experiment is approximately 25puM (i.e. an order of magnitude) lower than the concentration of serotonin. 10 Figure 3 provides a map of the molecular phylogeny of representative known mammalian 5-HT 3 sequences, representative known vertebrate and invertebrate nAChR sequences, the three putative receptor genes from ACeDB (F18G5.4.pep, F25G6.4/CE09640.pep and C31H5.3.pep) and the three related but distinct experimentally determined C. elegans 5-HT, sequences (F18, DIY 15 and DIAY). This molecular phylogeny positions the three C. elegans 5-HT, receptor subunits in a framework created by a representative selection of mammalian 5-HT, receptor sequences (including the newly discovered 5HT 3 ) and known nAChR sequences from an insect, nematode and human. The C. elegans genome project has assigned F18G5.4 as a hypothetical 20 acetylcholine receptor like protein and also as a "ligand gated ion-channel". The molecular phylogenetic analysis shows that, based on overall level of homology, F18G5.4 is the most similar of the available C. elegans sequences to previously identified mammalian 5-HT, receptors. The C. elegans genome project has assigned F25G6.4/CE09460 as a "ligand gated ion channel" and 25 C31H5.3 as a putative 5HT,. receptor. But the phylogeny of the present inventors shows that they are both as distantly related to currently known 5 HT3 and nAChR as these two classes are to each other. On the other hand, the bold amino-acid triplets indicate the sequence found at the DIy position in each branch of the tree. It can be seen that both CE09460 and C31H5.3 are 30 more similar to 5-HT, in respect of this character than are known nAChRs. Figure 4 shows the effects on serotonin-stimulated pharvngeal pumping of double-stranded RNA knockdown of the F18 messenger RNA in three separate and independent experiments. In each case, nematodes were fed according to Timmons. L. and Fire, A. (1998. Specific interference by 35 ingested dsRNA. Nature. 395: 854) on HT115 E. coli bacteria expressing either a portion of the F18 gene "F18" or the empty double-stranded ds RNA WO 01/61000 PCT/AUO1/00150 17 expression plasmid "pL44440" as a control. In the third experiment (Figure 4c), a second control comprising an irrelevant dsRNA being expressed'from the same plasmid was also included "pCB6". For each experimental treatment, individual nematodes are ordered along the X-axis according to 5 their rank by pumping rate. Note that the concentration of serotonin used to challenge the nematodes is 3.25mlMi in Figures 4a and 4b and 1mM in Figure 4c. Other effects of RNAi using F18, in addition to the depressed pumping rate, are noted in Example 5 below. For the experiments described below C. elegans of the Bristol N2 strain 10 (Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77: 71 94) were cultured at room temperature on HMS 174 E. coli bacteria (Campbell J.L. et a]. 1978. Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T77 DNA. Proc. Nat]. Acad. Sci. USA. 75: 2276-2280) on NGM agar (Sulston, J. and Hodgkin, J. 1988. 15 "Methods" in The nematode Caenorhabditis elegans. W.B. Wood pp 587-606. Cold Spring Harbor Laboratory Press, New York) unless otherwise described. Example 1: Pharmacological identification of a previously unknown 5-HT 3 receptor in C. elegans. which is responsible for controlling the rate and 20 strength of pharvngeal pumping. It is well-known that, among other effects. serotonin at micromolar to millimolar concentrations increases the rate and strength of nematode pharyngeal pumping (see Figure 1. which shows dose response curves for serotonin acting on pharvngeal pumping). 25 5-HTq-receptor class-selective aonists Much is known about the pharmacology of vertebrate 5-HT receptors and there are a number of compounds in clinical use that act on the serotonergic system. Although' relatively little is known about the pharmacology of invertebrate 5-HT receptors and there are some important 30 differences from vertebrates, it seems that there is broad correspondence between the compounds that are active on vertebrate receptors of a given sub class and invertebrate homologues. Studies were therefore conducted wherein selective 5-HT, 5-HT 2 and 5-HT 3 agonists were applied at known concentrations to agar on which C. 35 elegans was held in the absence of food and the effects on movement and pharyngeal pumping monitored over a period of 2-3 hours. The results for 5- WO 01/61000 PCT/AU01/00150 18

HT

1 , 5-HT 2 and 5-HT 3 -sElective agonists are shown in Table 1. A combination of two 5-HT 1 agonists each at 6 m1M had little or no effect'vn pharyngeal pumping. Movement was inhibited but most nematodes recovered the ability to move after transfer to a fresh agar plate. A 5-HT 2 5 specific agonist also failed to stimulate pumping. A 5-HT 3 -specific agonist (3mM) caused a dramatic stimulation of pharyngeal pumping similar to a maximal concentration of 5-HT. There was no mortality of nematodes in any of the treatments during the time-course of the experiment. Selective 5-HT 3 antagonists. 10 Studies were also conducted to investigate the ability of selective vertebrate 5-HT 3 antagonists to inhibit the 5-HT-induced increase in pharyngeal pumping. The two inhibitors chosen were ondansetron (a Glaxo Wellcome drug used to prevent chemotherapy-induced vomiting), which had been reported to be a much less potent inhibitor of the snail 5-HT 3 -like ion 15 channel than of the vertebrate 5-HT 3 -receptor (IC 50 of 10tM and 100 pM respectively) [Green, K.A. et a]. 1996, supra], and MDL 72222 (tropanyl dichlorobenzoate) which had an IC 5 1 of 14M on the snail's 5-HT gated conductance [Green, K.A. et al. 1996, supra]. When the inhibitors were applied individually at 100 4M in the 20 presence of 6.5 mMvi 5-HT they did not reduce the rate of pumping (not shown). However, 6.5 mM 5-HT is approximately 20x greater than the EC 50 (see Figure 1) and so may out-compete inhibitors, particularly if they do not have very high affinity for the invertebrate receptor subtype. In a second experiment, the two inhibitors were applied together. each at a concentration 25 of 325 pM and in the presence of 325 .M-5-HT (i.e. approximately the EC 50 ). There was total reversal of the 5-HT effect (see Table 2). In the absence of 5 HT, the effect of the two antagonists in combination was to stimulate pumping, although not to the same extent as 5-HT. In a third experiment, when the inhibitors were applied individually at an equimolar concentration 30 with 5-HT (i.e. both 5-HT and the inhibitor at 0.325 mm) they each significantly inhibited the rate of pharyngeal pumping (Table 3). MDL72222, which on its own completely reversed the effect of the 5-HT. appeared to be significantly more effective than ondansetron under these conditions. Neither of the inhibitors on their own stimulated pharyngeal pumping. 35 In another experiment, a dose response study was conducted to determine the effects of a range of doses of MDL72222 on the rate and WO 01/61000 PCT/AU01/00150 19 strength of pharyngeal F umping in the presence of 0.325mM serotonin (Figure 2). At this concentra don of 5-HT, the IC 50 for MDL72222 is approximately 30sM, i.e. 1Ox lower than the EC 50 for 5-HT. 5 Example 2: 5-HT 3 -selective antagonist is nematicidal for C. elegans and insecticidal for the sucking insect pest Mvzus persicae (Hemiptera: Aphididae) and antifeedant for the chewing insect pest Helicoverpa armigera (Lepidoptera: Noctuidae). The above data indicates that a 5-HT, receptor present in C. elegans 10 and also likely to be present in other nematodes which respond to serotonin in a similar fashion to C. elegans and is responsible for mediating increased rate (and strength) of pharngeal pumping in response to exogenous or endogenous serotonin. Because the pharvngeal pump is essential for nematode feeding and the maintenance of the nematode "hydrostatic 15 skeleton" [Brownlee, D.J.A. et a]. 1997, supra] and a previously established target organ for nematicides, an investigation was conducted to assess whether the 5-HT,-selective antagonists would function as nematicidal agents in a chronic exposure protocol. C. elegans nematodes were cultured on the HMS/174 strain of E. coli 20 [Campbell, J.L. et a]. 1978. Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA. Proc. Nat. Acad. Sci. USA. 75: 2276-2280] on NGM agar [Sulston, J. and Hodgkin, J. 1988, supra] in the wells of six well tissue culture cluster. 2 ml agar per well. MDL72222 was incorporated into the agar at the specified concentrations 25 using 565sl of acetone as a carrier. A control well contained 65pl of acetone only. Between five and 11 LI stage C. elegans were transferred to each well and the wells were incubated at 22'C for up to four weeks. Results are summarised in Table 4. All concentrations > 10sM MDL 72222 caused developmental delays. Concentrations of MDL 72222 down to 80 sM1 caused 30 40 % mortality within three days. At these concentrations, the nematodes died within a week and almost all their progeny were unable to hatch from the eggs. The bacterial food therefore remained uneaten for prolonged periods. Concentrations of MDL72222 down to 20sM caused 90-100% mortality 35 within 21 days and concentrations as low as 10gM and 5gM caused 50-100% mortality at 21 days. At the lowest concentrations of MDL72222. nematode WO 01/61000 PCT/AU01/00150 20 death was delayed until after the bacterial food had been consumed. At these concentrations, there were still clear differences from the nematodes ii the control wells, which formed dauer larvae on exhaustion of the food supply, whereas the nematodes exposed to even the lowest concentrations of 5 MDL72222 failed to respond properly to exhaustion of the food supply and died. The experiment shown in Table 3 was repeated with essentially identical results. Because of the nematicidal efficacy of the 5-HT 3 selective antagonist against C. elegans, 5-HT 3 -selective antagonists were tested to determine 10 whether they would have a similar effect on two species of insect pest, namely Helicoverpa armigera and Myzus persicae. When incorporated into artificial diet (Teakle, R.E. and Jensen, J.M. 1985. Heliothis punctiger, in Handbook of insect rearing. P. Singh and R.F. Moore, Vol. 2. pp 312-322, Elsevier. Amsterdam), by surface contamination, MDL 72222 had a mild 15 antifeedant effect on the growth and development of neonate H. armigera, which have biting and chewing mouthparts (Table 5). Ondansetron had no discernible effect of H. armigera (not shown). The effects of MDL 72222 on Green Peach Aphid (Myzus persicae) was also tested. The bioassay involved testing compounds at 3 different 20 concentrations (1mM, 100NI, 10M) in an artificial diet. Compounds that were not water-soluble were forced into solution using a non-toxic surfactant. For each compound, each concentration was tested in 10 replicate assays. In each replicate. 5 aphids were allowed to feed on the test solution in a Parafilm sachet for a period of 5 days. Survivorship and aphid growth rate 25 were compared among the test concentrations and a control containing only the artificial diet with surfactant. MDL 72222 caused dose-dependent mortality and significant reduction in weight gain in A. persicae (Table 6). Ondansetron had no discernible effect on M. persicae (not shown). It is clear that IDL 72222 is a more potent inhibitor of the invertebrate 30 5-HT, receptor than is ondansetron. It may, therefore, be concluded that agents that inhibit 5-HT 3 receptors of nematodes and those insects that have a feeding mechanism that involves a muscular pump (and therefore other invertebrates with similar mechanisms of feeding, maintaining their turgor pressure. or indeed attaching to their host 35 by oral suction), will be universally pesticidal in those species. It can also be concluded that the 5-HT 3 receptors and subunits thereof from nematodes and WO 01/61000 PCT/AU01/00150 21 other invertebrates represent ideal molecular targets for the screening, identification and optimisation of new, highly potent and highly invertebrate selective pesticides. 5 Example 3: Sequence Analysis. 1. It is well known that vertebrate aminergic receptors of the 7 transmembrane G-protein-linked superfamily have a DRY triplet of amino acids located close to one end of the 3rd transmembrane segment. Analyses conducted by the present inventors have shown that 5-HTA receptors from 10 four vertebrate species have a DIY conserved triplet. 2. In an eclustalw (Figure 3) alignment of representative known mammalian 5-HT 3 receptors, representative known mammalian, insect and nematode nAChRs and the three nematode 5-HT 3 receptor sequences of the present invention (previously assigned as hypothetical acetylcholine-like 15 receptor F18G5.4, putative 5-HT 3 receptor C31H5.3 and ligand gated ion channel F25G6.4/CE09640), the latter three fall outside either of the known sub-classes of mammalian 5-HT 3 receptor subunits. However, F18G5.4 is the closest sequence to the group of mammalian 5-HT 3 receptors, F25G6.4/CE09640 has a DIY sequence motif in the expected position, while 20 C31H5.3 has a DIAY (SEQ ID NO: 13) modified motif in the same position. Example 4: Cloning and construction of expression cassettes. The three sequences described in Example 3 above were identified in the C. elegans genome database by the automated algorithm "GeneFinder". 25 Since it is not therefore possible to conclude that these sequences are full length or authentic, nor that they are expressed in vivo in the form identifed by GeneFinder, the present inventors cloned and expressed the three cDNAs (DIY, F18 and DIAY) which are encoded in part by the sequences identified as F25G6.4/CE09640. F18G5.4 and C31H5.3 in the C. elegans genome 30 database. 1. Oligo-dT-primed cDNA was prepared from mixed life-stages of C. elegans. One portion of this was cloned into lambda gt10 to generate a cDNA library. Another portion was used as template for amplification of sequence-specific probes for each of the target genes. In each case, this was achieved using a 35 pair of unique sequence primers designed, on the basis of the cDNAs predicted by GeneFinder, to span a central region (0.5-1.0kb) of three WO 01/61000 PCT/AUO1/00150 22 predicted cDNAs. Although it would have been theoretically possible for this process to have failed due to -- rrors in the GeneFinder predictions, subsequent sequencing of the amplified products indicated that in all three cases, authentic fragments of the desired target sequences were obtained. 5 2. 1 2 P-labelled probes were prepared from the amplicons by random-priming and the resultant probes were used to screen and recover hybridising cDNA clones by standard plaque lift using high stringency hybridisation. 3. For DIY, a single large clone was recovered from the library. However, sequencing revealed that this contained a splice error which introduced a 10 number of in-frame stop codons. Two independent PCR amplicons were recovered from independent cDNA library pools, and a cDNA encoding the predicted coding sequence was reconstituted by ligation of a PCR fragment to the middle of the cDNA clone, thereby eliminating the splice error. 4. For DIAY, two independent large clones were recovered from the library. 15 Sequencing and comparison to the ACeDB genome sequence indicated that these were both authentic clones bearing the complete coding sequence. 5. For F18, a single large clone was obtained from the library and several shorter clones which confirmed the 3' sequence. However, the longer sequence did not encode a plausible cleavable signal peptide at the 5' end. A 20 search for additional sequences covering this region was instituted using anchored PCR with two nested F18-specific primers at the 3' end and a single lambda-phage-specific primer at the 5' end. Using independent pools from the cDNA library as template, two independent but identical amplicons were recovered which. when sequenced, predicted a plausible cleavable N 25 terminal signal peptide. A sequence encoding the predicted coding sequence was reconstructed by ligation of a fragment of one of the PCR amplicons to the 5' end of the cDNA clone. The authenticity of the experimentally-determined clones was confirmed by double-stranded sequencing and analysis of the sequence as 30 follows: - Comparison of the cDNA sequence with the C. elegans genomic sequence. - Presence of an open reading frame 1.5-2kb. - Predicted membrane topology for a subunit of a ligand-gated ion-channel. - Predicted N-terminal cleavable signal peptide. 35 - Sequence confirmed by multiple independent clones, either recovered from the cDNA library by screening and/or by PCR from independent cDNA pools.

WO 01/61000 PCT/AU01/00150 23 In each case, experimental cloning and sequencing of the cDNAs. revealed errors in the GeneFinder predictions of the three cDNAs, especially in the assignment of intron-exon boundaries. The authentic isolated cDNA corresponding in part to ACeDB predicted gene CE09640/F25G6.4 has been 5 designated "DIY' (SEQ ID NO: 5). The authentic reconstructed cDNA corresponding in part to the GeneFinder predicted cDNA C31H5.3 has been designated "DIAY" (SEQ ID NO: 6). Also, the authentic reconstructed cDNA corresponding in part to GeneFinder predicted cDNA F18G5.4 has been designated "F18" (SEQ ID NO: 4). 10 Each of the three cDNAs (DIY, F18 and DIAY) was inserted into the mammalian cell expression vector pcDNA3.1 (In-Vitrogen. Groningen, The Netherlands) to allow transfection of COS-7L cells and expression of each receptor subunit onto the cell surface. Each of the three cDNAs was also inserted into the related vector pcDNA3.1/mvc-His (In-Vitrogen, Groningen, 15 The Netherlands) to enable their expression as fusion proteins upstream of the C-myc and 6-His tags. Fusion proteins of this type have been shown not to interfere with expression of the mammalian 5-HTA subunit in HEK cells (Lankiewicz, S. et al., 2000. Phosphorylation of the 5-hydroxytryptamine (5-HT 3 ) receptor expressed in HEK293 cells. Receptors and Channels. 7: 9-15) 20 (also related LGIC receptor subunits in COS-7L cells: Johnson and Trowell, unpublished observations). Correct in-frame construction of these expression vectors was confirmed by sequencing. Example 5: Functional knockdown of F-18 and DIAY using the RNAi 25 technique. For each of the cDNAs F18, DIY and DIAY, the present inventors amplified a central portion of the cDNA of between 0.5-1.0kb using unique sequence PCR primers. The amplicon was ligated into the multiple cloning site of the plasmidic dsRNA expression vector pL4440 [Fire, A. et a]. 1998. 30 Patent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391. 806-811] and the plasmid was transfected into the HT115 host strain of E. coli. HT115, is genetically deleted for double-stranded ribonuclease and contains a T7 RNA polymerase under the control of an IPTG(isopropyl thiogalactoside)-inducible promoter 35 so that induction of the transfected bacteria with IPTG results in high level expression of the amplicon in the form of ds RNA.

WO 01/61000 PCT/AU01/00150 24 Axenic C. elegans Li stage were prepared by alkaline hypochlorite treatment of adult worms [Sulston J. and Hodgkin, J. 1988, supra] and were introduced onto an NGM agar petri dish with IPTG-induced bacteria expressing dsRNA from the gene of interest. The nematodes were allowed to 5 develop to the adult stage and were challenged with serotonin and the effects on the rate and strength of pharyngeal pumping were recorded by videomicroscopy. In three independent experiments, it was observed that F18 dsRNA caused a depression in both the rate of serotonin-stimulated pharyngeal 10 pumping (Figure 4 and Table 7) and the degree of opening/strength of contraction of the terminal bulb (Table 7). In the third experiment, the effectiveness of feeding of the nematodes was also assessed by challenging individual nematodes with serotonin in the presence of fluorescent beads and then observing the extent of bead ingestion. There was a visibly lower 15 number of beads in the guts of nematodes exposed to the F18 dsRNA than in controls and the beads did not penetrate as far into the guts of the former. Taken together, these results indicate that functional knockdown of F18 gene had a specific effect on pharyngeal pumping very similar to the effect of the 5-HT 3 specific antagonist MDL72222 and strongly indicates that 20 F18 encodes a subunit of the nematode 5-HT 3 receptor. The major difference in the effect was that, whereas MDL72222 completely suppresses pumping in all nematodes tested, in the case of the functional knockdown only a small number of worms were completely prevented from pumping, although the average pumping rate of the whole 25 population was below that of the control (Figure 4). The technique of RNAi knockdown by feeding is known to lack complete penetrance, especially in neurones [Schuske, K. et al. 2000. Snap-back RNA: in neurons. The W'Vorm Breeders' Gazette. 16: 181, and to be critically dependent on the induction protocol [Kamath, R.S. et al. 2000. Effectiveness of specific RNA-mediated 30 interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biology. 2: 1-10]. Therefore, to establish the degree of suppression of the F18 that had been achieved, individually identified worms from the F18 RNAi population and the control pL4440 plasmid population, whose rate of pumping had previously been established, were pooled in 35 groups of three and subjected to quantitative reverse transcriptase PCR in a Corbett Research Rotorgene thermal cycler so as to measure the levels of F18 WO 01/61000 PCT/AU01/00150 25 RNA. Comparison with standards indicated that even in control worms the abundance of F18 mRNA is below that which can be quantified in thres worms or fewer. However, it was possible to detect F18 message in all samples, indicating that suppression of F18 mRNA was not absolute. 5 One other difference that was noted between the F18 knockdown and normal worms was that there was a high incidence of visible anatomical defects in the pharynxes of the former. Preliminary high resolution microscopy indicates that this may be due to hypertrophy and degeneration of fine processes of the NSM motoneurones. 10 WO 01/61000 PCT/AU01/00150 26 0 '42 P. - 0) 0 Cac ElI El 2 E 00 m 0 0) 0D 0+ Cu C) + I CC CuY Cu C0C ~C 0 to cc -~ ~ -4-4 Cu' 0)) co ") (0)C u a C '-4- ca a 0 0o o~c C).g~ WO 01/61000 PCT/AU01/00150 27 0 04 0 - - 0U 0 0 cu0 0 w 0 a~ E E E E -H _) 0 en -.- H 0 W o co CU1 CD C Ui CUC) C) 0 0 6 0 r_ f . .00 0 &. l Cll C co 0 E cc L o616 I WO 01/61000 PCT/AU01/00150 28 VV 7a 0 0 o o 0 00 .P. EE 0 0 0 - 0 0 r. r.= - t 0 00 0 -0 0 0 Cd 0 0E E E s 0 0) 0 00 o 0 0 0 0 ~0 Io c z c E E E b = E oD 00J C1 0 0 + to vc*,o 0C) 0)0

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r.00 s-&I 00 C~ c s-I4 C, 00 E- -: c). C: WOO01/61000 PCT/AUO1/00150 29 00 c 00 a) C) 0 C 0 C r-, A A A rI A v V V 4 - - - - - - - - - W U'DC W L m 4 o cc~ ) ~ C . N LN 0)) CON4L WO 01/61000 PCT/AU01/00150 30 eq0 t a% 0 k ON 0 I1 m o Q) 0 t, 0 ) Z 0 z C' 00 co 0) $0 0 0 0 0o oD a 00 0 0lu 5i4 0~ 0 c .6..) cl~ (U CCl cu 0 0 l C 00 0 ~U ~ U i... ' WO 01/61000 PCT/AUOI/00150 31 "T 00 C) e W) m-1 co 0 00* 0 ca ca CD 00 C1 aCA cc o 0 CDe' ca clci E-4 cd WO 01/61000 PCT/AUO1/00150 32 - 0 0 z C\ N 0 \ m. tfl- - 'D 00 o 0 e- 0 0 "0 - a WO 01/61000 PCT/AU01/00150 33 Sequence listing: <110> Commonwealth Scientific and Industrial Research Organisation <120> 5-HT3 receptors of nematodes, polynucleotide molecules encoding same, and antagonists thereof <160> 13 <170> PatentIn Ver. 2.1 <210> 1 <211> 1908 <212> DNA <213> Caenorhabditis elegans <400> 1 atgatcatat gttattcgtg tctaaccgtc tccattcttc taaccattaa atttgtacca 60 tgtcgatttg ctggaattga acaccaaaat acgaaaagtc gtgtgcattt ctcgttgctg 120 gatagtagac aagaaaatga cactaatcac tttgagatag cagaagcaaa gttccagaaa 180 ccccacaatg aggaaaacac aataggtacg attacaaaat ttgctccatc ggtacaagaa 240 caacacagtt ctgcggtaat tccaatgccc cactttgacc agaaccggct tgagcaagct 300 cttcggatca agggctcaat tgatggaacc gaagaggctt tgtacaggtc tctactagat 360 catactgttt acgaaaaaga tgtgaggcca tgtatacatc actctcaacc aacaaatgtc 420 acatttggat ttcttctcaa tcagattgtg gaaatggatg aacgaaatca agctctaaca 480 accagaagct ggctgaatat caattggatg gatcctcgat tatcgtggaa tgaaagcctt 540 tggtctgaaa ttaaagcaat atatattcca catgcaagaa tctggaaacc cgatataatt 600 ctggtaaaca acgctatccg agaatactat gcatccctcg tctccaccga tgtaatggtc 660 acaagtgacg gaaacgtgac atggctgttt tccgcactat ttaggagttc ttgtccaata 720 cgggttcgat attatccatt cgatgatcaa caatgtgatc tgaaatttgc ctcctggtcc 780 catgatatca cagaaatcaa tctcgggttg aacacggaca aaggggattt gtcaagttat 840 atgaacaaca gcgaatttga tcttgtggat atgacggctg ttcgagaagt tgttacattt 900 ccatcggata ctaatagtga ttggccaata attgtgatac gaatacatat gcatagacgt 960 cctttgttct acgtatttaa tcatattgtt ccttgcgttc ttatttcatc aatggcagtt 1020 cttggtttcc tgatgccccc ggaaaccggc gagaaaatta acatgatcat aacaactttg 1080 ctctccatgg gtgtgtatct gcagtcaatc actgagtcaa tacccccaac atccgaaggt 1140 gttccattaa ttggaatgta ttacgtatct tctcttctta tagtttacct agcaacatgc 1200 gtaaatgcaa tcactcttaa catgcacagg aatggtgcag ctaatcaggg aaggcacgtg 1260 cctgcgtgga tgcagaagtg gattctgggg tacttggcca ctttcatgag aatgccaata 1320 agagaacccg atagtatagc attgctaaaa gcgtcacaga gcaaaaagtc aactattcgg 1380 agaagctcaa tacttcgaga tttgaaaagg gtgaaaaata tgtcaaacgt tagagcaaaa 1440 tcaaaagagc aaaatgcaaa tcgagagtgc gagtgcacgg acccacctgt gcatatctac 1500 gcagagtcca tcatgagctg cctggcagca gacacaaaac ctatgaacgg gtcaactatt 1560 agagaagatt ttgcaagtga aagcacattt cttggacgcg ttgttagtga tggcataatg 1620 ccaagaataa gtgcttcatc caactctgtg ctgacagaat tcgaaacaag atttagacgg 1680 atattaaaaa gggtttaccg aagtcttcag caacatgaaa tacgagaaga aattcttgac 1740 gaaagatctc gaattcaatg gcagtggcaa caacttgcat ctgtcgttga tcgactttta 1800 ctatgtcttt tttgcactgc aacactgttc acaatcatct gcctcctaat tgtacctgta 1860 gcataccgtg ataacgactc aatgttgtca ttcctcaatt ttttctga 1908 <210> 2 <211> 1440 <212> DNA WO 01/61000 PCT/AU01/00150 34 <213> Caenorhabditis elegans <400> 2 atgctcttgc ccattttatt gcattttttg cttctaatca cccaattaaa tggctcacca 60 gcagaagtac ggcttatcaa tgatcttatg tcaggatatg ttcgtgagga aagaccaaca 120 cttgatagtt caaagccagt tgttgtcagt ttgggagtct ttttgcaaca gattattaac 180 ttgtccgaaa aagaggaaca gctggaagta aatgcctggc ttaagttcca atggagagat 240 gaaaatttac gatgggaacc gactgcttat gagaacgtga cagatctaag acatccaccg 300 gatgctctat ggactcccga tatccttctt tataatagtg tcgattcgga gtttgattcg 360 tcgtataaag taaatctggt taattatcat acgggaaaca ttaattggat gccaccagga 420 atattcaaag tatcgtgtaa attggatatt tattggtttc catttgatga acaagtttgt 480 tattttaagt ttggctcatg gacgtatact cgtgataaga ttcaactaga aaagggtgat 540 tttgatttct ccgagttcat tccaaacggg gaatggatta taatagatta tcgaacaaat 600 attactgtga aacaatatga atgttgtccc gagcagtatg aagatatcac ttttacgcta 660 catttacgac ggagaacttt atactattcc ttcaatttaa ttgctccagt tcttttaaca 720 atgatactgg ttattttggg ctttactgtt agccctgaaa cttgcgaaaa agttggactt 780 cagatctctg tctctcttgc catatgcatt ttcctcacaa taatgagtga actgacacct 840 caaacatcag aagctgttcc acttcttgga gtattcttcc acacttgcaa cttcatttcc 900 gttttagcca cttctttcac agtttatgtg caaagttttc artttcgaaa ccaacatgta 960 cacgaacgga tggatttctg gatgaggttc attctcttgg agtggtcacc gtggctattg 1020 cgaatgaaaa tgccggatag agaaaataac tttcagacac tgacagaaag ttggaaggga 1080 aggaatcgaa gagaatctat ggcaagaaca gcgttcgaat atgcagatgg accggttaca 1140 cagatacatt ccatgggaat tatgttgaaa gataattttg aagagcttat ttatcaagtt 1200 aaacaggaga agattgctga tgaaaaagga attgagagat tgcgggtgtt acagaagatt 1260 tacgatcatg taaagatgat ccgagaacat gacgatgaca atgatgaaga cagtcgagta 1320 gctcttgaat ggagatttgc tgcaattgtc gtcgatcgtc tgtgccttct tgccttctcc 1380 ttactcatcg tcgtcgtctc catcatcatt gctttacgtg caccgtatct tttcgcttaa 1440 <210> 3 <211> 1665 <212> DNA <213> Caenorhabditis elegans <400> 3 atgaggagaa ggttcgaaat cggcatcgca ttctttttcg cactttttca agtgatatgg 60 acgggtgacc atgaacgtag actatatgca aaatrggcgg aaaactacaa caaattggcg 12C agacctgttc gaaatgaaag tgaagctgta gtagttcttc ttgggatgga ttatcaacaa 180 attttggata ttgacgaaaa acatcaaata atgaattcaa gtgtttggtt acggatgtca 240 tggacagatc attacttgac atgggatcca tcagagtttg gaaatatcaa agaagttcat 300 ttaccaatca ataatatcta gaaacctgat gttcttctct acaatagtgt tgatcaacag 360 tttgatagta catggcccgt taatgctgtt gttttgtaca cgggaaacgt aacgtggatt 420 cctccagcca tcattcgatc aagttgtgct attgacatag catattttcc atttgatact 480 caacattgta ctatgaagtt cggttcctgg acatattctg gttttttcac tqatctcatt 540 aacacaacaa tatctccagc cacttataaa ccaaatggag aatgggaatt acttggctta 600 acgtcgcaac gctcgatatt tttctatgaa tgctgcccgg agccatatta tgatgtcacg 660 tttactgttt caattaggag gagaactctc tattatggat tcaacttatt gctcccatgt 720 atgctcattt cctcactggc tttgttgagt ttcacacttc cagctgattg tggagagaaa 780 ctgaatttag gcgtcacaat cttcatgtct ctttgcgttt ttatgattat ggttgctgaa 840 gcaatgcctc aaacaagtga tgcacttcca ttaattcaaa tctatttctc gtgcataatg 900 ttccaagttg gtgcatcagt ggtggccact gtgattgcat tgaactttca tcatcgatca 960 ccagaacagt acaagcctat gaacaaattt ttgaaaactc ttcttctggg ctggcttcca 1020 acacttcttg gcatggaacg tcctgatgtt cttgaacttt ctgtacatgg agcacattat 1080 gcgtctgaca ataaaaaaaa acaacgtcaa tacctaatag aagtggagag acatattcta 1140 acccgtccaa atggaaatgg acattcagca gttgataaag cagtgcatct tgacttatca 1200 actggtaatc cacactctga tgctaaaaaa tcatcacctt ctccaaaacg aacaagtgct 1260 tcaataatgg gtatgactgg attgccaaca actcaaatga atggagcatt ggattcttca 1320 WO 01/61000 PCT/AUO1/00150 35 attaataaat atacttgtac aaaagttacg cgtccactgg aaaacggttc agcaacaata 1380 aatcacaaat catcacctca aataaatcca atcaataaca ataatatcta taaatgtgca 1440 aacaaccaaa agactcaatt cgaagatcgt cattttcatc atattctgaa tgagcttcgt'1500 gttatatcag ctcgtgtgag aaaagaagaa gcaatgcatg cacttcaagc tgattggatg 1560 tttgcaagtc gagttgtaga tcgggtttgt tttcttgctt tttcagcatt tctcttcatg 1620 tgcactgcta ttatttctta taatgccccg catttatttg tataa 1665 <210> 4 <211> 2138 <212> DNA <213> Caenorhabditis eiegans <400> 4 tgtccagtcg acgggccctc aattcccccc gtaaatattc tttatgatca tatgttattc 60 gtgtctaact gtctccattc ttctaaccat taaatttgta ccatgtcgat ttgctggaat 120 tgaacaccaa aatacgaaaa gtcgtgtgca tttctcgttg ctggatagta gacaagaaaa 180 tgacactaat cactttgaga tagcagaagc aaagttccag aaaccccaca atgaggaaaa 240 cacaataggt acgattacaa aatttgctcc atcggtacaa gaacaacaca gttctgcggt 300 aattccaatg ccccactttg accagaaccg gcttgagcaa gctcttcgga tcaagggctc 360 aattgatgga accgaagagg ctttgtacag gtctctacta gatcatactg tttacgaaaa 420 agatgtgagg ccatgtatac atcactctca accaacaaat gtcacatttg gatttcttct 480 caatcagatt gtggaaatgg atgaacgaaa tcaagctcta acaaccagaa gctggctgaa 540 tatcaattgg atggatcctc gattatcgtg gaatgaaagc ctttggtctg aaattaaagc 600 aatatatatt ccacatgcaa gaatctggaa acccgatata attctggtaa acaacgctat 660 ccgagaatac tatgcatccc tcgtctccac cgatgtaatg gtcacaagtg acggaaacgt 720 gacatggctg ttttccgcac tatttaggag ttcttgtcca atacgggttc gatattatcc 780 attcgatgat caacaatgtg atctgaaatt tgcctcctgg tcccatgata tcacagaaat 840 caatctcggg ttgaacacgg acaaagggga tttgtcaagt tatatgaaca acagcgaatt 900 tgatcttgtg gatatgacgg ctgttcgaga agttgttaca tttccatcgg atactaatag 960 tgattggcca ataattgtga tacgaataca tatgcataga cgtcctttgt tctacgtatt 1020 taatcatatt gttccttgcg ttcttatttc atcaatggca gttcttggtt tcctgatgcc 1080 cccggaaacc ggcgagaaaa ttaacatgat cataacaact ttgctctcca tgggtgtgta 1140 tctgcagtca atcactgagt caataccccc aacatccgaa ggtgttccat taattggaat 1200 gtattacgta tcttctcttc ttatggtttg cctagcaaca tgcgtaaatg taatcactct 1260 taacatgcac aggaatggtg cagctaatca gggaaggcac gtgcctgcgt ggatacagaa 1320 gtggattctg gggtacttgg ccactttcat gagaatgtca ataagagaac ccgatagtat 1380 agcattgcta aaagcgtcac agagcaaaaa gtcaactatt cggagaagct caatacttcg 1440 agatttgaaa agggtgaaaa atatgtcaaa cgttagagca aaatcaaaag agcaaaatgc 1500 aaatcgagag tgcgagtgca tggacccact tgtgcatatc tacgcagagt ccatcatgag 1560 ctgcctggca gcagacacaa aacctatgaa cgggtcaact attagagaag attttgcaag 1620 tgaaagcaca tttcttggac gcgttgttag tgatggcata atgccaagaa taagtgcttc 1680 atccaactct gtgctgacag aattcgaaac aagatttaga cggatattaa aaagggttta 1740 ccgaagtctt cagcaacatg aaatacgaga agaaattctt gacgaaagat ctcgaattca 1800 atggcagtgg caacaacttg catctgtcgt tgatcgactt ttactatgtc ttttttgcac 1860 tgcaacactg ttcacaatca tctgcctcct aattgtacct gtagcatacc gtgataacga 1920 ctcaatgttg tcattcctca attttttctg attatcaaat acttgtttac atgttcttaa 1980 tgaaatttgc gaattatgga gaatatattt gctagaatca aattttcggg acttgtgtag 2040 tattggctga aaaattttta tccattttga acttttgata tgaccctttt tggttgcatt 2100 acgtttatga ccagttttta aaqcctaaaa aaaaaaaa 2138 <210> 5 <211> 1503 <212> DNA <213> Caenorhabditis elegans WO 01/61000 PCT/AU01/00150 36 <400> 5 tgatgctctt gcccatttta ttgcattttt tgcttctaat cacccaatta aatggctcac 60 cagcagaagt acggcttatc aatgatctta tgtcaggata tgttcgtgag gaaagaccad 120 cacttgatag ttcaaagcca gttgttgtca gtttgggagt ctttttgcaa cagattatta 180 acttgtccga aaaagaggaa cagctggaag taaatgcctg gcttaagttc caatggagag 240 atgaaaattt acgatgggaa ccgactgctt atgagaacat gacagatcta agacatccac 300 cggatgctct atggactccc gatatccttc tttataatag tgtcgattcg gagtttgatt 360 cgtcgtataa agtaaatctg gttaattatc atacgggaaa cattaattgg atgccaccag 420 gaarattcaa agtatcgtgt aaattggata tttattggtt tccatttgat gaacaagttt 480 gttattttaa gtttggctca tggacgtata ctcgtgataa gattcaacta gaaaagggtg 540 attttgattt ctccgagttc attccaaacg gggaatggat tataatagat tatcgaacaa 600 atattactgt gaaacaatat gaatgttgtc ccgagcagta tgaagatatc acttttacgc 660 tacatttacg acggagaact ttatactatt ccttcaattt aattgctcca gttcttttaa 720 caatgatact ggttattttg ggctttactg ttagccctga aacttgcgaa aaagttggac 780 ttcagatctc tgtctctctt gccatatgca ttttcctcac aataatgagt gaactgacac 840 ctcaaacatc agaagctgtt ccacttcttg gagtattctt ccacacttgc aacttcattt 900 ccgttttagc cacttctttc acagtttatg tgcaaagttt tcattttcga aaccaacatg 960 tacacgaacg gatggatttc tggatgaggt tcattctctt ggagtggtca ccgtggctat 1020 tgcgaatgaa aatgccggat agagaaaata actttcagac actgacagaa agttggaagg 1080 gaaggaatcg aagagaatct atggcaagaa cagcgttcga atatgcagat ggaccggtta 1140 cacagataca ttccatggga attatgttga aagataattt tgaagagctt atttatcaag 1200 ttaaacagga gaagattgct gatgaaaaag gaattgagag attgcgggtg ttacagaaga 1260 tttacgatca tgtaaagatg atccgagaac atgacgatga caatgatgaa gacagtcgag 1320 tagctcttga atggagattt gctgcaattg tcgtcgatcg tctgtgcctt cttgccttct 1380 ccttactcat cgtcgtcgtc tccatcatca ttgctttacg tgcaccgtat cttttcgctt 1440 aaaccaaatg ccttgagcaa tcaaataaaa ccatttcatt tccaaaaaaa aaaaaaaaaa 1500 aaa 1503 <210> 6 <211> 1915 <212> DNA <213> Caenorhabditis elegans <400> 6 tgtttgagca actctcaatg ccacgccacc aaggtcgaca aggatgagga gaaggttcga 60 aatcggcatc gcattctttt tcgcactttt tcgagtgaca tggacgggtg accatgaacg 120 tagactatat gcaaaattag cggaaaacta caacaaattg gcgagacctg ttcgaaacga 180 aagtgaagct gtagtagttc ttcttgggat ggattatcaa caaattttgg atattgacga 240 aaaacatcaa ataatgaatt caagtgtttg gttacggatg tcatggacag atcattactt 300 gacatgggat ccatcagagr ttggaaatat caaagaagtt cgtttgccaa tcaataatat 360 ctggaaacct gatgttcttc tctacaatag tgttgatcaa cagtttgata gtacatggcc 420 cgttaatgct gttgttttgt acacgggaaa cgtaacgtgg attcctccag ccatcattcg 480 atcaagttgt gctattgaca tagcatattt tccatttgat actcaacatt gtactatgaa 540 gttcggttcc tggacatatt ctggtttttt cactgatctc attaacacaa caatatctcc 600 agccacttat aaaccaaatg gagaatggga attacttggc ttaacgtcgc aacgctcgat 660 atttttctat gaatgctgcc cggagccata ttatgatgtc acgtttactg tttcaattag 720 gaggagaact ctctattatg gattcaactt attgctccca tgtatgctca tttcctcact 780 ggctttgttg agtttcacac ttccagctga ttgtggagag aaactgaatt taggcgtcac 840 aatcttcatg tctctttgcg tttttatgat tatggttgct gaagcaatgc ctcaaacaag 900 tgatgcactt ccattaattc aaatctattt ctcgtgcata atgttccaag ttggtgcatc 960 agtggtggcc actgtgattg cattgaactt tcatcatcga tcaccagaac agtacaagcc 1020 tatgaacaaa tttttgaaaa ctcttcttct gggctggctt ccaacacttc ttggcatgga 1080 acgtcctgat gttcttgaac tttctgtaca tggagcacat tatgcgtctg acaataaaaa 1140 aaaacaacgt caatacctaa tagaagtgga gagacatatt ctaacccgtc caaatggaaa 1200 tggacattca gcagttgata aagcagtgca tcttgactta tcaactggta atccacactc 1260 tgatgctaaa aaatcatcac cttctccaaa acgaacaagt gcttcaataa tgggtatgac 1320 WO 01/61000 PCT/AU01/00150 37 tggattgcca acaactcaaa tgaatggagc attggattct tcaattaata aatatacttg 1380 tacaaaagtt acgcgtccac tggaaaacgg ttcagcaaca ataaatcaca aatcatcacc'1440 tcaaataaat ccaatcaata acaataatat ctataaatgt gcaaacaacc aaaagactca'.1500 attcgaagat cgtcattttc atcatattct gaatgagctt cgtgttatat cagctcgtgt 1560 gagaaaagaa gaagcaatgc atgcacttca agctgattgg atgtttgcaa gtcgagttgz 1620 agatcgggtt tgttttcttg ctttttcagc atttctcttc atgtgcactg ctattatttc 1680 ttataatgcc ccgcatttat ttgtataatt ttttctaatt caatagagta agagtcaaga 1740 aattcatatc tcttgttgct tctttttaaa ttttacattt agagccaatt tgtgatttta 1800 agtacaaatg tatatcttta tttcgtcttt ttaaaataac atatacagtt tcaattgttt 1860 ttgctttgtt gtacatataa acaattatta aatttaaaaa aaaaaaaaaa aaaaa 1915 <210> 7 <211> 10 <212> PRT <213> Caenorhabdtttas elegant <400> 7 Met Ile Ile Cys Tyr Ser Cys Leu Thr Val 1 5 10 <210> 8 <211> 10 <212> PRT <213> Caenorhabditcs elegant <400> 8 Met Leu Leu Pro Ile Leu Leu His Phe Leu 1 5 10 <210> 9 <211> 10 <212> PRT <213> Caenorhabditis elegans <400> 9 Met Arg Arg Arg Phe Glu Ile Gly Ile Ala 1 10 <210> 10 <211> 635 <212> FRT <213> Caenorhabditgs elegant <400> 10 Met Ile Ile Cys Tyr Ser Cys Leu Thr Val Ser Le Leu Leu Thr le 1 5 10 15 Lys Phe Val Pro Cys Arg Phe Ala Gly Le Giu His Gin Asn Thr Lys 20 25 30 WO 01/61000 PCT/AU01/00150 38 Ser Arg Val His Phe Ser Leu Leu Asp Ser Arg Gin Glu Asn Asp Thr' 35 40 45 Asn His Phe Glu Ile Ala Glu Ala Lys Phe Gin Lys Pro His Asn Glu 50 55 60 Glu Asn Thr Ile Gly Thr Ile Thr Lys Phe Ala Pro Ser Val Gin Glu 65 70 75 80 Gin His Ser Ser Ala Val Ile Pro Met Pro His Phe Asp Gin Asn Arg 85 90 95 Leu Glu Gin Ala Leu Arg Ile Lys Gly Ser Ile Asp Gly Thr Glu Glu 100 105 110 Ala Leu Tyr Arg Ser Leu Leu Asp His Thr Val Tyr Glu Lys Asp Val 115 120 125 Arg Pro Cys Ile His His Ser Gin Pro Thr Asn Val Thr Phe Gly Phe 130 135 140 Leu Leu Asn Gin Ile Val Giu Met Asp Glu Arg Asn Gin Ala Leu Thr 145 150 155 160 Thr Arg Ser Trp Leu Asn Ile Asn Trp Met Asp Pro Arg Leu Ser Trp 165 170 175 Asn Glu Ser Leu Trp Ser Glu Ile Lys Ala Ile Tyr Ile Pro His Ala 180 185 190 Arg Ile Trp Lys Pro Asp Ile Ile Leu Val Asn Asn Ala Ile Arg Glu 195 200 205 Tyr Tyr Ala Ser Leu Val Ser Thr Asp Val Met Val Thr Ser Asp Gly 210 215 220 Asn Val Thr Trp Leu Phe Ser Ala Leu Phe Arg Ser Ser Cys Pro Ile 225 230 235 240 Arg Val Arg Tyr Tyr Pro Phe Asp Asp Gln Gin Cys Asp Leu Lys Phe 245 250 255 Ala Ser Trp Ser His Asp Ile Thr Glu Ile Asn Leu Gly Leu Asn Thr 260 265 270 Asp Lys Gly Asp Leu Ser Ser Tyr Met Asn Asn Ser Glu Phe Asp Leu 275 280 285 Val Asp Met Thr Ala Val Arg Glu Val Val Thr Phe Pro Ser Asp Thr 290 295 300 Asn Ser Asp Trp Pro Ile Ile Val Ile Arg Ile His Met His Arg Arg 305 310 315 320 Pro Leu Phe Tyr Val Phe Asn His Ile Val Pro Cys Val Leu Ile Ser 325 330 335 WO 01/61000 PCT/AU01/00150 39 Ser Met Ala Val Leu Gly Phe Leu Met Pro Pro Glu Thr Gly Glu Lys' 340 345 350 Ile Asn Met Ile Ile Thr Thr Leu Leu Ser Met Gly Val Tyr Leu Gin 355 360 365 Ser Ile Thr Glu Ser Ile Pro Pro Thr Ser Glu Gly Val Pro Leu Ile 370 375 380 Gly Met Tyr Tyr Val Ser Ser Leu Leu Met Val Cys Leu Ala Thr Cys 385 390 395 400 Val Asn Val Ile Thr Leu Asn Met His Arg Asn Gly Ala Ala Asn Gin 405 410 415 Gly Arg His Val Pro Ala Trp Met Gin Lys Trp Ile Leu Gly Tyr Leu 420 425 430 Ala Thr Phe Met Arg Met Ser Ile Arg Glu Pro Asp Ser Ile Ala Leu 435 440 445 Leu Lys Ala Ser Gin Ser Lys Lys Ser Thr Ile Arg Arg Ser Ser Ile 450 455 460 Leu Arg Asp Leu Lys Arg Val Lys Asn Met Ser Asn Val Arg Ala Lys 465 470 475 480 Ser Lys Glu Gin Asn Ala Asn Arg Glu Cys Glu Cys Met Asp Pro Leu 485 490 495 Val His Ile Tyr Ala Glu Ser Ile Met Ser Cys Leu Ala Ala Asp Thr 500 505 510 Lys Pro Met Asn Gly Ser Thr Ile Arg Giu Asp Phe Ala Ser Glu Ser 515 520 525 Thr Phe Leu Gly Arg Val Val Ser Asp Gly Ile Met Pro Arg Ile Ser 530 535 540 Ala Ser Ser Asn Ser Val Leu Thr Giu Phe Glu Thr Arg Phe Arg Arg 545 550 555 560 Ile Leu Lys Arg Val Tyr Arg Ser Leu Gin Gin His Giu Ile Arg Glu 565 570 575 Glu Ile Leu Asp Glu Arg Ser Arg Ile Gin Trp Gin Trp Gin Gin Leu 580 585 590 Ala Ser Val Val Asp Arg Leu Leu Leu Cys Leu Phe Cys Thr Ala Thr 595 600 605 Leu Phe Thr Ile Ile Cys Leu Leu Ile Val Pro Val Ala Tyr Arg Asp 610 615 620 Asn Asp Ser Met Leu Ser Phe Leu Asn Phe Phe 625 630 635 WO 01/61000 PCT/AU01/00150 40 <210> 11 <211> 479 <212> PRT <213> Caenorhabditis elegans <400> 11 Met Leu Leu Pro Ile Leu Leu His Phe Leu Leu Leu Ile Thr Gin Leu 1 5 10 15 Asn Gly Ser Pro Ala Glu Val Arg Leu Ile Asn Asp Leu Met Ser Gly 20 25 30 Tyr Val Arg Glu Glu Arg Pro Thr Leu Asp Ser Ser Lys Pro Val Val 35 40 45 Val Ser Leu Gly Val Phe Leu Gin Gin Ile Ile Asn Leu Ser Glu Lys 50 55 60 Glu Glu Gin Leu Glu Val Asn Ala Trp Leu Lys Phe Gin Trp Arg Asp 65 70 75 80 Glu Asn Leu Arg Trp Glu Pro Thr Ala Tyr Glu Asn Val Thr Asp Leu 85 90 95 Arg His Pro Pro Asp Ala Leu Trp Thr Pro Asp Ile Leu Leu Tyr Asn 100 105 110 Ser Val Asp Ser Glu Phe Asp Ser Ser Tyr Lys Val Asn Leu Val Asn 115 120 125 Tyr His Thr Gly Asn Ile Asn Trp Met Pro Pro Gly Ile Phe Lys Val 130 135 140 Ser Cys Lys Leu Asp Ile Tyr Trp Phe Pro Phe Asp Glu Gin Val Cys 145 150 155 160 Tyr Phe Lys Phe Gly Ser Trp Thr Tyr Thr Arg Asp Lys Ile Gin Leu 165 170 175 Glu Lys Gly Asp Phe Asp Phe Ser Giu Phe Ile Pro Asn Gly Glu Trp 180 185 190 Ile Ile Ile Asp Tyr Arg Thr Asn Ile Thr Val Lys Gin Tyr Glu Cys 195 200 205 Cys Pro Glu Gin Tyr Glu Asp Ile Thr Phe Thr Leu His Leu Arg Arg 210 215 220 Arg Thr Leu Tyr Tyr Ser Phe Asn Leu Ile Ala Pro Val Leu Leu Thr 225 230 235 240 Met Ile Leu Val Ile Leu Gly Phe Thr Val Ser Pro Glu Thr Cys Glu 245 250 255 Lys Val Gly Leu Gin Ile Ser Val Ser Leu Ala Ile Cys Ile Phe Leu WO 01/61000 PCT/AU01/00150 41 260 265 270 Thr Ile Met Ser Glu Leu Thr Pro Gin Thr Ser Glu Ala Val Pro Leu 275 280 285 Leu Gly Val Phe Phe His Thr Cys Asn Phe Ile Ser Val Leu Ala Thr 290 295 300 Ser Phe Thr Val Tyr Val Gin Ser Phe His Phe Arg Asn Gin His Val 305 310 315 320 His Glu Arg Met Asp Phe Trp Met Arg Phe Ile Leu Leu Glu Trp Ser 325 330 335 Pro Trp Leu Leu Arg Met Lys Met Pro Asp Arg Glu Asn Asn Phe Gin 340 345 350 Thr Leu Thr Glu Ser Trp Lys Gly Arg Asn Arg Arg Glu Ser Met Ala 355 360 365 Arg Thr Ala Phe Glu Tyr Ala Asp Gly Pro Val Thr Gln Ile His Ser 370 375 380 Met Gly Ile Met Leu Lys Asp Asn Phe Giu Glu Leu Ile Tyr Gin Val 385 390 395 400 Lys Gin Glu Lys Ile Ala Asp Glu Lys Gly Ile Giu Arg Leu Arg Val 405 410 415 Leu Gin Lys Ile Tyr Asp His Val Lys Met Ile Arg Glu His Asp Asp 420 425 430 Asp Asn Asp Glu Asp Ser Arg Val Ala Leu Glu Trp Arg Phe Ala Ala 435 440 445 Ile Val Val Asp Arg Leu Cys Leu Leu Ala Phe Ser Leu Leu Ile Val 450 455 460 Val Val Ser Ile Ile Ile Ala Leu Arg Ala Pro Tyr Leu Phe Ala 465 470 475 <210> 12 <211> 554 <212> PRT <213> Caenorhabditis elegans <400> 12 Met Arg Arg Arg Phe Giu Ile Gly Ile Ala Phe Phe Phe Ala Leu Phe 1 5 10 15 Arg Val Ile Trp Thr Gly Asp His Giu Arg Arg Leu Tyr Ala Lys Leu 20 25 30 Ala Glu Asn Tyr Asn Lys Leu Ala Arg Pro Val Arg Asn Glu Ser Glu 35 40 45 WO 01/61000 PCT/AU01/00150 42 Ala Val Val Val Leu Leu Gly Met Asp Tyr Gin Gin Ile Leu Asp Ile 50 55 60 Asp Glu Lys His Gin Ile Met Asn Ser Ser Val Trp Leu Arg Met Ser 65 70 75 80 Trp Thr Asp His Tyr Leu Thr Trp Asp Pro Ser Glu Phe Gly Asn Ile 85 90 95 Lys Glu Val Arg Leu Pro Ile Asn Asn Ile Trp Lys Pro Asp Val Leu 100 105 110 Leu Tyr Asn Ser Val Asp Gin Gin Phe Asp Ser Thr Trp Pro Val Asn 115 120 125 Ala Val Val Leu Tyr Thr Gly Asn Val Thr Trp Ile Pro Pro Ala Ile 130 135 140 Ile Arg Ser Ser Cys Ala Ile Asp Ile Ala Tyr Phe Pro Phe Asp Thr 145 150 155 160 Gin His Cys Thr Met Lys Phe Gly Ser Trp Thr Tyr Ser Gly Phe Phe 165 170 175 Thr Asp Leu Ile Asn Thr Thr Ile Ser Pro Ala Thr Tyr Lys Pro Asn 180 185 190 Gly Glu Trp Glu Leu Leu Gly Leu Thr Ser Gin Arg Ser Ile Phe Phe 195 200 205 Tyr Glu Cys Cys Pro Glu Pro Tyr Tyr Asp Val Thr Phe Thr Val Ser 210 215 220 Ile Arg Arg Arg Thr Leu Tyr Tyr Gly Phe Asn Leu Leu Leu Pro Cys 225 230 235 240 Met Leu Ile Ser Ser Leu Ala Leu Leu Ser Phe Thr Leu Pro Ala Asp 245 250 255 Cys Gly Glu Lys Leu Asn Leu Gly Val Thr Ile Phe Met Ser Leu Cys 260 265 270 Val Phe Met Ile Met Val Ala Glu Ala Met Pro Gin Thr Ser Asp Ala 275 280 285 Leu Pro Leu Ile Gin Ile Tyr Phe Ser Cys Ile Met Phe Gin Val Gly 290 295 300 Ala Ser Val Val Ala Thr Val Ile Ala Leu Asn Phe His His Arg Ser 305 310 315 320 Pro Giu Gin Tyr Lys Pro Met Asn Lys Phe Leu Lys Thr Leu Leu Leu 325 330 335 Gly Trp Leu Pro Thr Leu Leu Gly Met Glu Arg Pro Asp Val Leu Glu 340 345 350 WO 01/61000 PCT/AU01/00150 43 Leu Ser Val His Gly Ala His Tyr Ala Ser Asp Asn Lys Lys Lys Gln 355 360 365 Arg Gln Tyr Leu Ile Glu Val Glu Arg His Ile Leu Thr Arg Pro Asn 370 375 380 Gly Asn Gly His Ser Ala Val Asp Lys Ala Val His Leu Asp Leu Ser 385 390 395 400 Thr Gly Asn Pro His Ser Asp Ala Lys Lys Ser Ser Pro Ser Pro Lys 405 410 415 Arg Thr Ser Ala Ser Ile Met Gly Met Thr Gly Leu Pro Thr Thr Gln 420 425 430 Met Asn Gly Ala Leu Asp Ser Ser Ile Asn Lys Tyr Thr Cys Thr Lys 435 440 445 Val Thr Arg Pro Leu Glu Asn Gly Ser Ala Thr Ile Asn His Lys Ser 450 455 460 Ser Pro Gln Ile Asn Pro Ile Asn Asn Asn Asn Ile Tyr Lys Cys Ala 465 470 475 480 Asn Asn Gln Lys Thr Gln Phe Glu Asp Arg His Phe His His Ile Leu 485 490 495 Asn Glu Leu Arg Val Ile Ser Ala Arg Val Arg Lys Glu Glu Ala Met 500 505 510 His Ala Leu Gln Ala Asp Trp Met Phe Ala Ser Arg Val Val Asp Arg 515 520 525 Val Cys Phe Leu Ala Phe Ser Ala Phe Leu Phe Met Cys Thr Ala Ile 530 535 540 Ile Ser Tyr Asn Ala Pro His Leu Phe Val 545 550 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Conserved motif <400> 13 Asp Ile Ala Tyr 1 WO 01/61000 PCT/AU01/00150 44 It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the 5 invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (43)

1. An isolated polynucleotide molecule encoding an invertebrate

5-HT 3 receptor subunit consisting of a nucleotide sequence which shows greater 5 than 75% homology to any or all of the nucleotide sequences shown as SEQ ID NO: 1-6. 2. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which shows 10 greater than 85% homology to any or all of the nucleotide sequences shown as SEQ ID NO: 1-6. 3. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which shows 15 greater than 95% homology to any or all of the nucleotide sequences shown as SEQ ID NO: 1-6. 4. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which 20 substantially corresponds to any one of the nucleotide sequences shown as SEQ ID NO: 1-6. 5. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which 25 substantially corresponds to the nucleotide sequence shown as SEQ ID NO: 1.

6. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which substantially corresponds to the nucleotide sequence shown as SEQ ID NO: 2. 30

7. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which substantially corresponds to the nucleotide sequence shown as SEQ ID NO: 3. WO 01/61000 PCT/AU01/00150 46

8. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which substantially corresponds to the nucleotide sequence shown as SEQ ID NO: 4. 5 9. A polynucleotide molecule according to claim 1, wherein said polynucleotide molecule consists of a nucleotide sequence which substantially corresponds to the nucleotide sequence shown as SEQ ID NO: 5.

10. A polynucleotide molecule according to claim 1, wherein said 10 polynucleotide molecule consists of a nucleotide sequence which substantially corresponds to the nucleotide sequence shown as SEQ ID NO: 6.

11. An expression cassette or vector comprising the polynucleotide molecule according to any one of the preceding claims. 15

12. A mammalian, insect, plant, yeast or bacterial host cell transformed with at least one expression cassette or vector according to claim 11.

13. A host cell according to claim 12, wherein said host cell expresses 20 homomeric invertebrate 5-HT, receptors.

14. A host cell according to claim 12, wherein said host cell expresses heteromeric invertebrate 5-HT, receptors. 25 15. A host cell according to claim 13 or 14. wherein said 5-HT, receptors are expressed onto the surface of the host cell.

16. A method of producing 5-HT, receptors, comprising culturing a host cell according to any one of claims 11-15 under conditions enabling the 30 expression of said 5-HT, receptors and, optionally, recovering the expressed 5-HT3 receptors.

17. An invertebrate 5-HT3 receptor comprising at least one subunit which is characterised by an N-terminal amino acid sequence selected from the 35 following: WO 01/61000 PCT/AUO1/00150 47 MIICYSCLTV (SEQ ID NO: 7), MLLPILIFL (SEQ ID NO: 8) or MRRRFEIGIA (SEQ ID NO: 9), or a functionally equivalent fragment of said receptor, in a substantially pure form. 5

18. A receptor according to claim 17, wherein the at least one subunit has an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 10, 11 or 12. 10 19. An assay for identifying and/or assessing nematicidal compounds, said assay comprising contacting a 5-HT, receptor or a functionally equivalent fragment thereof according to claim 17 or 18, or a host cell according to claim 15 having 5-HT 3 receptors on its surface. with a candidate nematicidal compound under conditions enabling the activation of 5-HT receptors, and 15 detecting an increase or decrease in activity of said 5-HT 3 receptor(s) or functionally equivalent fragment thereof.

20. An assay according to claim 19, wherein the increase or decrease in activity of the 5-HT, receptor(s) or functionally equivalent fragment thereof is 20 detected by measuring changes in cell membrane potential or Ca" levels.

21. An assay according to claim 19 or 20, wherein the the 5-HT 3 receptor(s) or functionally equivalent fragment thereof is contacted simultaneously with the candidate nematicidal compound and a serotonergic ligand. 25

22. An assay for identifying and/or assessing nematicidal compounds, said assay comprising contacting a 5-HT, receptor or a functionally equivalent fragment thereof according to claims 17 or 18, or a cell according to claim 15 having 5-HT, receptors on its surface, with a predetermined amount of a 30 suitably labelled serotonergic ligand together with a predetermined amount of a candidate nematicidal compound under conditions wherein said serotonergic ligand and said candidate nematicidal compound competitively bind to the 5-HT, receptor(s) or functionally equivalent fragment thereof. and determining the amount of bound and/or unbound labelled serotonergic 35 ligand. WO 01/61000 PCT/AU01/00150 48

23. An assay according to any one of claims 19 to 22, wherein said serotonergic ligand is 5-hydroxytryptamine.

24. A probe comprising all or, at least, a 10 nucleotide part of any of the 5 nucleotide sequences shown as SEQ ID NO: 1-6.

25. An assay for identifying and/or assessing nematicidal, insecticidal and/or other pesticidal compounds, said assay comprising; (i) isolating a polynucleotide molecule from an invertebrate other 10 than Caenorhabditis elegans wherein said polynucleotide molecule comprises a nucleotide sequence which encodes a 5-HT, receptor subunit and represents a homologue of any or all of the nucleotide sequences shown as SEQ ID NO: 1-6, (ii) expressing said polynucleotide molecule to produce 5-HT, 15 receptors or functionally equivalent fragments thereof, (iii) contacting at least one of said produced 5-HT, receptors or functionally equivalent fragments thereof to a candidate nematicidal, insecticidal and/or other pesticidal compound under conditions enabling the activation of 5-HT receptors, and 20 (iv) detecting an increase or decrease in activity of said produced 5 HT 3 receptor(s) or functionally equivalent fragment(s) thereof.

26. An assay according to claim 25. wherein the increase or decrease in activity of the 5-HT, receptor(s) or functionally equivalent fragment thereof is 25 detected by measuring changes in cell membrane potential or Ca 2 levels.

27. An assay according to claim 25 or 26, wherein the the 5-HT 3 receptor(s) or functionally equivalent fragment thereof is contacted simultaneously with the candidate nematicidal compound and a serotonergic ligand. 30

28. An assay for identifying and/or assessing nematicidal, insecticidal and/or other pesticidal compounds, said assay comprising; (i) isolating a polynucleotide molecule from an invertebrate other than Caenorhabditis elegans wherein said polynucleotide molecule comprises 35 a nucleotide sequence which encodes a 5-HT 3 receptor subunit and WO 01/61000 PCT/AU01/00150 49 represents a homologue of any or all of the nucleotide sequences shown as SEQ ID NO: 1-6, (ii) expressing said polynucleotide molecule to produce 5-HT, receptors or functionally equivalent fragments thereof, 5 (iii) contacting at least one of said produced 5-HT, receptors or functionally equivalent fragments thereof with a predetermined amount of a suitably labelled serotonergic ligand together with a predetermined amount of a candidate nematicidal, insecticidal and/or other pesticidal compound under conditions wherein said serotonergic ligand and said candidate compound 10 competitively bind to the 5-HT, receptor(s) or functionally equivalent fragment(s) thereof, and (iv) determining the amount of bound and/or unbound labelled serotonergic ligand. 15 29. An assay according to any one of claims 25 to 28, wherein said serotonergic ligand is 5-hydroxytryptamine.

30. A nematicidal compound identified by an assay according to any one of claims 19 to 23. 20

31. A nematicidal, insecticidal and/or other pesticidal compound identified by an assay according to any one of claims 25 to 29.

32. A method of killing a helminth, said method comprising exposing said 25 helminth to an effective amount of a compound which alters the activity of a 5-HT 3 receptor of said helminth.

33. A method according to claim 32, wherein said compound inhibits stimulation of the 5-HT, receptor by a serotonergic ligand. 30

34. A method according to claim 32 or 33, wherein said effective amount of said compound is in the range of 0.1 to 10p.M.

35. A method according to any one of claims 32 to 33, wherein the 35 helminth is a nematode. WO 01/61000 PCT/AUO1/00150 50

36. A method according to any one of claims 32 to 35, wherein said compound is selected fror, odansetron and tropanyl dichlorobenzoate.

37. A composition for killing a helminth comprising an effective amount of 5 a compound which alters the activity of a 5-HT, receptor of a helminth.

38. A composition according to claim 37, wherein said compound inhibits stimulation of the 5-HT, receptor by a serotonergic ligand. 10 39. A composition according to claim 37 or 38, wherein said effective amount of said compound is in the range of 0.1 to 10tM.

40. A composition according to any one of claims 37 to 39, wherein the helminth is a nematode. 15

41. A composition according to any one of claims 37 to 40, wherein said compound is selected from ondansetron and tropanyl dichlorobenzoate.

42. A method of killing an insect, said method comprising exposing said 20 insect to an effective amount of a compound which alters the activity of a 5 HT, receptor of said insect.

43. A method according to claim 42, wherein said compound inhibits stimulation of the 5-HT 3 receptor by a serotonergic ligand. 25

44. A method according to claim 42 or 43, wherein said effective amount of said compound is in the range of 0.1 to 10 M.

45. A method according to any one of claims 42 to 44, wherein the insect is 30 a sucking insect or other insect with a muscular pump-based feeding mechanism.

46. A method according to any one of claims 42 to 45, wherein said compound is selected from odansetron and tropanyl dichlorobenzoate. 35 WO 01/61000 PCT/AU01/00150 51

47. An insecticidal composition comprising an effective amount of a compound which alters the activity of a 5-HT 3 receptor of an insect.

48. A composition according to claim 47, wherein said compound inhibits 5 stimulation of the 5-HT 3 receptor by a serotonergic ligand.

49. A composition according to claim 47 or 48, wherein said effective amount of said compound is in the range of 0.1 to 10pM. 10 50. A composition according to any one of claims 47 to 49, wherein the helminth is a nematode.

51. A composition according to any one of claims 47 to 50, wherein said compound is selected from ondansetron and tropanyl dichlorobenzoate. 15

52. An isolated polynucleotide molecule from an invertebrate other than Caenorhabditis elegans, wherein said polynucleotide molecule comprises a nucleotide sequence which encodes a 5-HT, receptor subunit and represents a homologue of any or all of the nucleotide sequences shown as SEQ ID NO: 20 1-6.