WO2002033405A1

WO2002033405A1 - Methods for identifying pesticidal compounds

Info

Publication number: WO2002033405A1
Application number: PCT/IB2001/002391
Authority: WO
Inventors: Richard Zwaal; Titus Kaletta; Marc Van Den Craen; Marc Logghe; Elke Smits; Wim Van Creikinge; Thierry Bogaert
Original assignee: Devgen Nv
Priority date: 2000-10-18
Filing date: 2001-10-15
Publication date: 2002-04-25
Also published as: AU2002218457A1

Abstract

The invention is concerned with methods for use in the identification of compounds having potential utility as pesticides. In particular, the invention relates to methods for use in identifying compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA).

Description

Methods for identifying pesticidal compounds

The invention is concerned with methods for use in the identification of compounds having potential utility as pesticides. In particular, the invention relates to methods for use in identifying compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) . Although a lot of effort has been made over the past few years in the development of novel pesticides there is still a great demand for new pesticides. One of the main problems facing the agrochemical industry at present is the development of pesticide resistance by target organisms. To handle problem, various resistance action committees have been set up within the Global Crop Protection Federation (GCPF, Avenue Louise 143, 1050 Brussels, Belgium) . The insecticide resistance committee (IRAC) , reports regularly on the emergence of new resistance of insects against insecticides. The results of a resistance survey carried out in 1996, published in "The Pest Manual, 11th edition, ed CDS Tomlin", by the British Crop Protection Council, 49 Downing Street, Farnham, Surrey, GU9 7PH, UK, indicating the problems that exist with insect resistance and hence the need to develop new insecticides.

The Fungicide resistance action committee (FRAC) has already indicated that well known fungi have already developed resistance to well known fungicides such as benzimidazoles, dicarboximides, phenyla ides, sterol biosynthesis inhibitors. In 1996 and 1994, the stobilurins and the anilinopyrimidines were introduced on the market as novel fungicides. At the time of publication of the 11th Edition of "The Pest Manual", ibid, no resistance has been observed against those to classes of compounds, but one may expect that in the near future fungi will also develop resistance against these fungicides.

The herbicide resistance action committee (HRAC) also publishes regularly the present status of herbicide resistance world wide. These publications can be found on HRAC publicity office, C/O David Nevill & Derek Cornes, Novartis protection AG, 4002, Basel Switzerland. The results of these surveys indicate that there is a need for novel herbicides. A similar pattern of emerging resistance is also observed for other classes of pesticides, particularly rodenticides, acaricides and nematocides. An overview of all such compounds with pesticide activity can be found in "The pest manual", ibid, and in references cited therein; Insecticides with Novel Modes of action, Mechanisms and application, Springer-Verlag Berlin, eds. I. Ishaaya, and^' D. Degheele. Recently, completely new insecticides have been isolated, such as paralysins (Chiou et al., Biochem. and Biophys. Res. Com. 1998 246:457-462), deoxyribonucleosides and derivatives (Balzarini et al, Mol. Pharmacology. 2000, 57:811-819) .

New pesticides should be developed to further protect food production, but should have a minimal impact on the health of human populations and domestic animals and a minimal impact on the ecosystem. Hence, there is a great demand for safer, more selective pesticides affecting only specifically harmful pest species . The present inventors have identified the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) as a potential target for pesticidal intervention. The SERCA proteins belong to the group of ATP-driven ion- motive ATPases, which also includes, amongst others, the plasma membrane Ca²⁺-transport ATPases (PMCA) , the Na⁺-K⁺-ATPases, and the gastric H⁺-K⁺-ATPases . SERCA proteins are present in all higher organisms, including pest species. The evolutionary conservation of SERCA proteins identifies these proteins as an interesting target for pesticidal intervention. Furthermore, it is known that inhibition or deletion of SERCA activity in a variety of organisms results in lethality, or at least a marked reduction in the vitality of the organism. In particular, the present inventors have shown that inhibition of SERCA activity in the nematode C. elegans results in lethality. Inhibition of SERCA activity, and hence depletion of endoplasmic reticulum calcium stores also results in a lowering of muscle relaxation and hence immobility and/or respiration deficiency.

The maintenance of high calcium concentrations in the ER is important for the proper synthesis of proteins, including translation, folding, glycosylation, processing and transport. Treatment of living organisms with chemicals that down-regulate or inhibit the activity of SERCA will hence have a negative effect on the welfare of these organisms. As such, SERCA inhibitors are potential pesticides or can be considered as basic compounds for the development of pesticides such as herbicides, insecticides and nematocides. It has been shown that SERCA function is essential in the intracellular trafficking of the

Notch receptor in drosophila (Periz et al., 1999 EMBO J; 5983-5993) . This study and others indicate that SERCA is an interesting target for pesticidal intervention. The inventors have developed generic screening methods which may be used to identify compounds which down-regulate SERCA activity and may therefore have the potential to kill pests. Several of these screens are performed in microscopic nematode worms such as Caenorhabdi tis elegans . C. elegans is a small roundworm that has a life cycle of only three days, allowing rapid accumulation of large quantities of individual nematodes. C. elegans may be used in the development of high throughput live animal compound screens in which nematodes are exposed to the compound under test and any resultant phenotypic and/or behavioural changes are recorded. The present inventors have developed a number of C. e-Ze ans-based screening methods which may be used to identify compounds which modulate the activity of SERCA. Furthermore, these C. elegans based screening methods may also be used to identify compounds which modulate the activity of other proteins in the SERCA pathway, such as proteins involved in the calcium homeostasis of the cell.

Therefore, in a first aspect the invention provides a method of identifying compounds having pesticidal activity, which method comprises: providing microscopic nematode worms expressing a pest SERCA protein, said protein being derived from a pest species, other than the C. elegans SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the microscopic nematode worm in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has pesticidal activity.

The method of the invention may be used to identify compounds which have pesticidal activity because they directly or indirectly affect the activity of the SERCA protein. Hence, the invention further provides a method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises: providing microscopic nematode worms expressing a pest SERCA protein, said protein being derived from a pest species, other than the C. elegans SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the microscopic nematode worm in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.

The preferred microscopic nematode species for use in the screening methods of the invention is Caenorhabd.i t Is elegans . It will, however, be appreciated that the methods may be carried out with other nematodes and in particular with other microscopic nematodes, preferably microscopic nematodes belonging to the genus Caenorhabdi tis including C. briggsae. As used herein the term "microscopic" nematode encompasses nematodes of approximately the same size as C. elegans, being of the order 1mm long in the adult stage. Microscopic nematodes of this approximate size are extremely suited for use in mid- to high-throughput screening as they can easily be grown in the wells of a multi-well plate of the type generally used in the art to perform such screening.

C. elegans occurs naturally in the soil but can be easily grown in the laboratory on nutrient agar inoculated with bacteria, preferably E. coll , or in liquid culture. Each worm grows from an embryo to an adult worm of about 1 mm long in three days or so. As it is fully transparent at all stages of its life, cell divisions, migrations and differentiation can be seen in live animals. Furthermore, although its anatomy is simple its somatic cells represent most major differentiated tissue type including muscles, neurons, intestine and epidermis. Accordingly, differences in phenotype which represent a departure from that of wild-type C. elegans are relatively easily observed and many phenotypic, physiological or biochemical characteristics of the nematode submit to quantitative measurement.

In the context of this application, the term "pest SERCA protein" encompasses any SERCA protein derived from a pest species. The term "pest species" encompass species recognised as such by one skilled in the art. Pest species include, but are not necessarily limited to, arthropods such as insects, ticks, mites, spiders and nematodes (excluding C. elegans for the purposes of this application) and also fungi, plants and rodents. The term "pest species" also encompasses parasitic pest species, including human parasites, and the term "compounds having pesticidal activity" is to be interpreted accordingly as encompassing compounds having anti-parasitic activity which may have utility in the pharmaceutical and/or veterinary fields. A non-exhaustive list of pest species is included in the accompanying Examples. Further lists of pest species can be found in "The Pest Manual", ed CDS Tomlin, BCPC.

The term "compounds having pesticidal activity" is to be interpreted as encompassing compounds which are lethal to one or more pest species as hereinbefore defined or lethal to the progeny of such a pest species. As aforesaid, this definition encompasses compounds having anti-parasitic activity.

The term "SERCA protein derived from a pest species" is intended to encompass any SERCA protein naturally expressed by a pest species, including naturally occurring splice variants, allelic variants and isoforms. Many species express more than one SERCA isoform and the scope of the invention is not restricted to any particular isoform.

The term "SERCA protein derived from a pest species" is also intended to encompass specific mutant versions of naturally occurring pest SERCA proteins, including, for example, mutant proteins engineered by directed mutagenesis techniques. Specific mutant pest SERCA proteins will advantageously retain near wild- type SERCA ATPase activity. Further examples of "SERCA proteins derived from a pest species" within the scope of the invention are chimeric proteins created by in-frame fusion of fragments of two or more SERCA proteins, at least one of which is a SERCA protein derived from a pest species. Chimeric proteins included within this definition include fusions of a pest SERCA protein and a C. elegans SERCA protein (see accompanying Examples) .

The microscopic nematode worm expressing the pest SERCA protein may, advantageously, be a transgenic worm containing a transgene comprising nucleic acid encoding the pest SERCA protein operably linked to a promoter. In the context of this application the term ^' "transgene" refers to a DNA construct comprising a promoter operably linked to a DNA sequence encoding the pest SERCA protein. The construct .may contain additional DNA sequences in addition to those specified above. The transgene may, for example, form part of an expression vector, such as plasmid vector. By the term "operably linked" it is to be understood that the promoter is positioned to drive transcription of the protein-encoding DNA fragment.

Methods of preparing transgenic C. elegans, including C. elegans carrying multiple transgenes, are well known in the art and are described, for example, by Craig Mello and Andrew Fire, Methods in Cell Biology, Vol 48, Ed. H.F. Epstein and D.C. Shakes, Academic Press, pages 452-480. A typical approach involves the construction of a plasmid-based expression vector in which a protein-encoding DNA of interest is cloned downstream of a promoter having the appropriate tissue or cell-type specificity. The plasmid vector is then introduced into C. elegans of the appropriate genetic background, for example using microinjection. In order to facilitate the selection of transgenic C. elegans a second plasmid carrying a selectable marker may be co-injected with the experimental plasmid.

Plasmid vectors are usually maintained in cells of transgenic C. elegans in the form of an extrachromosomal array. Although plasmid vectors are relatively stable as extrachromosomal arrays they can alternatively be stably integrated into the C. elegans genome using standard technology, for example, using gamma ray-induced integration of extrachromosomal arrays (methods in Cell Biology, Vol 48 page 425-480) . The DNA sequence encoding the pest SERCA protein may be any DNA sequence comprising the complete open reading frame of the corresponding pest SERCA gene, such as, for example, a fragment of genomic DNA or cDNA. A number of pest SERCA cDNA sequences are available from publicly accessible sequence databases such as the GenBank database. The number of sequences deposited in the publicly accessible sequence databases is increasing all the time and these sequences are derived from an increasing diversity of species. A list of database accession numbers is provided in the accompanying Examples. Using this sequence data it is a matter of routine to clone a corresponding cDNA using molecular biology techniques well known in the art (see ^ΛCurrent Protocols in Molecular Biology', Ed Ausubel et al . , John Wiley &

Sons, Inc) . Specific examples of the cloning of pest SERCA cDNAs based on sequence data accessed from the database are included herein.

The inventors have developed an approach to isolate SERCA cDNAs from various other pest species, in particular pest species for which no or limited sequence data is available through database sources. The inventors' method is generally applicable and comprises the following steps:

a) Prepare a multiple alignment of known pest SERCA protein sequences (for example using ClustalW software) ;

b) Identify blocks of homology (for example using the Block Maker software accessible via the Blocks WWW

Server at the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA http://blocks.fhcrc.org);

c) Design degenerate oligonucleotide primers to conserved regions of amino acid sequence (for example using CODEH0P (Rose, et al . , NAR 26: 1628-1635);

d) Perform PCR using pairs of degenerate primers on cDNA prepared from the pest species;

e) Clone PCR fragments into a suitable cloning vector (many vectors suitable for the cloning of PCR products are available commercially) ;

f) Isolate full length cDNA corresponding to the PCR fragment (for example using 5" and 3' RACE or cDNA library screening, techniques which are well known in the art) .

By way of illustration of this approach, a homology series of plant SERCA proteins used to identify degenerate primers and primer combinations to isolate SERCA cDNAs from plant pests is shown in the accompanying Figure 1. A more general homology series of SERCA proteins from more diverse species is shown in Figure 2. This alignment may be used to design degenerate primers useful in the isolation of SERCA proteins from more diverse pest species. A list of primers and primer combinations is included in the accompanying Examples.

The promoter part of the transgene may be any promoter which is capable of directing gene expression in the nematode. Preferably the DNA encoding the pest SERCA protein is operably linked to the promoter region of a SERCA gene. Most preferably the promoter region of the C. elegans sca-1 gene is used. The term promoter region' as used herein refers to a fragment of the upstream region of a given gene which is capable of directing a pattern of gene expression substantially identical to the natural pattern of expression of the given gene. When the screen is carried out using transgenic C. elegans, the promoter may, advantageously, be the promoter region of a C. elegans gene and may be a tissue-or cell type-specific promoter. With the use of a promoter of appropriate specificity, the pest SERCA protein can be expressed in all the cells of C. elegans, in a given type of tissue (i.e. all muscles), in a single organ or tissue (for example, the pharynx or the vulva) , in a subset of cell types, in a single, cell type or even in a single cell. Tissue-specific C. elegans promoters which may be used in accordance with the invention include the myo-2 promoter which directs gene expression in the pharynx, the myo-3 promoter which directs gene expression in the body wall muscles, the egl-15 and ceh-24 promoters which direct gene expression in vulva muscles. Other tissue-specific C. elegans promoters are well known to persons skilled in the art.

In order to screen for compounds which act specifically on the expressed pest SERCA protein, rather than the endogenous nematode SERCA protein, it is preferred to use nematodes which, at the same time as expressing the pest SERCA protein, exhibit no or substantially reduced activity of the endogenous nematode SERCA protein in one or more tissues or cell types. C. elegans has a single SERCA gene, which was identified by the C. elegans genome-sequencing consortium (see Science issue 282, 1998) . The C. elegans SERCA gene, designated sca -1 , is located on chromosome III on a cosmid named K11D9. On a physical level, the gene consists of seven exons that span an Open Reading Frame of 3.2 kb, resulting in a predicted protein of 1059 amino acids. The consensus alternative splice site that is present in the C-terminal end of mammalian SERCA genes is also present in C. elegans . This leads to a second isoform consisting of eight exons that span an ORF of 3.0kb, resulting in a protein of 1004 amino acids.

In the context of this application the term ^activity' used in relation to a SERCA protein refers to the calcium ATPase activity of the protein, unless otherwise stated. There are various ways in which the activity of the endogenous nematode SERCA protein can be substantially reduced or abolished. In one embodiment, this is achieved by introducing the transgene encoding the pest SERCA protein into a mutant strain which exhibits no or substantially reduced activity of the endogenous SERCA protein in one or more tissues or cell types. This mutant strain may carry a knock-out or loss-of-function mutation in the chromosomal SERCA gene. Alternatively, the mutation may abolish/reduce SERCA activity through a down-regulation of SERCA expression in one or more cell types or tissues or a defect in regulation of the activity of the SERCA protein.

C. elegans having a reduction-of-function mutation or a knock-out mutation in the sca-1 gene can be isolated using a classical non-complementation screen, starting with a heterozygote C. elegans strain carrying a mutant sca-1 allele on one chromosome and a recessive marker close to the wild-type sca-1 allele on the other chromosome. The nematodes are subjected to mutagenesis using standard techniques (EMS or

UV-TMP are suitable for this purpose) and the progeny is screened by eye for defects, especially in tissues which express SERCA. Since the screening is performed in the Fl generation, mutations will only give rise to a phenotype if the mutation occurs in the sca-1 gene (due to non-complementation) or if the mutation is dominant, which does not occur frequently. These two possibilities can be distinguished in subsequent generations. A newly introduced sca-1 mutation should be linked to the recessive marker. As a further control, DNA sequencing can be performed to determine the nature of the mutation.

An example of a C. elegans strain which carries a knock-out mutation in the sca-1 gene is strain okl 90, described in the accompanying Examples. A protocol for introducing a pest SERCA transgene onto an sca-1 knock-out genetic background is included in the accompanying examples.

In another embodiment, activity of the endogenous nematode SERCA protein can be reduced by specifically down-regulating the expression of the SERCA protein in one or more tissues using antisense techniques or double-stranded RNA inhibition (RNAi) . This can be achieved by transfection of the nematode, preferably C. elegans, with a vector that expresses either an antisense SERCA RNA or a double-stranded SERCA RNA. The antisense or double-stranded SERCA RNA should be capable of selectively inhibiting expression of the endogenous nematode SERCA protein but not the pest SERCA protein. Specific down-regulation of SERCA expression in different cell types or tissues of the nematode can be achieved by incorporating into the vector an appropriate tissue-specific promoter to drive expression of the antisense RNA or double stranded RNA in the required tissues. SERCA expression will be specifically down-regulated only in those tissues which express the antisense RNA or double stranded RNA. By way of example, the promoter region of the C. elegans sca-1 gene itself can be used to direct expression of an antisense RNA or double stranded RNA in all the cells and tissues of C. elegans which express endogenous SERCA. The C. elegans myo-2 promoter can be used to direct expression in the pharynx. The C. elegans myo-3 promoter can be used to direct expression in the body wall muscles. The use of antisense and double stranded RNA inhibition will be further understood with reference to the Examples included herein.

RNAi technology is well known in the C. elegans field as a tool for inhibiting expression of a specific target gene in C. elegans , as described by Fire et al . , Nature 391:801-811 (1998) and Timmins and Fire, Nature 395:854 (1998). The standard approach is based on injection of dsRNA directly into the worm. Alternative RNAi techniques which may be used to inhibit SERCA activity are described in the applicant's International patent application No. WO 00/01846. These techniques, which are based on delivery of dsRNA to C. elegans by feeding with an appropriate dsRNA or feeding with food organisms which express an appropriate dsRNA, may lead to a more stable RNAi phenotype than results from injection of dsRNA.

In a further embodiment, a pest SERCA-specific screen may be performed by using transgenic C. elegans expressing a pest SERCA protein which is resistant to a chemical inhibitor of SERCA activity, such as thapsigargin. The pest SERCA protein may be variant carrying a mutation in the thapsigargin binding site The mutation Phe259Val renders C. elegans SERCA resistant to inhibition with thapsigargin. Equivalent mutations may be introduced into transgenes encoding pest SERCA proteins using standard site-directed mutagenesis . An alignment of SERCA amino acid sequences, such as that shown in Figure 2, may be used to locate the amino acid residue in the pest SERCA protein which is equivalent to residue Phe 259 of C. elegans SERCA. Applying the SERCA inhibitor, for example thapsigargin, to transgenic C. elegans which express a resistant mutant pest SERCA will result in inhibition of the endogenous C. elegans SERCA only.

Thus, if the inhibitor is added to the screening assay in addition to the test compound, the screen will be specific for the pest SERCA.

The invention also encompasses an embodiment of the screening method in which the pest SERCA protein is specifically expressed in a tissue or cell type of the nematode which exhibits no or only minor background activity of the endogenous C. elegans SERCA protein. In this case it is not necessary to reduce/abolish activity of the endogenous nematode SERCA protein in order to screen selectively on the pest SERCA protein.

An example of a nematode tissue which exhibits little or no SERCA activity is the neurons. In a preferred embodiment the screen is performed using transgenic C. elegans in which expression of a pest SERCA protein is driven by a neuron-specific promoter. Examples of neuron-specific promoters which may be used in this embodiment of the invention are the unc- 119, ser-1, eat-18, acm-1, acm-3 and avr-14 promoters. Other suitable neuron-specific C. elegans promoters are known in the art.

The screening methods of the invention rely on detection of an indicator of SERCA activity in the presence or absence of a test compound. There are a number of different phenotypic, behavioural or biochemical indicators of SERCA activity in the nematode which can be used as the basis of the screening method. These include pharynx pumping efficiency, egg laying behaviour, mating behaviour, defecation behaviour, growth rate, movement behaviour, life/death of the nematode and intracellular Ca²⁺ concentration.

The inventors have observed that a reduction in SERCA activity in nematodes such as C. elegans results in various phenotypic and behavioural defects. Many of these defects can be used as basis of an assay to isolate compounds that alter the activity of SERCA, and also compounds which affect the activity of other components of the SERCA pathway, such as proteins involved in the calcium homeostasis of the cell. The main defects, and hence phenotypes, associated with reduced SERCA activity are related to muscle function e.g pharyngeal muscle, body wall muscle, vulva muscle, anal repressor muscle, and anal sphincter muscle, as illustrated by the RNAi experiments and thapsigargin inhibition experiments described in the accompanying examples. Screens based on the detection of phenotypic characteristics associated with reduced SERCA activity in these muscles can be used to identify compounds and genes that alter the activity of SERCA. In addition, other phenotypes, such as paleness, reduced growth, reduced progeny, protruding vulva and protruding rectum can be used to identify compounds and genes that alter the function of SERCA.

In one embodiment, the assay can be based on detection of pharynx pumping efficiency as an indicator of SERCA activity. If the starting nematode strain exhibits a near wild-type rate of pharynx pumping, then a decrease in the rate of pharynx pumping in the presence of a test compound can be used as an indicator of reduction of SERCA activity in the pharynx. In order to use pharynx pumping efficiency as an indicator of the activity of the pest SERCA protein, the pest SERCA protein must be expressed in at least the muscles of the pharynx. Activity of the endogenous nematode SERCA protein should also be abolished or substantially reduced in the pharynx muscles in order to confer specificity for the pest SERCA protein.

C. elegans feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and internalises them into the gut lumen. This process is performed by the muscles of the pharynx. The process of taking up of liquid and subsequently spitting it out, requiring contraction and relaxation of muscles, is called pharyngeal pumping or pharynx pumping.

Alterations in SERCA activity influence the pharyngeal pumping rate. In particular, inhibition of SERCA using thapsigargin causes a reduction in the rate of pharynx pumping. Measurement of the pumping rate of the C. elegans pharynx is hence a method to determine the activity of SERCA. Pharynx pumping efficiency can be conveniently measured by placing the nematodes in liquid containing a fluorescent marker molecule precursor, such as calcein-AM. Calcein-AM present in the medium is taken up by the nematodes and the AM moiety is cleaved off by the action of esterases present in the C. elegans gut, resulting in the production of the fluorescent molecule calcein. As the quantity of calcein-AM that is delivered in the gut is dependent on the pumping rate of the pharynx, and hence of the activity of SERCA, calcein fluorescence measured in the gut is a quantitative and qualitative measurement of the SERCA activity. It would be readily apparent to one skilled in the art that other types of marker molecule precursor which are cleavable by an enzyme present in the gut of C. elegans to generate a detectable marker molecule could be used instead of calcein-AM with equivalent effect. In a further embodiment, the assay can be based on detection of changes in the egg laying behaviour of the nematode or on detecting changes in the amount of progeny produced by the nematode as indicators of SERCA activity. For this embodiment, the nematode should express the pest SERCA protein in at least the vulva muscles. Activity of the endogenous nematode SERCA protein should be abolished or substantially reduced in the vulva muscles in order to confer specificity for the pest SERCA protein.

Defects associated with reduced SERCA activity in the vulva muscles include defects in the production and laying of eggs and hence a reduction in the number of progeny produced. Typically, nematodes with reduced SERCA expression in the vulva are not able to lay their eggs. The eggs thus hatch inside the mother, which then dies. These mothers are easy to recognize under the dissection microscope. As a consequence of the egg laying defect, less progeny are produced and hence the culture as a whole grows much more slowly. Defects associated with reduced SERCA activity have also been observed in the gonad, including the sheath cells and the spermatheca. These defects also result in reduced egg formation and hence a reduced egg laying phenotype.

One convenient way in which the egg production - I I

and egg laying behaviour of the nematodes can be monitored is by counting the number of resultant offspring produced. A variety of different techniques can be used for this purpose. For example, the offspring can be measured directly using the growth rate assay and/or the movement assay described below. Alternatively, specific antibodies and fluorescent antibodies can be used to detect the offspring. Any specific antibody that only recognizes eggs, or Ll or L2 or L3 or L4 stage nematodes, will only recognize offspring. By way of example, an antibody that recognizes an antigen on the surface of C. elegans Ll larvae has been described by Hemmer et al . , (1991) J Cell Biol , 115(5): 1237-47. Finally, the number of eggs or offspring in each well can be counted directly using a FANS device. The FANS device is a worm dispenser apparatus' having properties analogous to flow cytometers such as fluorescence activated cell scanning and sorting devices (FACS) and is commercially available from Union Biometrica, Inc,

Somerville, MA, USA. The FANS device, also designated a nematode flow meter, can be the nematode FACS analogue, described as fluorescence activated nematode scanning and sorting device (FANS) . The FANS device enables the measurement of nematode properties, such as size, optical density, fluorescence, and luminescence and the sorting of nematodes based on these properties.

In a still further embodiment, the assay can be based on detection of a change in the defecation behaviour of the nematode as an indicator of SERCA activity. This embodiment is particularly suitable for use when the nematode expresses the pest SERCA protein in the anal sphincter or the anal repressor. In this case, activity of the endogenous nematode

SERCA protein should be abolished/reduced in the anal sphincter or anal repressor in order to confer specificity for the pest SERCA protein.

A reduction in the SERCA activity in the anal sphincter and/or the anal repressor, for example following treatment with thapsigargin, results in nematodes which are constipated and also in nematodes with a protruding rectum. Changes in the defecation rate of the nematodes can therefore also serve as an indicator of SERCA activity.

Defecation rate can be measured using an assay similar to that described above for the measurement of pharynx pumping efficiency, but using a marker molecule which is sensitive to pH. A suitable marker is the fluorescent marker BCECF. This marker molecule can be loaded into the C. elegans gut in the form of the precursor BCECF-AM which itself is not fluorescent. If BCECF-AM is added to nematodes growing in liquid medium the nematodes will take up the compound which is then cleaved by the esterases present in the C. elegans gut to release BCECF. BCECF fluorescence is sensitive to pH and under the relatively low pH conditions in the gut of C. elegans (pH<6) the compound exhibits no or very low fluorescence. As a result of the defecation process the BCECF is expelled into the medium which has a higher pH than the C. elegans gut and the BCECF is therefore fluorescent. The level of BCECF fluorescence in the medium (measured using a fluorimeter on settings Ex/Em=485/550) is therefore an indicator of the rate of defecation of the nematodes. Defecation can also be measured using a method based on the luminescent features of the chelation of terbium by aspirin. The method requires two preloading steps, first the wells of a multi-well plate are pre-loaded with aspirin (prior to the addition of the nematodes) and second, bacteria or other nematode food source particles are pre-loaded with terbium using standard techniques known in the art. C. elegans are then placed.in the wells pre-loaded with aspirin and are fed with the bacteria pre-loaded with terbium. The terbium present in the pre-loaded bacteria added to the wells will result in a low level of background luminescence. When the bacteria are eaten by the nematodes the bacterial contents will be digested but the terbium will be defecated back into the medium. The free terbium will then be chelated by the aspirin which was pre-loaded into the wells resulting in measurable luminescence. The luminescence thus observed is therefore an indicator of nematode defecation. In a still further embodiment, the assay may be based on the use of growth rate as an indicator of SERCA activity.

It has been observed that a reduction in SERCA activity, for example using inhibition by thapsigargin or double stranded RNA inhibition, results in a reduction in the growth rate of a C. elegans culture. Growth rate of the culture as a whole is reduced because the nematodes produce fewer progeny and also because the few progeny that are produced show poor/delayed growth. Cultures of nematodes which produce many healthy progeny grow faster than cultures of nematodes with few and/or sick progeny. Hence measurement of the growth rate of a culture of C. elegans is in indication of the activity of SERCA in the individual nematodes of the culture.

Growth rate can be monitored by measuring the number of eggs or the number offspring present in the culture, by measuring the total fluorescence in the culture (this can be autofluorescence, or fluorescence caused by a transgene encoding a flourescent or luminescent protein) , but can also be measured using the movement screen described below. Alternatively, the growth rate of a culture of C. elegans can also be assayed by measuring the turbidity of the culture. In order to perform this turbidity assay' the nematodes are grown in liquid culture in the presence of E. coli or other suitable bacterial food source. As the culture of nematodes grows the food source bacteria will be consumed. The greater the number of nematodes in the culture, the more food source bacteria will be digested. Hence, measurement of the turbidity or optical density of the liquid culture will provide an indirect indication of the number of nematodes in the culture. By taking sequential measurements over a period of time it is possible to monitor the growth rate of the whole C. elegans culture.

As an alternative to the above-described methods, the growth rate and amount of progeny can be measured on a plate. Slow growing nematodes, nematodes with vulva defects and nematodes with gonad defects will produce less progeny within a certain time compared to nematodes which do not have these defects. Preferentially, the amount of offspring produced is scored on day five and on day eight. In experiments where the amount of offspring is reduced very drastically due to severe defects in the vulva, gonad or growth rate reduction, the offspring can also be scored at later time intervals.

In a still further embodiment, the assay may be based on detecting changes in the movement behaviour of C. elegans as an indicator of SERCA activity. This embodiment is particularly suitable for use when the nematodes express the pest SERCA protein in at least the body wall muscles. At the same time, activity of the endogenous nematode SERCA protein should also be abolished/reduced in at least the body wall muscles in order that the assay is specific for the pest SERCA protein .

SERCA is widely expressed in the muscles of C. elegans, including the muscles of the body wall. A reduction of SERCA activity in the body wall muscles gives rise to nematodes with movement defects. Thus, movement defects can be used as the basis of an assay in which the nematodes are contacted with a compound under test and any changes in the movement behaviour of the nematodes are observed as an indication of SERCA activity. Compounds which cause defective movement behaviour are scored as compounds capable of down-regulating the activity of SERCA.

Changes in the movement behaviour of the nematodes can obviously be detected by visual inspection, but as an alternative a number of non- visual approaches for analysing the movement behaviour of nematodes have been developed which can be performed in a multi-well plate format and are therefore suitable for use in high-throughput screening. Nematode worms that are placed in liquid culture will move in such a way that they maintain a more or less even (or homogeneous) distribution throughout the culture. Nematode worms that are defective in movement will precipitate to the bottom in liquid culture. Due to this characteristic of nematode worms as result of their movement phenotype, it is possible to monitor and detect the difference between nematodes that move and nematodes that do not move. Advanced multi-well plate readers are able to detect sub-regions of the wells of multi-well plates. By using these plate readers it is possible to take measurements in selected areas of the surface of the wells of the multi-well plates. If the area of measurement is centralized, so that only the middle of the well is measured, a difference in nematode autofluorescence (fluorescence which occurs in the absence of any external marker molecule) can be observed in the wells containing a liquid culture of nematodes that move normally as compared to wells containing a liquid culture of nematodes that are defective for movement. For the wells containing the nematodes that move normally, a low level of autofluorescence will be observed, whilst a high level of autofluorescence can be observed in the wells that contain the nematodes that are defective in movement. In an adaptation of the movement assay, autofluorescence measurements can be taken in two areas of the surface of the well, one measurement in the centre of the well, and on measurement on the edge of the well. Comparing the two measurements gives analogous results as in the case if only the centre of the well is measured but the additional measurement of the edge of the well results in an extra control and somewhat more distinct results.

As an alternative to the above-described embodiments of the assay which are all based on the observation of changes in phenotypic and/or behavioural characteristics of the nematode as an indicator of SERCA activity, the assay may be based on detection of intracellular Ca²⁺ levels as an indicator of SERCA activity in a given cell type or tissue. This may be accomplished using a marker molecule which is sensitive to changes in intracellular Ca²⁺ such as, for example, apoaequorin.

Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea ictoria. Recombinant apoaequorin, which is luminescent in the presence of calcium but not in the absence of calcium, is most useful in determining intracellular calcium concentrations and even calcium concentrations in sub- cellular compartments. Expression vectors suitable for expressing recombinant apoaequorin and, in addition, vectors expressing apoaequorin proteins which are targeted to different sub-cellular compartments, for example the nucleus, the mitochondria or the endoplasmic reticulum are available commercially (e.g. from Molecular probes, Eugene, OR, USA) . As SERCA is a endoplasmic reticulum-localized calcium pump, an apoaequorin that is targeted to the endoplasmic reticulum (hereinafter referred to as erAEQ) is particularly useful for developing assays for SERCA activity. The vector erAEQ/pcDNAI (Molecular Probes) contains an Igγ2b heavy chain gene from mouse, an HA1 epitope and a recombinant apoaequorin in fusion. The mouse gene targets the apoaequorin to the endoplasmic reticulum, and the apoaequorin is mutated to make it less sensitive to calcium, as the concentrations of this ion are relatively high in the endoplasmic reticulum. Although apoaequorin is the calcium sensor of choice, it would be apparent to persons skilled in the art that any other calcium sensor localized in the endoplasmic reticulum could be used with equivalent effect .

Plasmid expression vectors which drive expression of the ER-localized apoaequorin in C. elegans can be easily constructed by cloning nucleic acid encoding erAEQ downstream of a promoter capable of directing gene expression in one or more tissues or cell types of C. elegans, such that the promoter and the erAEQ- encoding sequence are operably linked. In a typical cloning procedure, the apoaequorin gene in fusion with the signals needed to locate the resulting protein to the endoplasmic reticulum was isolated from erAEQ/pcDNAI by EcoRI digestion and cloned into pBlue2SK. The erAEQ was then isolated as an EcoRI/Acc65I fragment by partial digestion and cloned in the vector pGK13 digested with the same enzymes. pGK13 is a plasmid vector containing a 2915bp fragment of the upstream region of the C. elegans sca-1 gene. Suitable promoters which may be included into an expression vector to drive erAEQ expression include the pharynx-specific promoter myo-2, the C. elegans sca-1 promoter which directs expression in a wide range of muscle tissues and the body wall muscle- specific promoter myo-3. The vectors can then be used to construct transgenic C. elegans according to the standard protocols known to those of ordinary skill in the art. Expression of erAEQ allows for the determination of the calcium levels in the endoplasmic reticulum of various C. elegans cells and tissues, using the protocols of the manufacturer of erAEQ, or minor modifications thereof. Alterations in SERCA activity influence the concentration of calcium in the endoplasmic reticulum as SERCA functions as an endoplasmic reticulum calcium pump. Hence the apoaequorin luminescence measured in the assay is directly related to SERCA activity.

To perform a compound screen using one of the aforementioned indicators of SERCA activity nematodes are exposed to a variety of test compounds and compounds are selected which induce a change in the chosen indicator of SERCA activity. In a typical compound screen a plurality of tests may be run in parallel containing different concentrations of the test compound. For comparison purposes a negative control (zero concentration of test compound) may be included. Automated measuring allows the assay to be performed in mid-to-high throughput format. The precise concentration of the candidate compound to be tested in the screening method may vary according to the nature of the compound and such factors as solubility etc. It is advantageous to test a range of concentrations of the candidate compound. Concentrations in the range of about 5 μM to about 2000 μM are generally observed to be suitable. In general it is desirable to select a concentration which produces a detectable change in the worm as compared to the appropriate negative control (i.e. worms not exposed to the compound) , ignoring non- specific effects. It is to be noted that the chosen concentration need not necessarily an amount which would be considered a pesticidal' dose.

It is not strictly essential to screen on a pest- derived SERCA protein in order to identify SERCA inhibitors having the potential to kill pests . Screens can also be performed using nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein. Compounds which inhibit the endogenous nematode SERCA protein may also inhibit SERCA proteins from pest species.

Therefore, in a second aspect the invention provides a further nematode-based screening method which does not require the use of a pest-derived SERCA protein. This method comprises steps of: providing microscopic nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the nematodes in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has the potential to kill pests.

This method may also be used to identify compounds which have pesticidal activity because they directly or indirectly affect the activity of the SERCA protein. Therefore, according to this aspect of the invention there is also provided a method of identifying compounds capable of down-regulating the activity of a sarco/endoplas ic reticulum calcium ATPase, which method comprises: providing microscopic nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the nematodes in the presence or absence of test compounds ; and thereby identifying compounds capable of down- regulating the activity of SERCA.

These screening methods are again most preferably carried out using C. elegans, although it will be appreciated that the methods could be carried out using other microscopic nematode species.

An example of a C. elegans strain which exhibits wild-type SERCA activity is the N2 strain, available from the C. elegans Genetic Center (CGC) at the University of Minnesota, St Paul, Minnesota, USA. In a preferred embodiment the screening method may be carried out using the N2 strain. The N2 strain has been particularly well characterised in the literature with respect to properties such as pharynx pumping rate, growth rate and egg laying capacity (see Methods in Cell Biology, Volume 48, Caenorhabditis elegans: Modern biological analysis of an organism, ed. by Henry F. Epstein and Diane C. Shakes, 1995 Academic Press; The nematode Caenorhabditis elegans, ed. by William Wood and the community of C. elegans researchers., 1988, Cold Spring Harbor Laboratory Press; C. elegans II, ed. by Donald L. Riddle, Thomas Blumenthal, Barbara J. Meyer and James R. Priess, 1997, Cold Spring Harbor Laboratory Press.). The screening methods may also be carried out using a C. elegans strain other than N2 which exhibits similar SERCA activity to N2. This may be a mutant strain or a transgenic strain.

A range of C. elegans mutants may be obtained from the C. elegans mutant collection at the C. elegans Genetic Center, University of Minnesota.

Alternatively, specific mutants may be generated by standard methods known in the art. Suitable methods are described by J. Sutton and J. Hodgkin in "The Nematode Caenorhabditis elegans", Ed. by William B. Wood and the Community of C. elegans Researchers CSHL, 1988 594-595; Zwaal et al, "Target - Selected Gene Inactivation in Caenorhabditis elegans by using a Frozen Transposon Insertion Mutant Bank" 1993, Proc. Natl. Acad. Sci. USA 90 pp 7431 -7435. A population of nematodes can be subjected to random mutagenesis by using EMS, TMP-UV or radiation (Methods in Cell Biology, Vol 48, ibid) . Several selection rounds of PCR may then be performed to select a mutant with a deletion in a desired gene. In a preferred embodiment, the screening methods may be carried out using a constitutive pharynx pumping strain of C. elegans .

Phenotypic, behavioural or biochemical indicators of the activity of the endogenous nematode SERCA protein which can be used as the basis of the screening method include pharynx pumping efficiency, egg laying behaviour, mating behaviour, defecation behaviour, growth rate, movement behaviour, life/death of the nematode and intracellular Ca²⁺ concentration. The methods described above for the measurement of these characteristics are equally applicable to this second aspect of the invention.

In a third aspect the invention provides a method of identifying compounds having pesticidal activity which is carried out in cultured cells as opposed to whole organisms. This method comprises steps of: providing cultured cells expressing a SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the cells in the presence¹ or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has pesticidal activity.

According to this aspect of the invention there is also provided a method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic ^' reticulum calcium ATPase, which method comprises: providing cultured cells expressing a SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the cells in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.

These screening methods may be collectively referred to hereinafter as the "cell culture" assays. In one embodiment of the cell culture assays, the cultured cells may be cells derived from a pest species which express the endogenous pest SERCA protein. This may be a cultured primary cell line or a continuous, transformed cell line. The cell line will be capable of growth in culture, preferably monolayer or suspension culture. Various examples of suitable cell lines derived from pest species are known in the art. Many of these are derived from insect species, for example Heliothis virescens (Lynn, Development and characterisation of insect cell lines, Cytotechnology, 20: 3-11, 1996). Methods of culturing insect cell lines are well known in the art and described, for example, by Maramorosch and Mclntosh, Arthropod cell culture systems, 1994, ISBN: 0849376424, and Lynn & Shapiro, New cell lines from Heliothis virescens: Characterization and susceptibility to baculoviruses, 1998, 72: 276-280.

The use of cell lines derived from a pest species allows screening on the endogenous pest SERCA protein expressed in the cell line. In further embodiments, the cell culture assays may be based on the use of cultured cells which have been engineered to express a pest SERCA protein. In particular, the assays may be carried out using eukaryotic host cells containing an expression vector comprising nucleic acid encoding the pest SERCA protein.

Suitable expression vectors will include a sequence of deoxynucleotides encoding the pest SERCA protein, including a start codon (usually AUG) and a termination codon for detachment of the ribosome, and also regulatory elements required for expression of the encoded SERCA protein in a eukaryotic host cell. Such regulatory elements may include a promoter region, preferably one which is recognised by RNA polymerase II, optionally one or more additional transcriptional regulatory elements (e.g. enhancer elements) and also a terminator sequence and downstream polyadenylation signal. The vector may also possess an origin of replication allowing replication in prokaryotic cells and one or more selectable markers, such as a gene for antibiotic resistance. A wide range of suitable expression vectors into which nucleic acid encoding the pest SERCA protein may be inserted are available commercially. The expression vector will preferably be a plasmid vector, although virus and phage-based vectors designed for protein expression in eukaryotic host cells may also be used. The eukaryotic host cells may be a cell line capable of growing in monolayer or suspension culture and will preferably not express high levels of an endogenous SERCA protein (i.e. the SERCA protein encoded in the genome of the host cell) . Fibroblast cell lines or epithelial cell lines are most preferred. Suitable cell lines include COS1, BHK21, L929, PC12, CV1, SWISS3T3, HT144, IMR32, HEPG2, MDCK, MCF7, HEK293, Hela, A549, SW48 and G361. However, this list is not exhaustive.

Methods of transfecting expression vectors into eukaryotic host cells are well known in the art (see ^λCurrent Protocols in Molecular Biology' , Ed Ausubel et al., John Wiley & Sons, Inc) . Most preferably the host cell will be stably or permanently transfected with the expression vector such that it is retained through many cell divisions. However, it is also within the scope of the invention to use cells which are transiently transfected with the expression vector.

As with the nematode-based screening methods, the cell culture assays rely on detection of an indicator of SERCA activity in the presence or absence of a test compound. Suitable indicators of SERCA activity in cultured cells include intracellular Ca²⁺ levels, in particular Ca²⁺ levels in the endoplasmic reticulum, and cell death or apoptosis.

Suitable methods for the measurement of intracellular Ca²⁺ levels in cultured cells are based on fluorescent calcium indicators excited by ultraviolet light, such as fura-2, indo-2, quin-2 or visible light such as fluo-3 and rhod-2 that are available from Molecular Probes, Eugene, USA. The acetoxymethyl esters can passively diffuse across cell membranes to avoid the use of invasive loading techniques. Once inside the cells, these esters are cleaved by intracellular esterases to yield cell-impermeant fluorescent calcium indicators.

These indicators can be used to perform screens in a high throughput set-up. The test compound is usually added directly prior to the fluorescent indicator, but the screen, can also be performed by pre-incubating the cells with the test compound for an incubation time of, for example, 1 min, 5 min, 10 min or 30 min. Fluorescence can be measured directly after the addition of the indicator, but it is preferred to take fluorescence measurements over a period of time, for example every 10 minutes for one hour. Fluorescence data from typical experiments show that measurements after 10 to 15 minutes are generally sufficient to determine the calcium levels in the cell.

Quantitative determination of Ca²⁺ levels in the endoplasmic reticulum of cultured eukaryotic cells can be carried out using the bioluminescent calcium indicator aequorin, or the recombinant form apoaequorin, available from Molecular Probes, Eugene, OR, USA. To target aequorin to specific organelles such as the cytoplasm or endoplasmic reticulum, cultured cell lines may be transiently or stably transfected with an aequorin expression vector containing the aequorin structural gene. Once cells have been transfected with aequorin, they are incubated in a medium containing the cell-permeant coelenterazine or one of its analogs that are available from Molecular Probes, Eugene, USA in order to reconstitute the aequorin complex. After formation of the active aequorin complex, intracellular Ca²⁺ levels are measured by assaying cells for light production using a luminometer.

Screens based on the use of aequorin may be performed in multiwell plates. The test compound can be added prior to the addition of the aequorin substrate, but can also be added in time intervals before or after the addition of the substrate. Luminescence can be measured directly after the addition of the substrate, but preferentially luminescence measurements are performed over time ranges every 10 minutes for one hour after addition of the substrate. Luminescence data from typical experiments show that measurements after 10 to 15 minutes are sufficient to determine Ca²⁺ levels in the endoplasmic reticulum. Yet another method for measuring intracellular Ca²⁺ levels is by use of green fluorescent based calcium indicator "cameleon". This method is described by Tsien et al, WO98/40477. The cameleon calcium indicator can be transiently or stably expressed in mammalian, plant, insect or other pest cell lines and fluorescence ratio imaging of cameleon allows time-dependent measurements of intracellular calcium levels (Allen GJ et al . , 1999. Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J. , 19:735-47).

Cameleon fluorescence can be measured directly after the addition of the test compound or fluorescence measurements can be taken at various time intervals after addition of the test compound. The cell culture assays may also be based on the use of cell death or apoptosis as an indicator of SERCA activity in the cell. Methods to determine cell death are well described by Barile Frank in Introduction to in vitro cytotoxicology: mechanisms and methods .1994. ISBN 0849386594. Most of the methods described therein can be performed using standard kits which are commercially available, for example from Molecular Probes or Boehringer Mannheim. Inhibition of SERCA activity leads to apoptosis which can easily measured using specific apoptotic labels as has been described by Smits et al. in WO 99/64586. A variation on the cell culture assay may be based on measurement of calcium levels in isolated microsomes rather than intact cultured cells. Techniques for isolation of microsomes are known to those skilled in the art. Isolated microsomes are placed in a solution containing radioactively labelled calcium and ATP. After 10 minutes incubation the amount of radioactivity inside the microsomes is measured in a beta-counter. This approach has been described by Dode, L. et al . , 1998. Structure of the human sarco/endoplasmic reticulum Ca²⁺-ATPase 3 gene. Promoter analysis and alternative splicing of the SERCA3 pre-mRNA. J. Biol. Chem. 273: 13982-13994. Once again the test compound can be added at several time intervals after the isolation of the microsomes, and prior or after the addition of the radioactive calcium.

The cell culture assays will preferably be carried out in multi-well plates of the type well known in the art for use in mid-to-high-throughput screening. In the case of cells engineered to express the pest SERCA protein, non-transfected host cells may also be exposed to the test compounds in order to control for expression of the endogenous host SERCA protein, i.e. to determine the selectivity of the assay for the pest SERCA protein. The non-transfected control cells may also be used to assess general toxicity of the test compounds.

The precise concentration of the candidate compound to be tested in the screening method may vary according to the nature of the compound and such factors as solubility etc. An initial test may be performed using a single concentration of 10 μM. Interesting compounds may then be re-tested to establish a dose-response curve, for example using concentrations of 300 μM, 10.0 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.01 μM and 0.003 μM and a zero concentration negative control. In general, a dose- response curve with concentrations between 300 μM and 0.001 μM is sufficient.

The above-described screening methods of the invention, both the nematode-based assays and the cell culture assays, may all be used to identify compounds which have pesticidal activity because of their ability to down-regulate the activity of SERCA proteins, particularly SERCA proteins derived from pest species. Included within the category of ^compounds which down-regulate SERCA activity' may be compounds which act directly on the SERCA protein, including SERCA inhibitors and antagonists. The screens may also identify compounds which act indirectly to down-regulate SERCA activity, for example by affecting regulation of SERCA activity or expression of the SERCA protein. In addition, the screens may also identify compounds that modulate the activity of other proteins in the SERCA pathway, such as proteins involved in the calcium homeostasis of the cell.

There is no limitation on the types of candidate compounds to be tested in the screening methods of the invention. Test compounds may include compounds having a known pharmacological or biochemical activity, compounds having no such identified activity and completely new molecules or libraries of molecules such as might be generated by combinatorial chemistry. Compounds which are DNA, RNA, PNA, polypeptides or proteins are not excluded. Compounds identified as having pesticidal activity using the nematode-based assay, particularly the assays which do not involve a target pest SERCA protein, may be re-tested in a cell culture assay, for example to assess toxicity of the compound or to assess the specificity of the compound for a pest SERCA protein.

The invention further provides compounds identified as having the potential to kill pests using the methods of the invention. Such compounds are potential pesticides or can be considered as lead compounds for the development of novel pesticides, including insecticides, herbicides, nematocides and rodenticides . Furthermore, compounds identified as having pesticidal activity against parasitic pest species using the screening methods described herein may have potential utility as anti-parasitic agents or as lead compounds in the development of anti-parasitic agents useful in the treatment of parasitic infections in humans and animals.

The invention will be further understood with reference to the following experimental Examples, together with the accompanying Figures in which:

Figure 1 is an alignment of SERCA cDNA sequences from plant species, indicating consensus sequences and primer locations.

Figure 2 is a general alignment of SERCA cDNA sequences, indicating consensus sequences and primer locations.

Figure 3 shows the complete nucleotide sequence of a plasmid construct comprising the Arabidopsis SERCA cDNA in the vector pcDNA3.

Figure 4 shows the complete nucleotide sequence of a plasmid construct comprising the Heliothis SERCA cDNA in the vector pcDNA3.

Figure 5 shows the complete nucleotide sequence of a plasmid construct comprising the Heliothis

SERCA cDNA cloned in the vector pDW2600 (containing the sca-1 promoter) .

Figure 6 shows the complete nucleotide sequence of a plasmid construct comprising the Arabidopsis SERCA cDNA cloned in the vector pDW2600 (containing the sca-1 promoter) .

Figure 7 shows the complete nucleotide sequence of the plasmid pDW2700.

Figure 8 shows the complete nucleotide sequence of the plasmid pDW2800.

Figure 9 shows the complete nucleotide sequence of the plasmid pDW2400.

Figure 10 shows the complete nucleotide sequence of the plasmid pDW2422.

Figure 11 shows the complete nucleotide sequence of the plasmid pDW2721, comprising DNA encoding GFP cloned into pDW2700.

Figure 12 illustrates the nucleotide sequence of the genomic fragment of C. elegans SERCA bounded by primers SERCA P4 and SERCA P8. Exon IV and exon V are shown in capitals, intron IV in lower case. The fragment deleted in okl90 is underlined.

Figure 13 shows the nucleic acid sequence of a 732bp EcoRI-Hindll fragment of C. elegans SERCA exon 5. This fragment was cloned into pGEM3 for use in RNA inhibition experiments. Figure 14 shows the nucleic acid sequence of a 11207bp Spel-Mlul fragment of cosmid K11D9. This fragment contains the complete C. elegans SERCA gene with 5631bp of upstream sequence, the entire coding region and 1088bp of downstream sequence. The fragment was cloned into pUCl8 to give plasmid pGKJ .

Figure 15 shows the nucleic acid sequence of a 5026 bp fragment of the upstream region of C. elegans SERCA, up to and including A of the initiating ATG.

Figure 16 shows the nucleic acid sequence of a 2915bp fragment of the upstream region of C. elegans SERCA, as found in plasmid pGK13.

Figure 17 shows the nucleic acid sequence of a 6612bp fragment of the C. elegans SERCA gene containing 5637bp of upstream sequence and ending in exon 4.

Figure 18 shows the nucleic acid sequence of the long isoform of the C. elegans SERCA cDNA.

Figure 19 shows the nucleic acid sequence of the C. elegans myo-2 promoter.

Figure 20 shows the nucleic acid sequence of the C. elegans myo-3 promoter.

Figure 21 shows the nucleic acid sequence of the C. elegans vulval muscle enhancer. This is an enhancer element from ceh-24 that directs gene expression in the vulval muscles (Harfe and Fire, 1998, Developmental 125: 421-429) Figure 22 shows a dose-response curve for thapsigargin produced using a liquid culture assay.

Figure 23 shows a dose response curve for thapsigargin produced using a plate assay.

Examples

General Methodology

Molecular biology work, such as cloning, PCR etc may be performed as described by Sambrook et al. Molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press or Ausubel et al . Current

Protocols in Molecular Biology, John Wiley & Sons, Inc or using minor modifications of the methods described therein.

Manipulations of C. elegans worms may be performed using techniques described in Methods in

Cell Biology, vol 84; Caenorhabditis elegans: modern biological analysis of an organism, ed. Epstein and Shakes, Academic Press, 1995, or using minor modifications of the methods described therein.

Example 2 Cloning and Expression:

Vectors pDW2700 general cloning vector containing C. elegans myo-2 promoter (Figure 7) .

pDW2800 general cloning vector containing C. elegans myo-3 promoter (Figure 8) .

pDW2400 general cloning vector containing C. elegans egl -15 promoter (Figure 9) . pDW2422 general cloning vector containing C. elegans ceh-24 promoter (Figure 10) .

pDW2721 cloning vector comprising DNA encoding GFP cloned into in pDW2700.

Cloning of pest SERCA cDNAs

A number of pest SERCA cDNA sequences are available in databases such as GenBank. Further sequences can be cloned using standard PCR technology. Pest SERCA cDNAs can be cloned into standard expression vectors to enable expression in C. elegans or in cultured mammalian cells. By way of example, the complete nucleotide sequences of plasmids that enable the expression of Heliothis insect SERCA and

Arabidopsis plant SERCA in C. elegans are shown in the accompanying Figures . These plasmids contain SERCA- encoding DNA cloned under the control of the C. elegans SERCA (sca-2) promoter. The complete nucleotide sequences of plasmid constructs comprising the

Heliothis SERCA cDNA and the Arabidopsis SERCA cDNA in the vector pcDNA3 are also shown.

Primers for cloning Arabidopsis and Heliothis SERCA in pcDNA3:

Arabidopsis SERCA

Forward primer: cgatggatccatggaagacgcctacgccag Reverse primer: CGATGGGCCCCTACTTGTCACGCCGGTCC

Heliothis SERCA

Forward primer: cgatggatccatggaggacgctcactcgaaatc Reverse primer : CGTAGGGCCCTTACAGCTTCCACGTCGGCTG Strategy for cloning novel pest SERCA cDNAs

1. Assemble multiple alignment of known pest SERCA protein sequences with ClustalW,

2. Make Blocks using program accessible at http: //blocks . fhcrc.org/blockmkr/,

3. Design primers using CODEHOP (Rose, et al. (NAR 26: 1628-1635),

4. Select primers from conserved regions,

5. PCR on pest cDNA using appropriate primer combinations ,

6. Clone PCR fragments into appropriate cloning vector,

7. Isolate full length cDNA sequence, for example using 3' or 5 ' RACE or by hybridisation techniques, e.g. cDNA library screening, using labelled cDNA fragments as probes.

Construction of chimeric SERCA proteins

The introduction of pest SERCA into C. elegans, the latter being a SERCA mutant such as okl90 or a wild-type strain where the endogenous SERCA is inhibited, for example by RNAi technology, will result in rescue of the mutant phenotypes, but maybe not to the full extent. This could be due, for example, to different kinetic properties of the C. elegans and pest SERCA proteins. Using chimeric fusion proteins will overcome this problem. A fusion protein may be constructed that has sufficient properties of the C. elegans SERCA for rescue of the mutant phenotype, and has those pest SERCA properties sufficient in a screen to select for compounds that alter the pest SERCA activity.

At least four types of fusion proteins are contemplated: 1) A fusion protein harboring the - terminal end of the C. elegans SERCA and the C-terminal part of a pest SERCA .

2) A fusion protein harboring the N-terminal part of a pest SERCA and the C-terminal part of the C. elegans SERCA. 3) A fusion protein harboring the C- and - terminal part of the C. elegans SERCA and an internal part of a pest SERCA.

4) A fusion protein harboring the C- and - terminal part of a pest SERCA and an internal part of the C. elegans SERCA.

Such fusion proteins can easily be constructed using standard molecular biology techniques.

Example 3 RNAi:

General strategy

Although primary RNAi experiments indicate that the level of expression the SERCA protein needs to be fine-tuned for the survival of the C. elegans nematode, strains in which the level of SERCA activity is reduced, in particular strains in which SERCA activity is reduced in a single tissue, are probably still viable. Due to the sensitivity of C. elegans to the level of SERCA activity this could result in a recognisable phenotype, such as reduced pharyngeal pumping, vulva muscle defects, and hence egg laying defects, anal repressor and anal sphincter defects, and hence defecation defects, and body wall muscle defects, and hence movement defects. The phenotypic defects in such strains can be complemented by expression of a pest SERCA protein in the appropriate tissues in order to restore SERCA function to substantially wild-type.

The expression levels of SERCA in C. elegans can be specifically reduced by using antisense technology or double stranded RNA inhibition. The use of antisense technology to specifically reduce expression of a given protein is well known. For the expression of antisense RNA in the worm, the non-coding strand of a fragment of the sca-2 gene can be expressed under the control of the sca-1 , myo-2 or myo-3 promoter or any other promoter. The expression of the antisense SERCA RNA will result in the inhibition of expression of SERCA.

Antisense technology can be used to control gene expression through triple-helix formation of antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion or the mature protein sequence, which encodes for the SERCA protein, is used to design an antisense RNA oligonucleotide of from 10 to 50 base pairs in length. The antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule into the protein (Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple-helix - see Lee et al . Nucl. Acids Res., 6:3073 (1979); Cooney et al . , Science, 241:456 (1988); and Dervan et al . , Science,

251: 1360 (1991), thereby preventing transcription and the production of the protein.

In order to perform an antisense experiment in C. elegans, an EcoRI-Hind III fragment of SERCA exon 5 was cloned antisense under the control of the myo-2 promoter, the myo-3 promoter, the SERCA promoter or the ceh-24 enhancer and injected into C. elegans. These vectors result in the expression of an antisense SERCA RNA, and hence in inhibition of SERCA activity. As an alternative to the antisense approach, the expression of a given gene in a cell can also be specifically reduced by introducing into the cell double stranded RNA corresponding to a region of the transcript transcribed from the gene. Double stranded RNA can be prepared by cloning an appropriate fragment into a plasmid vector containing opposable promoters. A suitable example is the pGEM® series of vectors from Promega Corporation, Madison, WI, USA, which contain opposable promoters separated by a multiple cloning site. When the plasmid vector is transformed or transfected into a host cell or organism which expresses the appropriate polymerases, RNA will be transcribed from each of the promoters. As the vector contains two promoters oriented in the opposite sense, complementary sense and antisense transcripts will be transcribed which will combine to form double stranded RNA. The injection of double stranded RNA in C. elegans has previously been described (Fire et al, Potent and Specific Genetic Interference by Double- Stranded RNA in C. elegans 1998, Nature 391, 860-811) .

Inhibition of expression of C. elegans SERCA (sca-1) using RNAi.

732 bp EcoRI-Hindlll fragment from C. elegans SERCA exon 5 (SEQ ID NO: 1) was PCR amplified and cloned into the vector pGEM3 (PROMEGA corporation,

Madison, WI, USA) . RNA was in vitro transcribed from both strands using standard procedures. The generated double stranded RNA was injected into C. elegans (see Fire at al., 1998, Nature 391:806-811). This resulted in the following phenotypes: 50% of the progeny of the injected animals were embryonic lethal, while the other 50% were early larval lethal. This indicates that SERCA function is vital for C. elegans . In conclusion, inhibition of the expression of SERCA in all tissues results in embryonic or early larval lethality of the nematode. Inhibition of SERCA using RNAi feeding technology

Improved RNAi methods which lead to more stable RNAi phenotypes exist and are described, for example in International patent application No. WO 00/01846. More particularly, an RNAi technology has been developed and tested in which dsRNA can be delivered by feeding the nematode dsRNA or by feeding nematodes with DNA. pGN4 was constructed by cloning the Hindlll - EcoRI fragment of SERCA cloned in vector pGNl using these same restriction sites. This is the same fragment as was used for in vi tro transcription and dsRNA injection, described above.

HT115(DE3) bacteria (Fire A, Carnegie Institution, Baltimore, MD) were transfected with pGN4 (and controls with pGNl) and seeded on plates containing IPTG and ampicillin resulting in a high expression of dsRNA by the bacteria. N2 and nuc-1 (el392) adult nematodes were put on these plates and allowed to lay eggs and the progeny was followed over time. The progeny mostly looked healthy during the larval stages, but the adults (and some of the L4) had a starved appearance (nuc-1 more pronounced then N2) . Pharynx pumping was irregular and slower then normal, and the growth rate was somewhat reduced. This example indicates that a stable RNAi phenotype useful in assay development and compound screening can be developed using feeding. As described in co-pending application No. WO 00/01846, other possibilities and variants can be used to create a C. elegans SERCA RNAi phenotype.

The use of RNAi technology allows the production of C. elegans strains in which the activity of the endogenous SERCA protein is abolished/substantially reduced without the construction of a C. elegans SERCA mutant. E. coli HT115 has the following characteristics which make it a useful host cell for high level expression of dsRNA: HT115 (DE3) : F- mcrA mcrb I (rrnD-rrnE) 1 λ- rncl 4 : : trl O (DE3 lysogen: lacUV5 promoter-T7 polymerase) ; host for IPTG inducible T7 polymerase expression; Rnaselll-.

Other host strains suitable for expression of dsRNA could be used with equivalent effect.

Example 3 Isolation of SERCA mutants:

Construction of a C. elegans sca-1 mutant.

The following strategy may be used to isolate a nematode that is mutated in the sca -1 gene, using standard selection procedures well known in the art.

A population of nematodes are mutagenized, preferentially using UV-TMP, and grown for two generations. The mutagenized worms are distributed per 500 over approximately 1152 plates and grown for an additional two generations. DNA is isolated from a fraction of the worms from each of these plates and used as a template for PCR selection to select for an sca-2 gene that has a deletion. From a plate with worms, of which some have been demonstrated to contain an sca-1 deletion, new plates are started with fewer worms. Further rounds of PCR selection finally result in the isolation of a heterozygote C. elegans carrying a mutation in the sca-1 gene (see Jansen et al., 1997, Nature Genetics 17:119-121). As experiments have shown that the expression level of SERCA is important for the survival of the nematode it is possible that this strategy may result only in the isolation of partial knock-out mutations as heterozygote C. elegans carrying a severe knock-out mutation in the sca-1 gene may not viable. In this situation, strategy 1 based on extrachromosomal expression can be used to isolate severe knock-out mutations.

Analysis of a C. elegans mutant (designated ok!90) C. elegans strain okl90 which is mutated in the sca-1 gene was kindly provided by R. Barstead (Oklahoma, USA) . This strain can be purchased from the same supplier or from the C. elegans Genetic Center, Minnesota, USA (see above) . Heterozygous animals show no defect, but their homozygous progeny die as Ll . The lethal phenotype can be rescued by reintroduction of the C. elegans gene by injection of pGK7.

Using standard PCR protocols the genomic region of okl 90 around the deleted area was cloned in the following way:

A nested PCR was performed on C. elegans genomic DNA using the following primer pairs:

Outer: SERCA P2 CGAAGAGCACGAAGATCAGACAG SERCA P8 GAGAGGCGGTTGGTTTGGG

Inner: SERCA P4 CCGTTCGTCATCCTTCTCATTC

SERCA P7 CGACAGATGGACCGACGAGC

Analysis of the nested PCR product by agarose gel electrophoresis showed that the PCR product in the okl90 strain harbors a deletion of 1.7 kbp. (The wild-type PCR product from SERCA P4 - SERCA P7 would be 3.4 kbp but the observed okl90 PCR product was only 1.7 kbp) . To enable detailed analysis of the deleted region the PCR product was cloned into the pCR-XL-TOPO vector (Invitrogen, The Netherlands) . The resulting plasmid was designated pK04. This cloned fragment was then sequenced revealing the exact coordinates of the deleted region. One of the breakpoints of the deletion occurred in the intron between exon IV and exon V, the other in exon V, deleting a total of 1702 bp of which 1690 bp represent coding sequence.

The nucleotide sequence of the genomic fragment of C. elegans sca-1 bounded by primers SERCA P4 and SERCA P8 is shown in Figure 12. Exon IV and exon V are shown in capitals, intron IV in lower case. The fragment deleted in okl90 is underlined.

Example 4 Construction of C. elegans strains for use in screening:

Rescue of an sca-2 mutant C. elegans using a pest SERCA cDNA The following strategy may be used to introduce a pest SERCA transgene onto an sca-2 (okl90) mutant genetic background in C. elegans .

The starting C. elegans strain is an sca-1 (okl90) /qCl heterozygotic strain. The heterozygous strain is used as an okl90/qCl strain is viable, whilst both okl90/okl90 and qCl/qCl are lethal. The qCl allele is a balancer, and is well known in the area of C. elegans genetics .

The following constructs are required: a) DNA encoding heterologous pest SERCA or heterologous pest /C. elegans SERCA chimera under control of the C. elegans SERCA promoter ( sca-1 promoter) . Other more general promoters able to drive expression of SERCA could be used with equivalent effect .

b) Marker cassette eg. pDW2721 (GFP) or rol-6. For the GFP marker cassette the myo-2 promoter is chosen to prevent interference with the read out in the pharynx pumping assay. Using this promoter GFP is only expressed in the pharynx.

The pest SERCA and marker cassettes are transformed into the worm using standard C. elegans techniques.

Development :

Rescue the SERCA mutant phenotype of sca-1 (okl90) /qCl with heterologous SERCA. Select for wild type phenotype combined with stable fluorescent or roller phenotype (depending on the chosed marker) .

Screening:

Rescue of the sca-1 mutation by expression of a pest SERCA protein results in wild-type phenotypes of pharynx pumping, movement, egg laying, defecation, mating etc. These characteristics can therefore be used as indicators of SERCA activity to perform screens on the pest SERCA target, based on detection of changes in these phenotypes.

C. elegans expressing thapsigargin-resistant pest SERCA

The starting C. elegans strain may be wild-type C. elegans (N2 strain) or a selected mutant strain.

Required constructs: a) DNA encoding heterologous pest SERCA or pest

SERCA/C. elegans SERCA chimera which is resistant to inhibition by thapsigargin under the control of the C. elegans SERCA (sca-2) promoter.

b) Marker cassette eg. pDW2721 (GFP) or rol-6. Development:

The expression of a pest SERCA or pest SERCA/C. elegans SERCA chimera which is resistant to inhibition by thapsigargin results in rescue of the lethal phenotype induced by lethal doses of thapsigargin.

Screening:

The screen is performed in the presence of a lethal dose of thapsigargin. In the presence of thapsigargin the strain exhibits substantially wild-type pharynx pumping, movement, egg laying, defecation, mating etc. These characteristics can therefore be used as indicators of SERCA activity to perform screens on the pest SERCA target, based on detection of changes in these phenotypes.

C. elegans expressing heterologous pest SERCA in a tissue in which expression of the endogenous C. elegans SERCA protein is low or absent. The starting C. elegans strain may be wild-type

C. elegans (N2 strain) or a selected mutant strain.

Required constructs: a) DNA encoding heterologous pest SERCA or pest SERCA/C. elegans SERCA chimera under the control of the C. elegans unc-119 promoter or any other neuronal promoter.

Development: The strain exhibits ectopic expression of a pest SERCA protein or pest SERCA/C. elegans SERCA chimera in one or more neurons of C. elegans . The phenotype is evaluated and a characteristic selected to form the basis of a screen. Example 5 Inhibition of endogenous C. elegans SERCA by compounds :

Several compounds are known to inhibit the function of SERCA, such as cyclopiazonic acid, cyproheptadine, thapsigargin, 2,5-di

(tert-butyl) -1, 4-benzohydroquinone, 2.4-benzoquinone, and vanadate. Other compounds are known to activate the activity of SERCA, such as diethylether, gingerol, and 1- (3, 4-dimethoxyphenyl) -3-dodecanone. Still other compounds have a dual activity, they stimulate SERCA at low concentrations, but inhibit at high concentrations, such as phenothiazines, and pentobarbital .

Using two kinds of assays, the optimal concentration of compounds that inhibit the activity SERCA has been determined. The first assay is designated the drop or plate assay in which the nematodes are fed E. coli strains pre-loaded with the compound. In a second assay, the compound is administrated to the worm in liquid culture.

Plate assay

A standard plate drop assay is performed according to the following protocol. 4ml NGM agar

(see "The nematode C. elegans" Ed. by William B. Wood and the Community of C. elegans Researchers, CSHL Press, 1988, pg589) is into 3cm plates and seeded with approximately 5μl of an E. coli overnight culture and grown preferably for one week at room temperature.

Approximately lOμl of test compound dissolved in DMSO or other suitable solvent is pipetted onto the bacterial lawn so that the lawn is covered completely. After overnight soaking in or compound, one C. elegans (L4 stage) per plate is put onto the bacterial lawn. Plates are incubated at 21°C and checked after some hours. Plates are checked again after 4 days for phenotypes of the FI progeny (control shows all stages up to gravid hermaphrodites) .

Thapsigargin at various concentrations (5 μM, 2.5 μM and 1.25 μM) causes the nematode to stop pharynx pumping within 10 min. Within an hour the worms restart pumping, although at a low level. The worms are pale and thin and have a slow and irregular movement, with an increased amplitude. No plate drop response is observed, and the worms show poor backing, reduced pumping and strong constipation. The worms have a defective gonad with only very few eggs, and a protruding vulva. Some worms also have a protruding rectum. Progeny reaches L2 stage only after four days, and the brood size is very small. Lower concentrations of thapsigargin (0.5 μM, 0.25 μM, 0.125 μM) still cause reduced brood size.

2,5-di-tert butylhydroquinone at a concentration of 500 μM resulted in pale, starved, thin worms with slow movement, defective gonad, constipated and reduced brood size.

Cyclopiazonic acid at a concentration of 500 μM resulted in nematodes that lay still or move slowly after one hour. The worms showed strong avoidance and after 24 hours they look starved, pale and thin, with only a few eggs in the body, a defective gonad, and reduced brood size. A delayed growth of the FI generation was observed.

Thapsigargicin at 500 μM, 125 μM, 31 μM, 10 μM, 5 μM resulted in nematodes with similar phenotypes to those described above for thapsigargin at 5 μM, 2.5 μM, 1.25 μM. Lower concentrations of thapsigargicin (3 μM and 1.5 μM) caused a slightly reduced brood size. Thapsigargin-epoxide did not result in a clear observable effect, even at the highest concentration tested (1 mM drop, 5 μM end concentration) .

1, 4-benzoquinone did not result in a clear observable effect, even at the highest concentration tested (100 mM drop, 500 μM end concentration) .

Liquid culture assay Thapsigargin at 100, 50 and 20 μM resulted in small worms which show slow and loopy movement. They had a protruding vulva, and no progeny (or no progeny that grows up) were observed. At lower concentrations of 10 μM and 5 μM a reduced number of progeny and delayed growth could be observed.

2,5-di-tert butylhydroquinone at a concentration of ImM resulted in progeny exhibiting delayed growth and the worms were observed to be thinner than ^λnormal' worms. Cyclopiazonic acid at a concentration of ImM resulted in pale, thin worms with a slow movement and a very strongly reduced brood size. At lower concentrations of 0.5mM, growth delay was observed.

Thapsigargicin at 1000 μM, 250 μM, 62.5 μM and 16 μM concentrations resulted in small worms with slow and loopy movement, a protruding vulva, and no progeny (or no progeny that grows up) were observed. At lower concentrations of 10 μM, delayed growth and reduced progeny were observed. The effect of thapsigargin on progeny of wild-type strains was tested with the liquid assay: On an average of 12 worms, the number of progeny for the different concentrations is summarized in Figure 22. The effect of thapsigargin was also tested on progeny of wild-type strains using the plate assay: On an average of 12 worms the number of progeny at different concentrations is summarized in Figure 23. The effect of thapsigargin on the production of progeny was determined for a number of different C. elegans strains. The numbers of progeny produced following thapsigargin treatment was counted for an average of 15 animals, the results are summarised as follows : unc-31: control : 132 0.5 mM : 35 1 mM : 5,6 srf-3: control: 50 1 mM : 18,3

The effect of thapsigargin on pharynx pumping behaviour was also determined. In wild-type worms, all animals stopped pumping after 10 minutes. In mutant strain unc-31 at a concentration of 1 mM thapsigargin, all worms stopped pumping after 10 minutes, some start again after half an hour, but pumping is only one third of normal speed. In summary, the above experiments demonstrate that inhibition of C. elegans SERCA activity using thapsigargin or other chemical inhibitors of SERCA results in worms with recognisable phenotypic characteristics, including reduced growth, reduced rate of pharynx pumping and reduced numbers of progeny. These phenotypic changes can be used as the basis of a screen for other compounds which inhibit the activity of the endogenous C. elegans SERCA protein.

Example 7 C. elegans screening technology:

Distribution of nematodes, and dilution of compounds. The following is a basic protocol for performing a compound screen in 96 well plates.

Preferentially, synchronized worms are used. The production of large amounts of synchronized worms has been described in (Methods in cell biology, Vol. 48, ibid) . After the worms have grown to the preferred stage, they are washed in M9 buffer prior to further use, and re-suspended in an assay buffer (40mM NaCl, 6mM Kcl, ImM CaCl₂, ImM MgCl₂) . (10 X M9 buffer: 30g KH₂P0₄, 60 g Na₂HP0₄, 50 g NaCl, 10 ml MgS04 IM, made up to 1 litre with H₂0) . Other buffers than M9 buffer can be suitable for this purpose. The worms are then diluted and resuspended in semi-soft agar (final concentration of 0.25% low melting agarose in M9 buffer) . This procedure results in an equal, homogenous and stabilised suspension of the nematodes. Other polymers than low melting agarose can be used in this procedure. The presence of a homogenous worm suspension facilitates the equal distribution of the worms in the multi-well plates, but is not essential. Any other method that results in a homogenous distribution of the nematodes worms over the wells will be useful. More specifically, the use of a worm dispenser will result in even a better, and hence a more equal distribution of the worms over the wells of the multi-well plate.

The worms are distributed in the multi-well plates using electronic 8 channel pipettes. In a preferred set-up of this experiment 40 +/- 5 worms are added to every well of the microtiter plate.

Compounds are dissolved in DMSO. Any other solvent can be used for this purpose, but most selected compounds appear to be soluble in DMSO. The compounds are added in the wells at various concentrations. The concentration of the DMSO should not be too high and preferentially should not exceed 1%, more preferentially the concentration of the DMSO should not exceed 0.5% and even more preferentially, the concentration of the DMSO is lower than 0.3%.

General pharynx pumping assay.

Depending on the specific assay which it is desired to perform, different C. elegans strains can be used. Screens to select for compounds inhibiting the pumping rate of the C. elegans pharynx are preferably performed with mutant C. elegans strains which have a constitutively pumping pharynx. Wild-type worms can also be used in this screen, but the mutants worms are preferred. Other C. elegans mutants can be used in this screen to select for inhibitors of pumping. The selected mutant C. elegans with the constitutively pumping pharynx pumps medium into the gut at a constant rate and reduction/rescue of this phenotype can easily be scored, which facilitates the detection and selection of compounds.

The pumping rate of the pharynx is measured indirectly by adding a marker molecule precursor such as calcein-AM to the medium and measuring the formation of marker dye in the C. elegans gut. Calcein-AM is cleaved by esterases present in the C. elegans gut to release calcein, which is a fluorescent molecule. The pumping rate of the pharynx will determine how much medium will enter the gut of the worm, and hence how much calcein-AM will enter the gut of the worm. Therefore by measuring the accumulation of calcein in the nematode gut, detectable by fluorescence, it is possible to determine the pumping rate of the pharynx.

Compounds that alter the pumping rate of the pharynx will result in more or less uptake of the calcein-AM and hence in more or less fluorescent signal. Moreover, using a multi-well plate reader, the fluorescence can be measured rapidly and quantitatively, resulting in a fast, quantitative high throughput screening method for the identification of compounds with potential pharmacological activity.

To perform the pharynx pumping screen with calcein-AM, a concentration of between 1 and lOO M calcein-AM is added into the medium. Preferably 5 to lOμM calcein-AM is used. Fluorescence is measured using a multi-well plate reader (Victor2, Wallac Oy, Finland) with following settings: Ex/Em = 485/530.

This measurement of the pharynx pumping rate by detecting the accumulation of a marker molecule is not limited to calcein-AM. Other precursors can be used and thus the assay as described here can be changed to be suitable for other precursors. The precursor can be cleaved by esterases, but could also be a substrate for other enzymes in the nematode gut. Furthermore, the marker molecule should not necessary be a fluorescent molecule, but can be a molecule detectable by other methods. Most of these precursor substances are commercially available or could be synthesized according to methods known in the art. Some examples are:

With a fluorescent read out:

-Esterases substrates: Calcein-AM, FDA, BCECF-AM

-Alkaline phosphatase substrates: Fluorescein diphospate (FDP)

-Endoproteases; Aminopeptidase substrates: CMB-leu

With a luminescent read out:

-alkaline phosphatase substrates: AMPPD

With a colour read out. -Glucuronidase substrates: X-gluc

Other target enzymes present in the gut for which substrates can be found or developed are DNAses,

ATPases, lipases and amylases. An overview of various marker molecules, mainly fluorescent can be found in "Handbook of fluorescent probes and research chemicals, molecular probes, ed. by R. P. Haughland" Example 8 Inhibition of SERCA in cultured mammalian cells :

COS cells have been transfected with erAEQ/pCDNAi provided by Molecular probes, to investigate the influence of calcium modulation of thapsigargin in these cells. Transfection was performed using the Lipofectamine Plus reagent (Life technloglogies, Inc) according to the standard protocol supplied by the manufacturer. Cell lysis was performed as described in "The Molecular Sampler Kit" provided by Molecular Probes, to determine the best substrate. Experiments show that for COSI cells coelentazine hep is the best substrate (data not shown) . For other cells the most suitable substrate would need to be determined by experiment.

Tests were repeated in multiwell plates, without cell lysis. The transfected cells were treated with thapsigargin and aequorin fluorescence was measured directly. A clear variation was observed between cells treated with thapsigargin and cells that have not been treated. Measurements in a high throughput format can be made from 5 minutes to at least 45 minutes after contacting the cells with the appropriate test compound.

GenBank accession numbers of SERCA cDNAs Arabidopsis thaliana: Q9SWS8 ,004987 ,023087. Artemia sanfranciscana: ATC_ARTSF. Aspergillus niger: AAF37300. Bacillus halodurans: BAB06234. Bacillus subtilis: 034431. Bos taurus: AAF64433. Caenorhabditis elegans: Q9XTG6. Candida albicans : CAB87245. Drosophila melanogaster: ATC1_DR0ME , Q9VNR2. Dunaliella bioculata: ATCl DUNBI . Gallus gallus: Q9YGL9 ,ATC1_CHICK, B40812.

Heliothis virescens: 096696.

Homo sapiens: 060900 ,ATC1_HUMAN.

Leishmania mexicana: 009489. Lycopersicon esculentum: Q42883.

Makaira nigricans: ATC1_MAKNI .

Methanobacterium thermoautotrophicum: 027560 ,

Mus musculus: Q64517 ,ATC2_M0USE.

Mycobacterium tuberculosis: CTPF_MYCTU. Neurospora crassa: Q9UUY0.

Oryctolagus cuniculus: ATC2_RABIT.

Oryza sativa: BAA90510 ,004938.

Paramecium tetraurelia: CAB96170 ,061073.

Patinopecten yessoensis: 096039. Placopecten magellanicus : 077070.

Plasmodium berghei: Q27764.

Plasmodium falciparum: ATC_PLAFK.

Procambarus clarkii: 017314.

Pseudomonas aeruginos: AE004572_6. Rana esculenta: ATC1_RANES .

Schistosoma mansoni: 096527 , 0.21119.

Schizosaccharomyces pombe : 059868.

Synechococcus sp.: ATCL_SYNP7.

Synechocystis sp. : Q59999. Synechocystis sp: SSPMA1_1.

Trichomonas vaginalis: Q95060.

Trypanosoma brucei: ATC_TRYBB.

Trypanosoma cruzi: 096608.

Ureaplasma urealyticu: AE002123_6. Zea mays: AAF73985. LIST OF PEST SPECIES:

94 Brown rust, wheat IPuccinia recondita Uredinales iPuccinia

95 jBrown soft scale Coccus hesperidum I Homoptera- Coccidae Coccus 96(Brown spot, peanut Mycosphaerella arachidis Dothidiales Mycosphaerella 971 Brown spot, rice jCochliobolus miyabeanus Dothidiales Cochliobolus 98jBrown stripe, sugar cane Bipolaris stenospila Hyphales Bipolaris 88. Brown-banded cockroach Supella longipalpa Dictyoptera: Blattidae Supella

99JBuffalo fly Haematobia irritans exigua Diptera: Muscidae Haematobia OOlBuffaϊograss Brachiaria mutica (= Panicum Gramineae Brachiaria

01 Bugs Heteroptera Hemiptera Heteroptera

02 Bulb mites Rhizoglyphus callae, R robini Acari: Acaridae Rhizoglyphus

03 iBulb scale mite Steneotarsonemus laticeps Acari: Tarsonemidae Steneotarsonemus

04 Bullrushes Typha spp. Typhaceae Typha

05 j Bunt, stinking smut Tilletia caries Ustilaginales Tillet

06 j Burrowing nematode Radopholus similis Nematoda: Tylenchidae Radopholus 07JButt rot, conifers Heterobasidion annosum Aphyllophorales Heterobasidion 08! Buttercups Ranunculus spp. Ranunculaceae Ranunculus

09 Cabbage looper Trichoplusia ni Lepidoptera: Noctuidae Trichoplusia

10 Cabbage root fly [ Delia radicum Diptera: Anthomyiidae DeliaA 7^"~L 11 ] Cabbage seed weevil JCeutorhynchus assimilis Coleoptera: Ceutorhynchus 12 (Cabbage stem weevil Ceutorhynchus quadridens Coleoptera: Ceutorhynchus 131 Cabbage white butterflies Pieris spp. Lepidoptera- Pieridae Pieris

14jCalifornian red scale Aonidiella aurantii Homoptera: Diaspididae Aonidiella

15 J Canadian pondweed Elodea canadensis Hydrocharitaceae Elodea

16 Canary grass, awned Phalaris paradoxa Gramineae Phalaris

17 Canary grasses Phalaris spp. Gramineae Phalaris lδiCanker, apple, pear Nectria galligena Nectriaceae Nectria 19,Capsid bugs Miridae Heteroptera Miridae 20 Carmine spider mite Tetranychus cinnabarinus Acari: Tetranychidae Tetranychus 21 Carpenter ants Camponotus spp. Hymenoptera: Camponotus 22 Carpet beetles Anthrenus spp. Coleoptera: Dermestidae Anthrenus

23 [Carrot fly Psila rosae Diptera: Psilidae Psila

24 j Carrot leaf blight^"" 'Alternaria dauci Hyphales Alternaria

25 i Cat flea ______ ._ __r I Ctenocephahdes felis Siphonaptera: Pulicidae Ctenocephahdes

26|Cattle biting louse Bovicola bovis Phthiraptera: Bovicola 27 [Cattle tail louse JHaematopinus quadripertusus jPhthiraptera: JHaematopinus 28lCereaϊϊeafbeetle ^" lOulema melanopus i Coleoptera: lOulema 29!Chamomiles jAnthemis spp. jCompositae Anthemis 30JCharlock ^~ i Sinapis arvensis ¹ Cruciferae Sinapis 31 ϊ Cherry leaf spot Blumeriellajaapii j Helotiales Blumeriella 32 'Chicken mite Dermanyssus gallinae I Acari: Dermanyssidae Dermanyssus

33 j Chigoe flea Tunga penetrans j Siphonaptera: Pulicidae Tunga

34jChinch bug _ Blissus leucopterus ILygaeidae Blissus

35 (Chrysanthemum leaf miner Phytomyza syngenesiae j Diptera: Agromyzidae j Phytomyza

36] Chrysanthemum leaf miner parasitoid Dacnusa sibirica fHymenoptera: ( acnusa

37 Chrysomelid beetles Cluysomelidae (Coleoptera [Chrysomelidae

38 |CΪgarette beefle ^~~_ iLasioderma serricorne [Coleoptera: Anobiidae _jLasioderma 39; Citrus aphid I Aphis citricola j Homoptera: Aphididae [Aphis

422 [Northern leaf blight, maize Helminthosporium turcicum Hyphales Helminthosporium

423 Nutgrass Cyperus rotundus Cyperaceae Cyperus I

424 Nutsedges Cyperus spp. Cyperaceae j Cyperus

^"425 Oat, sterile Avena sterilis Gramineae i Avena j

426 Oats (wild and cultivated) Avena spp. Gramineae j Avena ι

427 Ol ^' World bolϊworm Helicoverpa armigera Lepidoptera Noctuidae 'Helicoverpa [

428 [Olive fruit fly Dacus oleae Diptera: Tephritidae Dacus 29 Onion couch Arrhenatherum elatius var. Gramineae Arrhenatherum j 430 Onion thπps Thπps tabaci Thysanoptera: Thnpidae Thrips '

431 Oriental tobacco budworm Helicoverpa assulta Lepidoptera- Noctuidae Helicoverpa , 432 Ox warble fly Hypoderma bovis Diptera. Oestπdae Hypoderma

433 Pacific rat Rattus hawaiiensis Rotentia: Muridae Rattus ^""434 Pale persicaria Polygonum lapathifohum Polygonaceae Polygonum ^! 435 Palm rat Rattus tiomanicus Rotentia: Muridae Rattus 436 Panic grasses Panicum spp. Gramineae Panicum 437 Paralysis tick Ixodes holocyclus Acari- Ixodidae Ixodes t438 Parasitic yeasts Candida spp. Endomycetales Candida

439 Pea cyst nematode Heterodera goettingiana Nematoda: Heterodera j 440 Pea weevils Sitona spp. Coleoptera: Sitona j

441 Peach leaf-curl Taphrina deformans Taphrinales Taphrina 442^" Peach-potato aphid Myzus persicae Homoptera Myzus 443 Pear leaf blister moth Leucoptera malifohella Lepidoptera- Lyonetudae Leucoptera

, 444 Pear rust Gymnosporangium fuscum Uredinales 1 Gymnosporangium

445 Pear rust mite Epitrimerus pyri Acari: Eriophyidae Epitrimerus

446 [Pearly green lacewing Chrysoperla carnea Neuroptera: Chrysopidae Chrysoperla

447 Penicillium rots Penicillium spp Hyphales Penicillium , 448 Persicaria Polygonum persicaria Polygonaceae Polygonum 44~9 Petunia Petunia spp Solanaceae Petunia _t

450 Pharaoh's ant Monomorium pharaoms Hymenoptera. Monomorium

45l [Pheιdole ants Pheidole megacephala Hymenoptera: Pheidole • 452 'Pickerel weed Monochoria vaginalis Pontederiaceae Monochoπa j 453jPimpernef, false Lindernia procumbens Scrophulariaceae Lindernia |

J 454 Pine processionary caterpillar Thaumetopoea pityocampa Lepidoptera: Thaumetopoea

^! 455 Pink bollworm Pectinophora gossypiella Lepidoptera: Gelechiidae Pectinophora

456 Plantains Plantago spp Plantaginaceae Plantago ; 457 Planthoppers Nilaparvata spp. Homoptera: Delphacidae Nilaparvata 458 Plum rust Tranzchelia pruni-spinosi Uredinales Tranzchelia 459 Pod rot, cocoa Monilia roreri Hyphales Monilia 460 Pollen beetle Meligethes aeneus Coleoptera: Nitidulidae Meligethes | 46l^" Pondweed, American Potamogeton distinctus Potamogetonaceae Potamogeton 462 Pondweeds Potamogeton spp. Potamogetonaceae Potamogeton ;

463 Poppies Papaver spp. Papaveraceae Papaver | 464 Post-harvest rot Fusarium coeraleum Hyphales Fusarium ι ϊ 465 Post-harvest rots Rhizopus spp. Mucorales Rhizopus | 1 466 Post-harvest rots Sclerotium spp. Agonomycetales Sclerotium ' 1 467 Potato aphid Macrosiphum euphorbiae Homoptera: Aphididae j Macrosiphum < i 468 Potato cyst nematodes iGlobodera spp. Nematoda. JGlobodera

519 ] Root rot, brassicas Phytophthora megasperma [Peronosporales [Phytophthora

520 Root rot, cereals, grasses Cochliobolus sativus 1 Dothidiales l Cochliobolus

521 Root rot, tomato Colletotrichum coccodes [Melanconiales Colletotrichum

J522 Root rots, various hosts Aphanomyces spp. iSaproiegniales Aphanomyces

A²³ Root rots, various hosts Phoma spp. jDeuteromycotina Phoma

524 Root rots, various hosts Pythium spp. [Peronosporales Pythium

525 Root rots, various hosts Rhizoctonia spp. Stereales Rhizoctonia Al7 Root-knot nematodes Meloidogyne spp. Nematoda Meloidogyne

. £18 Root-lesion nematodes Pratylenchus spp. Nematoda: Pratylenchus _526 Rose aphid Macrosiphum rosae [Homoptera: Aphididae Macrosiphum J527 Rose thrips Thrips fuscipennis Thysanoptera: Thripidae Thrips _528 Rot, various crops Phytophthora palmivora j Peronosporales Phytophthora 529 Rots of stems, storage organs, etc., Sclerotinia sclerotiorum Helotiales Sclerotinia

531_ Rots, various hosts Pellicularia spp. Tulasnellales _ Pellicularia

[532 Rots, various hosts Sclerotium rolfsii Agonomycetales Sclerotium

J530 Rots, various hosts (Imperfect fungi Fusarium spp. Hyphales Fusarium

533 Rough Cocklebur Xanthium strumarium Compositae Xanthium 34 Roughseed bulrush Scirpus mucronatus Cyperaceae Scirpus 535 [Rubbery rot, potatoes Geotrichum candidum Hyphales Geotrichum

J536 fRunch ^" Raphanus raphanistrum Cruciferae (Raphanus

5371 Rush, flowering Butomus umbellatus iButomaceae JButomus

538 Russian thistle Salsola kali (Chenopodiaceae TSaisola 540 Rust fungi Uredinales JBasidiomycotina [Uredinales Rust mite, apple Aculus schlechtendali [Acari: Eriophyidae Aculus

_542 ^" Rust mites ._l jAculus spp. _' Acari: Eriophyidae Aculus 543^" Rust mites [Phyllocoptruta spp. j Acari: Eriophyidae Phyllocoptruta 539 JRust, beet crops _ ^■Uromyces betae [Uredinales (Uromyces

545 [Rust, roses jPhragmidium mucronatum [Uredinales iPhragmidium

546 [Rust, soya i Phakopsora pachyrhizi Uredinales J Phakopsora

547 (Rust, various hosts jPuccinia spp., Uromyces spp. Uredinales jPuccinia 544 i Rust-red flour beetle j Tribolium castaneum Coleoptera: Tribolium

_548 Ryegrass, italian ! Lolium multiflorum Gramineae Lolium

_549 Ryegrass, perennial [Lolium perenne Gramineae Lolium

5~50 Ryegrasses .Lolium spp. Gramineae Lolium

^"55l San Jose scale [Comstockaspis perniciosus Homoptera: Coccidae Comstockaspis 552JSaprophytic fungi IPaecilomyces spp. Hyphales Paecilomyces 553 Saramatta grass j Ischaemum rugosum Gramineae Ischaemum 5541 Saw-toothed grain beetle [Oryzaephilus surinamensis 1 Coleoptera: Cucujidae Oryzaephilus 555 |Sawflies iDiprion spp. [Hymenoptera: Diprion

556 [Scab, apples Venruria inaequalis (Dothidiales Venturia

557[Scab, cereals [Gibberella spp. (= various jHypocreales Gibberella 558 j Scab, citrus [έlsinoe fawcettii ! Dothidiales Elsinoe 559 [ Scab, pears jVenturia pirina Dothidiales Venturia 560 j Scabies mites, etc. I Sarcoptes spp. Acari: Sarcoptidae Sarcoptes 56 l ^'ι Scale insect [Didesmococcus brevipes [Homoptera: Coccidae Didesmococcus 562 [Scale insects [Coccus spp. j Homoptera: Coccidae Coccus 563 [Scale insects [Coccidae, Diaspidae, [Homoptera Coccidae,

Preferred Primer combinations for PCR of plant SERCA cDNA:

1-6, 1-7, 1-8, 1-9, 2-7, 2-8, 2-9, 3-7, 3-8 ,3-9 ,4-9 ,5-9

Preferred Primer combinations to isolate SERCA cDNA from all origins

2-6, 2-12, 2-9, 10-12, 10-9, 11-12, 11-9

Claims

Claims :

1. A method of identifying compounds having pesticidal activity, which method comprises: providing microscopic nematode worms expressing a pest SERCA protein, said protein being derived from a pest species, other than the C. elegans SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the nematode worm in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has pesticidal activity.

2. A method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises: providing microscopic nematode worms expressing a pest SERCA protein, said protein being derived from a pest species, other than the C. elegans SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the microscopic nematode worm in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.

3. A method as claimed in claim 1 or claim 2 wherein the microscopic nematode worm is C. elegans.

4. A method as claimed in any one of claims 1 to 3 wherein the pest species is an invertebrate.

5. A method as claimed in claim 4 wherein the invertebrate is an arthropod.

6. A method as claimed in claim 5 wherein the arthropod is an insect.

7. A method as claimed in claim 4 wherein the invertebrate is a nematode other than C. elegans .

8. A method as claimed in any one of claims 1 to 3 wherein the pest species is a rodent.

9. A method as claimed in any one of claims 1 to 3 wherein the pest species is a plant.

10. A method as claimed in any one of claims 1 to 3 wherein the pest species is a fungus.

11. A method as claimed in any one of the preceding claims wherein the C. elegans is transgenic C. elegans containing a transgene comprising nucleic acid encoding the pest SERCA protein operably linked to a promoter.

12. A method as claimed in any one of claims 1 to 11 wherein the microscopic nematode exhibits no or substantially reduced activity of the endogenous nematode SERCA protein in one or more tissues or cell types .

13. A method as claimed in claim 12 wherein the nematode have a mutant genetic background.

1 . A method as claimed in claim 13 wherein the nematode is a mutant which exhibits no or substantially reduced expression of the endogenous nematode SERCA protein in one or more cell types or tissues.

15. A method as claimed in claim 14 wherein the nematode is mutant C. elegans which carries a loss-of- function or knock-out mutation in the chromosomal C. elegans sca-1 gene.

16. A method as claimed in claim 15 wherein the C. elegans have okl90 genetic background.

17. A method as claimed in claim 12 wherein expression of the endogenous nematode SERCA protein is inhibited in one or more tissues or cell types using double-stranded RNA inhibition.

18. A method as claimed in claim 12 wherein the nematodes are treated with a SERCA inhibitor to reduce the activity of the endogenous SERCA protein and the pest SERCA protein is resistant to inhibition by the said SERCA inhibitor.

19. A method as claimed in claim 18 wherein the pest SERCA protein is modified so as to be resistant to inhibition by the said SERCA inhibitor.

20. A method as claimed in claim 19 wherein the SERCA inhibitor is thapsigargin and the pest SERCA protein carries a thapsigargin resistance mutation.

21. A method as claimed in claim 20 wherein the thapsigargin resistance mutation is a single amino acid substitution equivalent to a Phe259Val substitution in the C. elegans SERCA protein.

22. A method as claimed in claim 11 wherein the transgene comprises nucleic acid encoding the pest SERCA protein operably linked to a promoter which is capable of directing tissue or cell-type specific gene expression in a tissue or cell type which exhibits no or background activity of the endogenous nematode SERCA protein.

23. A method as claimed in claim 22 wherein the nematode is C. elegans and the promoter is capable of directing specific gene expression in one or more C. elegans neurons.

24. A method as claimed in claim 23 wherein the promoter is the unc-119 promoter.

25. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is pharynx pumping efficiency.

26. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is egg laying behaviour.

27. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is mating behaviour.

28. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is Ca²⁺ concentration in one or more tissues or cell types.

29. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is defecation behaviour.

30. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is growth rate.

31. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is movement behaviour.

32. A method as claimed in any one of claims 1 to 24 wherein the indicator of SERCA activity is life/death of the C. elegans .

33. A method of identifying compounds having the potential to kill pests using the nematode worm C. elegans, which method comprises: providing microscopic nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein; and ^"' detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the nematodes in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has the potential to kill pests.

34. A method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises: providing microscopic nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the nematodes in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.

35. A method as claimed in claim 33 or claim 34 wherein the microscopic nematodes are C. elegans .

36. A method as claimed in claim 33 or claim 34 wherein the microscopic nematode strain is a wild-type strain.

37. A method as claimed in claim 33 or claim 34 wherein the microscopic nematode strain is a mutant strain.

38. A method as claimed in claim 37 wherein the mutant strain is a constitutive pharynx pumping mutant.

39. A method as claimed in any one of claims 33 to 38 wherein the indicator of SERCA activity is pharynx pumping efficiency.

40. A method as claimed in any one of claims 33 to 38 wherein the indicator of SERCA activity is egg laying behaviour.

41. A method as claimed in any one of claims 33 to 38 wherein the indicator of SERCA activity is mating behaviour.

42. A method as claimed in any one of claims 33 to 38 wherein the indicator of SERCA activity is Ca²⁺ concentration in one or more tissues or cell types .

43. A method as claimed in any one of claims 33 to 38 wherein the indicator of SERCA activity is defecation behaviour.

44. A method as claimed in any one of claims 33 to 38 wherein the indicator of SERCA activity is growth rate.

45. A method as claimed in any one of claims 33 to 38 wherein the indicator ot Si-iKUA activity is movement behaviour.

46. A method as claimed in any one of claims 33 to 38 wherein the indicator of SERCA activity is life/death of the C. elegans.

47. A method of identifying compounds having pesticidal activity, which method comprises: providing cultured cells expressing a SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the cells in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has pesticidal activity.

48. A method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises: providing cultured cells expressing a SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the cells in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.

49. A method as claimed in claim 48 or claim 49 wherein the cultured cells are derived from a pest species .

50. A method as claimed in claim 48 or claim 49 wherein the pest species is an insect.

51. A method as claimed in claim 48 or claim 49 wherein the cultured cells are eukaryotic host cells containing an expression vector comprising nucleic acid encoding a SERCA protein.

52. A method as claimed in claim 51 wherein the host cells are a cell line capable of growing in monolayer or suspension culture.

53. A method as claimed in claim 52 wherein the host cells are COSI, BHK21, L929, PC12, CVl, S ISS3T3,

HT144, IMR32, HEPG2, MDCK, MCF7, HEK293, Hela, A549, SW48 or G361.

54. A method as claimed in any one of claims 51 to 53 wherein the SERCA protein is a pest SERCA protein.

55. A method as claimed in any one of claims 47 to 54 wherein the indicator of SERCA activity is intracellular Ca²⁺ concentration.

56. A method as claimed in claim 55 wherein the indicator of SERCA activity is Ca²⁺ concentration in the endoplasmic reticulum.

57. A method as claimed in any one of claims 47 to 54 wherein the indicator of SERCA activity is cellular apoptosis.

58. A method of identifying compounds having pesticidal activity, which method comprises: isolating microsomes from cultured cells expressing a SERCA protein; and measuring Ca²⁺ levels in the microsomes in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has pesticidal activity.

59. A method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises: isolating microsomes from cultured cells expressing a SERCA protein; measuring Ca²⁺ levels in the microsomes in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.

60. A method as claimed in claim 58 or claim 59 wherein the cultured cells are derived from a pest species.

61. A method as claimed in claim 58 or claim 59 wherein the cultured cells are eukaryotic host cells containing an expression vector comprising nucleic acid encoding a pest SERCA protein.

62. A compound identified as having pesticidal activity using a method according to any one of claims 1 to 61.