Molecular and cellular biology of helminth parasites IV.

FOR ORAL PRESENTATIONS Nematode genomics and informatics: leveraging C. elegans PAUL W. STERNBERG. CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA, CA HHMI, Caltech and WormBase C. elegans has emerged as a model organism, not only for classical biology but also for genomics and bioinformatics. With the emergence of additional genome informatic resources, the C. elegans and parasitic helminth communities can collaborate to greatly accelerate helminth research. WormBase (www.wormbase.org) now includes sequences of other Caenorhabditis species as well as Brugia malayi and Pristionchus pacificus. Besides genome and EST sequences, WormBase curates a variety of experimental data such as microarrays, gene expression and regulation, phenotypes, and cell function. Many features of WormBase can accommodate biological information about other species. WormBook (www.wormbook.org) is an open access online review that is indexed in PubMed. Textpresso (www.textpresso.org) is an ontologybased, full-text search engine for biological literature that was developed by WormBase but has been extended to Drosophila, Arabidopsis, rat, and neuroscience, among others. Sentences are indexed according to categories of terms, for example, a “Reporter Gene” category includes “GFP,” “luciferase,” “Cyan Fluorescent Protein,” and so forth, while a human disease gene category includes all names of human diseases. A pilot Textpresso-for-nematodes has been developed and help in developing the search categories is sought. WormBase handles Gene Ontology (GO; www.geneontology.org) annotations for C. elegans and can help process annotation for other species; GO annotations are used in essentially every gene expression profiling experiment. A data integration pipeline to predict genetic interactions based on phenotypic, expression and interaction data in multiple species now includes C. elegans, Drosophila and mouse (www.GeneOrienteer.org). In addition to these resources, three types of genomic experiments will be discussed: gene expression profiling during larval/juvenile growth and arrest, gene expression profiling of single cells using short-read sequencing technology, and comparative genomic analysis of cis-regulatory sequences. ABSTRACTS ~ FRIDAY 12 SEPTEMBERS ~ FRIDAY 12 SEPTEMBER Parasitic helminth genome sequencing M. BERRIMAN, WELLCOME TRUST SANGER INSTITUTE, HINXTON, CB1 3PT, UK The availability of complete genome sequence data from both parasites and their mammalian hosts provides a unique opportunity to take a genomic approach to explore host-/pathogen biology. The helminth genomes initiative at the Wellcome Trust Sanger Institute is aiming to produce reference genomes from a wide phylogenetic spectrum of helminths. Currently Schistosoma mansoni, Haemonchus contortus, Echinococcus multilocularis and Strongyloides ratti, are in active sequencing and additional projects for Nippostrongylus brasiliensis, Onchocerca volvulus, Ascaris lumbricoides, Globodera pallida, and Trichuris muris are just starting. Although, high levels of polymorphism and the lack of inbred lines remain serious problems, steps are being taken to decrease the complexity of the assembly task and produce higher quality drafts. These include using whole genome amplified material from single worms or using new, higher throughput (and lower cost), sequencing technologies to resolve haplotypes from complex mixtures. Data from the projects is made available through two main interfaces: GeneDB – a gene centric database of reference genomes – and the TDR targets database – designed to allow users to prepare their own prioritised and sorted list of molecular targets for drugs discovery, based on available validation criteria. 1 www.genedb.org 2 www.tdrtargets.org The Genome Project of Taenia solium A. GARCIARUBIO, R.J. BOBES, J.C. CARRERO, M.A. CEVALLOS, K. ESTRADA, J.L. FERNÁNDEZ, G. FRAGOSO, P. GAYTÁN, V.M. GONZÁLEZ, M.V. JOSÉ, L. JIMÉNEZ, M.S. JUÁREZ, A. LANDA, C. LARRALDE, J.L.IMÓN-LASSON, L. MENDOZA, J. MORALES-MONTOR, E. MORETT, A. SÁNCHEZ, E. SCIUTTO, X. SOBERÓN, P. DE LA TORRE,, V. VALDÉS, J. YÁNEZ. AND J..P. LACLETTE* INSTITUTO DE INVESTIGACIONES BIOMÉDICAS, INSTITUTO DE BIOTECNOLOGÍA, CENTRO DE CIENCIAS GENÓMICAS, FACULTAD DE MEDICINA AND FACULTAD DE CIENCIAS, UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO, MEXICO, D.F. A consortium of key laboratories at the National Autonomous University of Mexico is carrying out a full genomic project for Taenia solium. This project will provide powerful resources for the study of taeniasis/cysticercosis. The nuclear DNA content estimated through cytofluorometry on isolated cyton nuclei as well as two probabilistic calculations based on shotgun sequenced genomic clones, resulted in a size estimate for the haploid genome of about 130 Mb. A combined strategy with 454 and capillary sequencing is under process. So far, we have achieved 10X coverage by 454 pyrosequencing and 4.5X coverage by capillary sequencing. Results indicate that T. solium genome is not highly repetitive (< 7%). One small 53 bp tandem-repeat and different tetranucleotide repeats represented 0.5% and 4.5% of the genome, respectively. Current assemblage still shows tens of thousands contigs, however, several estimates suggest that 99% of the genes are already included. Besides genomic sequencing, more than 34,000 ESTs have been obtained: 14,113 from adult cDNA libraries and 9,157 from larval libraries, which have been made public through GenBank. Additional 12,200 5’ end sequenced ESTs from a larval full-Length cDNA library are already available. Unique genes were identified by clustering all EST-fragments with an assembler (minimus). We have identified around 7,000 “genes”, some of them are highly expressed in both adult and larvae stages. Thus, there are 349 “genes” with 10 or more sequences that account for 50% of all transcripts. Approximately one third (2,038) of the 7,000 genes have a significant match in SwissProt and about 27% of the genes have no match in SProt + TREMBL, and could constitute new genes. Gene identification in progress is allowing to elucidate some metabolic traits, signal transduction cascades and other physiological routes present in this tapeworm. The consortium for the T. solium genome project wishes to use this International Congress on Helminth Parasites to make a call for collaborative research. 1 This project is supported by a special grant IMPULSAUNAM. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 1S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 1 Sequence of Meloidogyne hapla: A compact nematode genome for plant parasitism D. M. BIRD, C. H. OPPERMAN AND THE M. HAPLA SEQUENCING/ANNOTATION TEAM. CENTER FOR THE BIOLOGY OF NEMATODE PARASITISM, NC STATE UNIVERSITY, RALEIGH NC, USA. Root-knot nematodes (Meloidogyne spp.) are widely distributed throughout temperate and tropical regions and are responsible for yield losses exceeding 10% on food and fiber crops. They also render plants more susceptible to drought stress and are a major contributing factor to a looming world food crisis. We have established Meloidogyne hapla as a tractable model plant-parasitic nematode amenable to forward and reverse genetics. Based on sequencing from multiple libraries (3, 6, 8 kb shotgun libraries; 40 kb fosmids) we have obtained an assembled draft sequence with 10.4X coverage that spans ≥ 98% of the genome in 1,523 scaffolds. At 54 Mb, M. hapla represents not only the smallest nematode genome yet completed, but also the smallest metazoan, and defines a platform to elucidate mechanisms of parasitism by what is the largest uncontrollable group of plant pathogens worldwide. Ab initio gene discovery tools converge on an estimate of 14,494 protein-coding genes (freeze Mh1.1g). More than 20% of these gene models have been confirmed by analysis of transcripts, and over half encode proteins with highly significant matches to proteins in GenBank (<1.0e). The M. hapla genome encodes significantly fewer genes and also shows overall compression of the genome compared to C. elegans. Disparity in gene number between parasite and free-living nematode is particularly striking in gene families. In some instances, most notably the G-protein coupled receptors, this family seems to have expanded in C. elegans to nearly 10-fold over M. hapla. Conversely, M. hapla encodes functions unique to its role as a plant-parasite, with some of these genes apparently acquired horizontally from bacteria and/or plants during its evolutionary history. Some of these genes (such as those encoding pectate lyase, an enzyme required for digestion of carbohydrates in the plant cell wall) appear to have undergone subsequent expansion and diversification; M. hapla encodes 22 pectate lyases. Although M. hapla and C. elegans diverged more than 500 million years ago, many developmental and biochemical pathways, including those for dauer formation and RNAi are strikingly conserved. Understanding how these generalized features of nematode biology have been adapted for a parasitic life-style is of particular interest to our group. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 1S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 1 The genome of Pristionchus pacificus and implications for the evolution of parasitism ROBBIE RAE, GILBERTO BENTO & RALF J SOMMER MAX-PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY, TÜBINGEN, GERMANY Pristionchus pacificus represents a nematode that shares with Caenorhabditis elegans many technical features and has been developed as a model system in evolutionary developmental biology. Many important differences on the genetic and molecular level of development, in particular vulva development, have been identified. Intriguingly, Pristionchus also occupies a distinct ecological niche. Pristionchus nematodes are associated with scarab beetles and have a necromenic life style: that is, worms invade the beetle as dauer larvae and wait for the insect's death to feed on the developing microbes on the carcass. It has been suggested that a close association with other organisms as seen in necromenic nematodes represent a pre-adaptation towards true parasitsm. With 169-Mb and 29,000 predicted protein-coding genes the P. pacificus genome is substantially larger than the genomes of C. elegans and the human parasite Brugia malayi. Comparative analysis with C. elegans revealed an elevated number of genes encoding cytochrome P450 enzymes, glucosyl-transferases and ABC transporters that were experimentally validated and confirmed. P. pacificus contains cellulase and diapausin genes and cellulase activity is found in P. pacificus secretions, the first time cellulases have been identified in nematodes beyond plant parasites. The increase in detoxification and degradation enzymes is consistent with the Pristionchus life-style and is a pre-requisite for parasitism. Thus, comparative genomics of three ecologically distinct nematodes offers a unique opportunity to investigate the correlation between genome structure and lifestyle. Current studies involve developmental and genetic analyses of various traits associated with the specific ecological setting of P. pacificus, such as dauer formation, insect recognition by olfaction, mouth form dimorphism and bacterial association. Specificity of the Pristionchus beetle association is achieved by nematode interception of the insect sex pheromone/sex attractant system. For example, P. maupasi recognizes its cockchafer host by olfaction towards phenol, one of the cockchafers ́ sex attractants, whereas P. pacificus identifies the oriental beetle host by recognition of the sex pheromone (Z)-7-tetradecen-2-one. In contrast, analysis of the Pristionchus associated bacteria did not reveal any specificity so that the ultimate causation of the high specificity of the nematode beetle association remains open. Current analyses investigate a potential competetion between Pristionchus and entomopathogenic nematodes. Pristionchus, unlike many nematodes can feed on bacteria, fungi and other nematodes. This is because Pristionchus nematodes show a mouth dimorphism between a teethless bacterial feeding stenostomatous form and an eurystomatous form with teeth that can feed on fungi and nematodes as well. Genetic analysis suggests that the mouth dimorphism represents a case of phenotypic plasticity and that the ratio of the two forms is influenced by environmental factors, such as starvation. The genetic regulation of the mouth dimorphism is currently investigated and shares several genes with other plastic nematode traits. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 1S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 1 Genetics in the parasitic nematode genus Strongyloides ALEXANDER G. EBERHARDT, LINDA NEMETSCHKE, WERNER E. MAYER, BASSIROU BONFOH, MARK E. VINEY, ADRIAN STREIT. DEPARTMENT OF EVOLUTIONARY BIOLOGY, MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY, TUEBINGEN, GERMANY; 2 CENTRE SUISSE DE RECHERCHE SCIENTIFIQUE EN COTE D‘IVOIRE, ABIDJAN, COTE D‘IVOIRE; SCHOOL OF BIOLOGICAL SCIENCES, UNIVERSITY OF BRISTOL, UK The nematode genus Strongyloides consists of parasites that live as parthenogenetic females in the small intestines of their hosts. In addition to producing parasitic offspring, they can also form a facultative free-living generation with males and females (for review see Viney and Lok, 2007). While some species of Strongyloides, like S. ratti, and the human parasite S. stercoralis, employ an XX/XO sex determining system, S. papillosus, a parasite of farm ruminants, contains no true X chromosome. For this species it has been suggested that males are the product of a sex specific chromatin diminution event which leads to the elimination of a large portion of one of the homologues of a chromosomal pair during the single mitotic oocyte maturation division (for review see Streit, 2008). Contrary to earlier reports that were based on cytological observations, males of S. ratti (Viney et al., 1993) and S. papillosus (Eberhardt et al., 2007) do contribute genetic information to the next generation. We have generated molecular genetic markers for S. ratti and S. papillosus and we are analyzing their inheritance and linkage both genetically and molecularly (FISH). We have shown that in both species recombination within chromosomes occurs. All this indicates that these two species undergo standard sexual reproduction and are therefore amenable to classical genetic analysis and mapping. One of our goals is to complement the ongoing S. ratti whole genome sequencing with a genetic linkage map for this species. We are particularly interested in genetic differences between the two species, which relate to the different sex determining systems. The ability to reliably distinguish between different species is an indispensable prerequisite for comparative studies. Generally all Strongyloides found in farm ruminants were considered to belong to the species S. papillosus, first described as a parasite of sheep. We have analyzed the sequences of the 18S rDNA and of three protein-encoding genes from Strongyloides individuals isolated from sheep and cattle from Germany, Mali and the USA. Our data clearly indicate that there exist at least two different, genetically isolated, sympatric populations that therefore should be considered different, relatively closely related, species. One species was the predominant Strongyloides in cattle and was found in this host only. The other species was the only species we found in sheep but did also occur in cattle at low numbers. Eberhardt, A.G., Mayer, W.E., Streit, A. (2007) Int. J. Parasitol. 37:989-1000. Streit, A. (2008) Parasitol. 135:285-294. Viney, M.E., Lok, J.B. (2007) In: Community, T.C.e.R. (Ed.), WormBook, http://www.wormbook.org. Viney, M.E., Matthews, B.E., Walliker, D. (1993) Proc. R. Soc. Lon. B 254:213-219. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 2S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 2 Developing Haemonchus contortus as a model parasite for forward genetic studies of anthelmintic resistance JOHN GILLEARD, NEIL SARGISON , LIBBY REDMAN, FIONA WHITLOW, AND FRANK JACKSON. 1 DEPT OF COMPARATIVE BIOLOGY AND EXPERIMENTAL MEDICINE, FACULTY OF VETERINARY MEDICINE, UNIVERSITY OF CALGARY, CANADA DIVISION OF INFECTION AND IMMUNITY, FACULTY OF VETERINARY MEDICINE, UNIVERSITY OF GLASGOW, 3 MOREDUN INSTITUTE, EDINBURGH, UK Genetic analysis has been central to the progress made in many areas of biological research ranging from model organism biology to human medicine. However, although there are many potential applications, genetic approaches have been under-exploited in parasitic helminth research with studies being largely limited to a relatively small number of population genetic studies. Although there has been significant progress in the use of forward genetic analysis to map genes associated with traits such as drug resistance and virulence in parasitic protozoa, similar approaches have yet to be applied to parasitic helminths. However, many parasitic helminth species should be amenable to forward genetic analysis and the vast amount of sequence data currently being generated from parasitic helminth genome projects provides major new opportunities in this area. The parasitic nematode of sheep, H. contortus is a good system in which to explore parasite genetics for a number of reasons. The ability to surgically transplant adult worms into the sheep abomasum, together the parasite’s extremely high fecundity makes it possible to undertake genetic crosses. The availability of characterized, genetically differentiated drug resistant and susceptible isolates and the increasing amount of genome sequence available provides unprecedented opportunities for the analysis of genetic crosses. We have developed panels of autosomal and X-linked microsatelletie markers and used them to study the basic genetics of this parasite to provide a framework for forward genetic analysis. We have shown female and male worms have XX and XO karyotypes respectively and that polyandrous mating occurs. A single female can contain the progeny of up to four male worms providing an additional mechanism of generating genetic diversity. We have developed an approach to produce highly inbred lines for genetic crossing and have validated the inbreeding process using genetic markers. We have also undertaken a series of backcrosses between the susceptible isolate MHco3(ISE) (the isolate being used for the genome sequencing) and two different ivermectin resistant isolates. Five generations of backcrossing these resistant isolates against MHco3(ISE) in the presence of ivermectin selection have been performed to introgress resistance genes from the two genetically divergent isolates into the MHco3(ISE) (susceptible) genetic background. The backcrossed isolates will be a valuable resource for genetic mapping and other molecular studies to identify ivermectin resistance loci. Expression of potential control targets of parasitic nematodes using Caenorhabditis elegans C. BRITTON, A.B. ROBERTS, L MURRAY AND D.P. KNOX. DIVISION OF VETERINARY INFECTION AND IMMUNITY, UNIVERSITY OF GLASGOW VETERINARY SCHOOL, GLASGOW, UK AND DIVISION OF PARASITOLOGY, MOREDUN RESEARCH INSTITUTE, EDINBURGH, UK. Parasitic nematodes of livestock are a significant economic and welfare problem. Anthelmintic drugs are currently used to control infection, however the efficacy of these is decreasing due to the emergence of drug resistant nematodes and alternative controls are needed. For the sheep parasitic nematode Haemonchus contortus, a number of native proteins and protein complexes extracted from the parasite can induce significant protective immunity. However, protection trials using recombinant forms of these proteins expressed in bacteria, yeast or insect cells have demonstrated that the recombinant proteins are far less effective. This could be for a number of reasons, including the possibility that not all components of the native extract are present in the recombinant vaccines, as well as the difficulties associated with protein folding and correct glycosylation using standard expression systems. We are using Caenorhabditis elegans to express potential control targets of Haemonchus, with the aim of producing these in a form with similar conformation and glycosylation to the native proteins. We have expressed H. contortus cathepsin L cysteine protease in C. elegans and shown that the parasite gene can rescue C. elegans cathepsin L (cpl-1) mutants. The C. elegans expressed protease is active and glycosylated. We are now testing expression of previously identified protective proteins including the gut aminopeptidase H11 and cathepsin B protease gene family. Different C. elegans promoters and 3’UTRs are being used to try to optimise expression levels. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 2S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 2 Functional analysis of Ostertagia ostertagi vaccine candidates in Caenorhabditis elegans GELDHOF P., VISSER A. , MEYVIS Y. , HOUTHOOFD W. , BRITTON C. , VERCRUYSSE J. , CLAEREBOUT E. . LABORATORY OF PARASITOLOGY, FACULTY OF VETERINARY MEDICINE, GHENT UNIVERSITY, BELGIUM, DEPARTMENT OF BIOLOGY, GHENT UNIVERSITY, BELGIUM, INSTITUTE OF COMPARATIVE MEDICINE, UNIVERSITY OF GLASGOW, GARSCUBE STATE, GLASGOW, UK. Previous vaccination trials against the abomasal nematode Ostertagia ostertagi in cattle have demonstrated the protective capacity of a protein fraction termed ES-thiol. Mass spectrometric analysis revealed that this fraction is highly enriched for two activation-associated secreted proteins (ASP-1 and -2) and contains, amongst others, a translationally controlled tumor protein (TCTP). The biological role of these molecules in O. ostertagi and other nematodes is still unclear. Therefore, the objective of this study was to further unravel the function of these molecules by using the model organism Caenorhabditis elegans. Database searches indicated that C. elegans contains around 36 ASP encoding genes of which VAP-1, T05A10.4 and T05A10.5 show the highest sequence similarity with the O. ostertagi ASPs. VAP-1 is a two domain ASP, transcribed in the amphid sheath cells and is upregulated during the aging of the worms. RNAi mediated knockdown of vap-1 results in a significant extension of the lifespan of adult worms and has an effect on the stress response of the worms. Although T05A10.4 and T05A10.5 were predicted to be 2 separate genes, RT-PCR experiments showed that they are actually part of the same gene encoding for a second two domain ASP in C. elegans. The gene is expressed in the excretory duct cell and in some hypodermal cells of the tail. The localization and the observed phenotypic effects suggest a role of these ASPs in signal transduction. TCTP on the other hand seems to be a single-copy gene in nematodes. In O. ostertagi, the transcription pattern of TCTP indicated an upregulation in adult female worms and eggs. These results were confirmed by Western blotting and immunolocalisation, which showed that TCTP was highly present in the developing eggs of both O. ostertagi and C. elegans. RNAi mediated knockdown of tctp in C. elegans rendered a 90 % reduction in egg production, indicating a crucial role of this protein in nematode egg production and development. Functional expression of parasitic SLO-1 in Caenorhabditis elegans slo-1 knockout mutants C. WELZ* AND N. FISCHER, M. GUEST, L. M. HOLDEN-DYE, A. HARDER, T. SCHNIEDER, AND G. VON SAMSON-HIMMELSTJERNA. INSTITUTE FOR PARASITOLOGY, UNIVERSITY OF VETERINARY MEDICINE HANNOVER, GERMANY, SCHOOL OF BIOLOGICAL SCIENCES, UNIVERSITY OF SOUTHAMPTON, UK; BAYER HEALTHCARE AG, MONHEIM; GERMANY The calcium-gated potassium channel SLO-1 is of major importance for the effects of the new anthelmintic drug emodepside on the locomotory behaviour of Caenorhabditis elegans. In a recent study, C. elegans slo-1 functional null mutants were shown to be highly resistant to emodepside, as they could be grown on agar plates containing 1 μM emodepside, a concentration, which immobilises wild-type worms. Expression of SLO-1 driven by either the neuronal snb-1 promotor or the muscle-specific myo-3 promotor reconstituted the susceptibility of the knockout mutants to emodepside. In the present study, we identified homologues of slo-1 in the canine hookworm Ancylostoma caninum, the barber pole worm in sheep, Haemonchus contortus, and the bovine trichostrongyle Cooperia oncophora. The identities of the amino acid sequences were 96 to 98 % with each other and 88 % with C. elegans SLO-1. We expressed the slo-1 coding sequences of A. caninum and of C. oncophora in the C. elegans null mutant slo-1(js379). As promotors the neuronal snb-1 promotor, which was also used in the previous study on C. elegans, and the putative C. elegans slo-1 promotor (3 kb in size) were used. To determine susceptibility to emodepside, successfully transfected worms were incubated on agar plates containing emodepside and the locomotory behaviour was evaluated by counting the number of body bends per minute. Expression of SLO-1 from parasitic nematodes, driven by the snb-1 promotor, in the C. elegans null mutant slo-1(js379) significantly increased susceptibility of the worms to emodepside. Furthermore, expression of SLO-1 under the C. elegans slo-1 promotor in the slo-1 (js379) mutant background restored the sensitivity of the mutant to emodepside to the same level as wild-type worms. Experiments were carried out with emodepside concentrations between 1 nM and 10 μM, and the observed effects were dose-dependent. These data confirm that emodepside signals through the calcium-activated potassium channel of both C. elegans and parasitic nematodes and the experiments provide C. elegans strains expressing the parasite target for further functional and pharmacological analysis. Acknowledgement: Thanks to Lawrence Salkoff for the snb-1 promotor and to the C. elegans Genetics Centre, Washington, for the mutant strain slo-1(js379) ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 2S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 2 Transgenesis in Strongyloides stercoralis and S. ratti: prospects for transposon-mediated gene transfer and applications in studies of gene function, immunology and sensory neurobiology JAMES B. LOK. DEPARTMENT OF PATHOBIOLOGY, SCHOOL OF VETERINARY MEDICINE, UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, PA, USA At Hydra 2005 we reported that regulated expression of a reporter transgene fusing the era-1 promoter to the gfp coding sequence could be achieved in Strongyloides stercoralis by incorporating the 3’ UTR for era-1 into the construct. Since then, we have demonstrated that the Ss-era-1 3’ UTR can act as a multi-purpose terminator for reporter transgene constructs with promoters driving either ubiquitous reporter expression (Ssrps-21) or cellor tissue-specific expression in such anatomical sites as the intestinal epithelium (Ss-era-1), body wall muscle (Ss-act-2) and amphidial neurons (Ss-gpa-3). We have demonstrated that F1 transgenic S. stercoralis are capable of infecting gerbils and maturing to parasitic females in the intestine while maintaining transgene expression. Transgene DNA can be detected in progeny of transgenic S. stercoralis to the F5 generation, but to date, none of our constructs is expressed after the F1 generation. Recently, we found that Strongyloides ratti, can be transformed, and exhibits celland tissue-specific transgene expression using constructs developed for S. stercoralis. We recently used our transgenesis system to investigate the function of the forkhead transcription factor encoded in S. stercoralis fktf-1. This factor is orthologous to DAF-16, which regulates dauer development and lifespan in C. elegans. Expression of recombinant FKTF-1 fused to GFP in S. stercoralis reveals that, like its C. elegans ortholog, this transcription factor is localized in cell nuclei of larvae destined for development to the dauer-like L3i. Moreover, expressing a putative dominant loss-of-function mutant FKTF-1 results in severe defects in pharyngeal morphology in transgenic S. stercoralis, suggesting a role for FKTF-1 in pharyngeal development. With an eye toward achieving heritable transgene expression, we are investigating transposons as means of integrating constructs into the genome of Strongyloides spp. To this end, we are evaluating an exogenous transposon, PiggyBac, which is capable of integrating DNA sequences into the genome of Schistosoma mansoni, and an endogenous transposon, designated SMART-1, which we have recently discovered in S. stercoralis. We are assessing the utility of transgenic S. ratti in studies of the immune response to infection in mice, where this parasite establishes a patent infection of low intensity and short duration. We have synthesized constructs designed to express avian ovalbumin (OVA) under the control of various tissue-specific promoters and are evaluating the OVA-specific response to worms carrying these transgenes in mice adoptively transferred with OVA-specific CD4 T cells. Finally, we are studying the feasibility of genetically targeted neuronal ablation in Strongyloides spp. In C. elegans, expression of caspases or mutant channel forming proteins under the control of neuronor other cell-specific promoters has been applied in this manner. We intend to express such lytic or apoptotic factors under the control of neuron-specific promoters such as Ss gpa-3 as a means of assessing functions of chemosensory neurons in Strongyloides spp. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 3S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 3 Regulation and function of fktf-1 in transgenic Strongyloides stercoralis MICHELLE L. CASTELLETTO, HOLMAN C. MASSEY JR., JAMES B. LOK SCHOOL OF VETERINARY MEDICINE, UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, PA, USA Strongyloides stercoralis has an unusual life cycle consisting of alternating free-living and parasitic generations. When appropriately triggered via external stimuli or internal genetic mechanisms, the post-parasitic larvae can develop either into a free-living generation, which can be cultured in vitro, or directly into the infective third stage. The developmental switch in post-parasitic L1s, the free-living generation and the morphological similarities to Caenorhabditis elegans make S. stercoralis an attractive model organism to study the development of parasitic infective larvae. Insulin-like signaling is one well-characterized mechanism directing C. elegans' larval development. Specifically, it negatively regulates the function of daf-16, a forkhead transcription factor that, when active, directs the development of the arrested third-stage larva. fktf-1, an S. stercoralis ortholog of daf-16, is hypothesized to regulate larval development in a similar manner to daf-16. Free-living S. stercoralis females were transformed with transgenes encoding gfp reporters under the direction of an fktf-1β promoter. Transgenic first-stage larvae were scored for GFP expression as well as tissue and subcellular localization. 60% of transgenic larvae showed GFP expression in either the hypodermis or the pharyngeal procorpus, 30% had expression in both sites. A GFP::FKTF-1b fusion protein under direction of the fktf-1β promoter was localized to the nuclei of hypodermal cells in transgenic larvae. In contrast, when GFP alone was expressed from the transgene it was cytoplasmic in hypodermal cells. fktf-1b, like daf-16, has three Akt/PKB phosphorylation consensus sites plus a fourth overlapping site in the forkhead domain. When phosphorylated, the transcription factor is inactivated and exported from the nucleus. We mutated two phosphorylation sites to phospho-mimetics and found that localization of the GFP::FKTF-1b(S238E/T240E) fusion protein was cytoplasmic in hypodermal cells, as opposed to nuclear with the wild-type sequence. In order to investigate the function of fktf-1b using transgenesis, we created a dominant-repressor construct to antagonize the function of the endogenous fktf-1b. First, alanine mutations were made in the Akt/PKB consensus sites to create a protein unable to be exported from the nucleus. Second, the transactivation domain was replaced with a repressor domain from C. elegans' pie-1. Finally, gfp was fused to the 5' end to easily visualize the protein. Transgenic larvae expressing the dominant-repressor version of GFP::FKTF-1b show defects in overall growth as well as defects in pharyngeal and intestinal morphology. The intestines are shrunken and appear to have a loss of granularity compared to age-matched larvae. The larvae survive as L1s, however they fail to grow or to develop into L3is. Using transgenesis, we have found that fktf-1b is active predominantly in tissues remodeled during infective larval development namely the hypodermis and pharyngeal procorpus, is localized to cell nuclei in larvae developing into L3is, and appears to be regulated posttranscriptionally via phosphorylation in a similar manner to daf-16. Furthermore, transgenic larvae expressing a dominant-repressor form of FKTF-1b have altered pharyngeal morphology and fail to develop into infective larvae. These findings support the hypothesis that fktf-1b regulates larval development in Strongyloides stercoralis. Analysis of transcription and mRNA processing in Brugia malayi using a homologous transient transfection system THOMAS R. UNNASCH The recent completion of the sequence of the B. malayi genome has provided a wealth of information regarding gene structure in this parasite. However, sequence data alone provide little information regarding the mechanisms that filaria use to regulate gene expression. Reverse genetic techniques, such as transfection, have proven to be quite useful in the study of the mechanisms of gene regulation in other organisms. We have developed a method to transiently transfect B. malayi, and have used this method together with a luciferase reporter assay system to begin to dissect the cis-acting factors that are necessary for transcription in B. malayi. An analysis of several B. malayi promoters using this system has revealed that the core promoter domains of this organism appear to lack many of the features that characterize a typical eukaryotic promotor. In addition, we have used the transient transfction system to study trans-splicing in vivo in B. malayi, and have demonstrated that trans splicing is dependent on cis-acting elements that are encoded downstream of the promoter domain. These studies provide evidence to demonstrate that transient transfecion may be a useful tool to study both mRNA processing and gene regulation I the filarial parasites. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 3S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~ SESSION 3 Integration of reporter transgenes into Schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus KRISTINE J. KINES, MARIA E. MORALES, VICTORIA H. MANN, GEOFFREY N. GOBERT, PAUL J. BRINDLEY. DEPARTMENT OF MICROBIOLOGY, IMMUNOLOGY & TROPICAL MEDICINE, GEORGE WASHINGTON UNIVERSITY MEDICAL CENTER, WASHINGTON DC, USA; BIOMEDICAL SCIENCES PROGRAM, TULANE UNIVERSITY HEALTH SCIENCES CENTER, NEW ORLEANS, LOUISIANA USA; DIV OF INFECTIOUS DISEASES, QUEENSLAND INSTITUTE OF MEDICAL RESEARCH, BRISBANE, AUSTRALIA The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomules were exposed to virions of VSVGpseudotyped MLV after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCRbased approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic and others, and also near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomules and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomules with increased activity observed after one to two weeks in culture. These findings indicate the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward towards germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes. ABSTRACTS ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~SESSION 3S ~ SATURDAY 13 SEPTEMBER ~ DAY 1 ~SESSION 3 Cestode stem cells: their role in host-induced larval development and their utilization to achieve parasite transgenesis K. BREHM. INSTITUTE OF HYGIENE AND MICROBIOLOGY, JULIUS-MAXIMILIANS-UNIVERSITY, WÜRZBURG, GERMANY A population of totipotent stem cells, called germinal cells or neoblasts, is central to the developmental biology of flatworms. In free-living planarians, these cells are crucially involved in regeneration. In parasitic flatworms, a role of totipotent stem cells in asexual proliferation of larvae as well as ‘never-ending’ growth of tapeworms has been discussed. Investigating the role of germinal cells in larval development of the model cestode Echinococcus multilocularis, we were recently successful in isolating germinal cells and in establishing primary cultures that are devoid of contaminating host cells. When incubated in the presence of host hepatocytes, physically separated through a trans-well system, or hepatocyte supernatant, the germinal cells proliferated and formed cell aggregates which contained central cavities surrounded by the growing germinal layer. After six weeks of cultivation, complete and fully infective metacestode vesicles were present, indicating that the ‘metamorphosis’ of the oncosphere towards the metacestode within the host’s liver (i.e. the initial phase of infection) can be mimicked in vitro by our system. Several hormones and cytokines which are present in high concentrations in the liver, such as insulin, significantly stimulated metacestode formation from germinal cells. Furthermore, isolated germinal cells expressed surface receptor kinases of the insulin-, EGF-, and FGFreceptor families which we have shown to be capable of interacting with the corresponding host cytokines. Signalling pathways that act downstream of receptor kinases, such as the MAP kinase cascade, were also stimulated within germinal cells in the presence of host insulin, EGF and FGF. Taken together, these data indicate that germinal cells, which are delivered to the host liver by the oncosphere, are able to sense the cytokine milieu of this host organ which finally results in germinal cell activation and metacestode formation at this specific site in the intermediate host. The ability to generate infective parasite tissue from single cells now also allows the production of fully transgenic parasites once the methodology to introduce foreign DNA into germinal cells is available. Toward this end, we have successfully used transfection reagents to transiently express a heterologous reporter protein in cultivated germinal cells. Furthermore, we were able to stably introduce foreign DNA into the genome of germinal cells using heterotypic lentiviruses, which also resulted in heterologous reporter gene expression. Heterotypic lentiviruses and the in vitro germinal cell cultivation system are currently used in our laboratory to produce the first fully transgenic strain of E. multilocularis. In conjunction with the E. multilocularis whole genome sequencing project that is currently in an advanced stage, the established methods of in vitro larval and germinal cell cultivation as well as the ability to produce fully transgenic parasites will greatly facilitate molecular studies on E. multilocularis development and host-parasite interaction. Since the methods to in vitromanipulate E. multilocularis cells should principally also be applicable to other tapeworms, the entire field of cestode molecular biology is expected to profit from our results. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4 Signaling molecules involved in gonad differentiation of Schistosoma mansoni C. G. GREVELDING, T. QUACK, S. BECKMANN, J. KNOBLOCH, C. DISSOUS AND C. BURMEISTER INSTITUTE FOR PARASITOLOGY, JUSTUS-LIEBIG-UNIVERSITY GIESSEN, GERMANY; INSTITUTE FOR ANIMAL DEVELOPMENTAL AND MOLECULAR BIOLOGY, HEINRICH-HEINE-UNIVERSITY DÜSSELDORF, GERMANY; UNITÉ INSERM 547, INSTITUT PASTEUR, LILLE CEDEX, FRANCE. Schistosomes are the only members of the trematodes which live dioeciously. The sexual maturation of the female essentially depends on signals from the male inducing mitoses and differentiation processes in the vitellarium. Here, vitelline cells develop for the production of eggs, which are responsible for pathogenesis in the final host. Upon separation from the male, mitoses and vitelline cell differentiation in the female decline, and egg production stops. These effects are reversible upon re-pairing. Although known for a long time, the molecular basis of this phenomenon is poorly understood. In an approach to identify molecules involved in the sexual maturation of schistosome females, we found among other signaling molecules Src and Syk tyrosine kinases (TK), and the transforming growth-factor β receptor 1 (TGFβ-R1) to play important roles during gonad differentiation. The Src-kinase inhibitor Herbimycin A, or the TGFβ-R1-kinase inhibitor blocked the activity of these signaling molecules in adult females kept in vitro. As revealed by the DAPI-staining/BrdU-incorporation technique, inhibitor treatment significantly reduced mitoses in paired females. In addition, egg production declined. A combined treatment with both inhibitors led to an additive effect indicating that at least two pathways cooperatively activate mitotic activity and egg production in paired females. To unravel signalling cascades, in which the schistosome Src-kinase SmTK3 is involved, we searched for its binding partners by Yeast-Two-Hybrid (Y2H) analyses. For this we generated an Y2H-cDNA library on the basis of poly(A)-RNA of male and female S. mansoni. Using SH3-domain constructs with or without the N-terminally located unique site of SmTK3 as baits for library screening identified the diaphanous homolog SmDia as the most prominent “downstream” binding partner. With the SH2 domain as bait to screen for potential “upstream” interaction partners, we identified the epidermal growth factor substrate protein 8 (SmEps8), a known multifunctional signaling molecule in eukaryotes. For characterization we used deletion constructs of SmEps8 and provide evidence for an atypical binding between SmTK3 and SmEps8 in Y2H interaction-studies. Since an Eps8-typical binding-domain for the epidermal growth-factor receptor (EGFR) exists also in SmEps8, we have started to investigate its binding activity to the known EGFR of S. mansoni (SER). Besides this, in situ hybridization experiments have demonstrated the transcription of the SmEps8 gene in the reproductive organs of both genders, which corresponds to the transcript localizations of SmTK3 and SmDia. Since homologs of Eps8, diaphanous, and Src kinases have been shown to be involved in receptor signalling, cytoskeleton remodelling and cell proliferation in other eukaryotes, and since the schistosome homologs SmTK3, SmDia and SmEps8 interact in Y2H assay and colocalize in the reproductive organs of S. mansoni, we conclude that these molecules fulfil cooperative functions during gonad differentiation in this parasite. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4 Developmental genes in the life cycle of a parasitic flatworm P. D. OLSON, THE NATURAL HISTORY MUSEUM, LONDON, U.K. Tapeworms have exploited the regenerative and proliferative abilities of stem cells to extraordinary ends and provide many excellent models for understanding the genetic basis of morphological disparity and adaptation. From an evolutionary perspective, their typically segmented form is seen to have evolved in a step-wise fashion, first through the serial repetition of their hermaphroditic reproductive organs (ie. ‘proglottization’) followed subsequently by their somatic compartmentalization (ie. ‘segmentation’). Whether these processes are novel or share a genetic programme homologous with other lophotrochozoans (eg. annelids and molluscs) or with the more basal, strobilate cnidarians (eg. jellyfish) is a question that can inform both flatworm development as well as the degree of genomic conservation in the animal kingdom. To address this, and to resolve more basic outstanding questions such as their correct anteroposterior (AP) orientation (ie. is the scolex in fact anterior?) and the possible change in polarity during metamorphosis from larval to adult form, information from Hox, ParaHox and other homeobox-containing genes is being examined. Using a fully segmented, adult model tapeworm (Hymenolepis), homeodomains of anterior (Hox1/Lab; Hox4/Dfd), central (Lox4/AbdA) and posteriorclass (Post-1/2) Hox genes were recovered and their full transcripts characterized by PCR RACE. Full-length genes were then amplified, cloned and used as templates for reverse transcribing digoxigenin-labelled riboprobes, and the spatial expression patterns of the genes in adult worms examined by whole-mount in situ hybridization. Initial efforts achieved highly specific patterns for two of the genes that implicate potentially direct roles in the processes described above: the posterior gene Post-2 is seen expressed in bands at the leading (ie. growing) margin of each segment, indicative of having a role in the process of segmentation, whereas expression of the central gene Lox4 is associated strictly with the developing ovaries and thus either promotes ovarian development or alternatively, is being expressed by the ovaries themselves. It is thus implicated in playing a role in proglottization and, depending on the relationship between cause and effect, may be responsible for differences in the timing of development of male and female systems and for the loss or gain of hermaphroditism. Alternatively, Lox4 may be acting to coordinate development in response to the maturing ovaries. Ongoing work aims to examine the expression of these and other homeoboxes throughout the complex ontogeny of cestodes and to arbitrate on cause and effect by inferring the functions of the genes using knockdown techniques. Once understood in the model system, this work can be extended to non-model groups exhibiting fundamentally different body plans and reproductive strategies and in this way be integrated with our existing knowledge of tapeworm phylogeny. The work also provides inroads for examining the roles of homeoboxes in parasitic flatworms more broadly and thus the ability to contrast these roles with those found in free-living groups whose regenerative abilities have made them long-serving models in developmental biology. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4 The Syk-kinase SmTK4 from Schistosoma mansoni: upstream interaction partners and functional aspects in oogenesis and spermatogenesis S. BECKMANN*, J. HIRZMANN, C. DISSOUS, AND C. G. GREVELDING 1 INSTITUE FOR PARASITOLOGY, JUSTUS-LIEBIG-UNIVERSITY, GIESSEN, GERMANY. 2 UNITÉ INSERM 547, INSTITUT PASTEUR, LILLE CEDEX, FRANCE. The parasitic helminth Schistosoma mansoni is the causing agent of schistosomiasis, one of the most prevalent parasitic infections in the world. Schistosomes, which represent the only family within the trematodes being dioecious, show unique biological features. Among these is the sexual maturation of the female, which depends on a constant pairing contact with the male. Upon pairing the reproductive organs of the female differentiate, a prerequisite for egg production leading to pathogenesis. Between the two genders, molecular communication processes occur, which probably include signal transduction cascades. During our attempts to identify signaling molecules from S. mansoni we isolated the Syk-family kinase SmTK4, which is tissue-specifically transcribed in the ovary and in the testes of adult schistosomes. In other biological systems, Syk-kinases are members of signaling cascades controlling differentiation processes. To elucidate signaling cascades in which TK4 may be involved, we searched for interaction partners by yeast two-hybrid analyses. First evidence is provided that SmTK4 is a member of a kinase complex interacting with the Src-kinase SmTK3 and with the newly detected Src/Abl-kinase SmTK6 from S. mansoni. Furthermore, an interaction of these three cellular tyrosine kinases with the cytoplasmatic part of the schistosome receptor tyrosine kinase SmVKR (SmRTK1) was demonstrated. In situ hybridization confirmed colocalization of the cellular kinases and the receptor kinase in reproductive organs. These results lead to the postulation of a receptor tyrosine kinasemediated signaling pathway in the ovary and testes, probably involved in cell proliferation and/or differentiation. In addition to the yeast two-hybrid experiments we performed inhibitor and RNAi experiments to investigate the function of SmTK4 in the reproductive organs. Treatment of adult schistosomes with a Syk kinase-specific inhibitor showed clear phenotypes in the ovary and the testes by confocal laser scanning microscopy. These results corresponded to data obtained by an RNAi approach aiming to knock-down SmTK4 transcription. From the phenotypic analyses we conclude that SmTK4 plays a central role in spermatogenesis and oogenesis of S. mansoni. These data strengthen the concept that specific tyrosine-kinase inhibitors may be suitable candidates for novel chemotherapeutic strategies to fight schistosomes. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 4 A diverse family of Kunitz inhibitors from Echinococcus granulosus involved in host-parasite cross-talk in echinococcosis M. FLÓ, M. MARGENAT, S. GONZÁLEZ, R. M. MAIZELS, G. SALINAS, B. ÁLVAREZ, C. FERNÁNDEZ. CÁTEDRA DE INMUNOLOGÍA, FACULTAD DE QUÍMICA AND LABORATORIO DE ENZIMOLOGÍA, FACULTAD DE CIENCIAS, UNIVERSIDAD DE LA REPÚBLICA, URUGUAY, INSTITUTE OF IMMUNOLOGY AND INFECTION RESEARCH UNIVERSITY OF EDINBURGH, UNITED KINGDOM. The successful establishment of a parasite relies, to a great extent, on the efficiency of secreted molecules acting through high affinity interactions with host counterparts at the initial phases of infection. We present a molecular and functional description of a family of Kunitz inhibitors (whose prototype is the trypsin inhibitor from bovine pancreas), predicted to be secreted by E. granulosus larval worms (protoscoleces), and provide evidence supporting their participation in parasite establishment in the duodenum of its definitive dog host. Kunitz inhibitors were found to predominate among protoscolex secreted proteins: eight such molecules (EgKU1 to EgKU-8) were identified while surveying the larval transcriptome. The predicted mature peptides contain a single ‘Kunitz domain’: about 50 amino acids cross-linked by three conserved disulphide bonds. EgKU-1 – EgKU-8 differ in MW (7-10 kDa), pI (5-10) and anti-proteinase site. Consequently, they could show diverse specificities if acting as serine protease inhibitors. Importantly, their expression is induced by exposure to pepsin/H, a stimulus the worms naturally encounter upon ingestion by the dog. In addition, EgKU-1/EgKU-4, EgKU-3/EgKU-8 and EgKU-6/EgKU-7 represent three pairs of close paralogues that would have arisen from recent gene duplications. Interestingly, putative orthologues of EgKU-3, EgKU-4 and EgKU-8; and of EgKU-2 were respectively found among E. multilocularis and Taenia solium ESTs. Furthermore, genes encoding all eight members of the family were identified in the E. multilocularis draft genome. These sequences indicate that the genes encoding the pairs of paralogues are closely located in the genome. Detailed kinetic studies carried out with EgKU-1 and EgKU-8 purified to homogeneity from protoscolex lysates showed that EgKU-8 is a powerful slow tight-binding trypsin inhibitor. Remarkably, EgKU-8 displayed higher affinity towards dog trypsin than towards the bovine enzyme. In contrast, no inhibition of a panel of serine proteases was observed with EgKU-1. Interestingly, molecular modelling revealed a clear structural similarity between EgKU-1 and the snake venom dendrotoxins, which are Kunitz proteins that selectively block specific subtypes of voltage–gated K channels. We are currently studying whether EgKU-1 is indeed capable of acting in a similar way. We are also functionally characterising other members of the family using the corresponding recombinant proteins. Recombinant EgKU-3, in particular, was found to be a potent inhibitor of chymotrypsins. Finally, we are analysing the presence of Kunitz proteins in the secretions of protoscoleces exposed to different stimuli, using ‘intensity fading’ MALDI-TOF MS, a methodology elegantly combining affinity purification and MS. So far, this approach has confirmed the presence of EgKU-3 and EgKU-8 in pepsin/H-treated protoscolex secretions. This family of secreted cestode molecules would provide a striking example of protein evolution, similar to the one described in animal toxin multigene families, where natural selection has diversified the duplicated genes, allowing the emergence not only of paralogues whose products would interact with paralogous proteins (i. e. specific inhibitors of particular enzymes); but also of a new function associated with the same molecular scaffold. We discuss the significance of the family in terms of putative targets of the Kunitz inhibitors in the protoscolex establishment scenario. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 5S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 5 TGF-beta and hedgehog signalling pathways in Trichinella spiralis B. CONNOLLY*, E. M. SHEILS, M. W. ROBINSON AND D. H MASSIE. INSTITUTE OF MEDICAL SCIENCES, UNIVERSITY OF ABERDEEN, ABERDEEN, SCOTLAND, UK. The recent release of genome sequence information is revolutionising the study of helminth parasites by providing important datasets for comparative genomics that will allow us to analyse the signalling pathways that regulate nematode parasite development. Much of our current knowledge of nematode signalling pathways is based on the study of the free-living model Caenorhabditis elegans. While most are conserved (e.g TGF-beta pathway), a number of pathways are missing in C. elegans, e.g. the JAK/STAT signalling pathway, or are incomplete. Most strikingly, the hedgehog signalling pathway in C. elegans is modified and lacks a bone-fide Hedgehog ligand. Studies indicate that other chromadorean nematodes (e.g. Brugia malayi) are likely to be similar to C. elegans. The recent availability of the draft genome sequence of the enoplean Trichinella spiralis has presented an opportunity to study signalling pathways in a basal nematode. We have undertaken an analysis of the hedgehog and TGF-beta signalling pathways in T. spiralis and have identified a gene encoding a bone-fide hedgehog ligand. Data on the Hedgehog expression and localisation will be presented. In addition we have identified 5 genes encoding TGF-beta-like ligands, three of which belong to the BMP subfamily and are closely related to the vertebrate BMP2/4, BMP5/8 and BMP3 proteins. The remaining two proteins clearly belong to the TGF-beta/activin subfamily. To date only one member (DAF-7) of this subfamily has been identified in other nematode species, including C. elegans and there is some support for grouping one of the Trichinella proteins with the nematode Daf-7-like proteins. In contrast, the second protein shares significant amino acid identity with myostatin, a TGF-beta ligand involved in regulating muscle mass in adult vertebrates. The identification of a myostatin in Trichinella is unique among the nematodes to date and raises the possibility that it may play a role in altering gene expression in skeletal muscle cells postinfection.TGFbeta signaling in schistosomes TGFbeta signaling in schistosomes EDWARD J. PEARCE & TORI C. FREITAS, DEPARTMENT OF PATHOBIOLOGY, UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, PA 19104, USA. Schistosomes are complex metazoan pathogens that belong to an early diverging branch of the Bilateria, the Lophotrochozoans. Little is known of the molecular basis of cell to cell communication in animals in this group, but targeted studies and genome sequencing efforts have revealed that, not surprisingly, schistosomes contain some of the intercellular signaling systems that are found in higher order metazoa. Amongst these, we are particularly interested in the transforming growth factor beta (TGFbeta) pathways. Members of the TGFbeta superfamily can be split into two main subfamilies based on sequence homology and the different downstream pathways they activate, namely, the TGFbeta/activin/nodal subfamily and the BMP/GDF/MIS (bone morphogenetic protein/growth and differentiation factor/Muellerian inhibiting substance) subfamily. The basic TGFbeta signaling mechanism involves the binding of the extracellular TGFbeta homologue to a heterodimeric receptor complex resulting in the activation of specific cytoplasmic proteins (Smads) that translocate to the nucleus to influence transcriptional responses. Several components of the TGFbeta signaling pathway have been characterized from S. mansoni including TGFbeta receptors SmRK1 (also known as SmTbRI) and SmRKII (also known as SmTbRII); several Smad proteins, and one homologue of the TGFbeta subfamily, SmInAct. Analysis of the Wellcome Trust’s Sanger Institute’s S. mansoni genome sequence database has revealed that schistosomes possess at least two additional Type 1 receptors, one additional Type 2 receptor and one homologue of the BMP subfamily (SmBMP). With the isolation of the BMP homologue from S. mansoni, we now have representatives from each major subfamily of the TGFbeta superfamily in this parasitic flatworm. The current state of the S. mansoni genome sequence suggests SmBMP and SmInAct are the only two TGFbeta-like ligands present. The functions of SmInAct and SmBMP, as deduced from studies using RNAi and specific inhibitors of TGFbeta signaling, will be discussed. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 5S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 5 The nicotinic acetylcholine receptors of Ascaris suum S.M. WILLIAMSON, L. BROWN , A. ROBERTSON , R. MARTIN , T. WILLIAMS , D. WOODS , D.B. SATTELLE , A.J.WOLSTENHOLME 1. 1 DEPT. BIOLOGY AND BIOCHEMISTRY, UNIVERSITY OF BATH, UK 2 MRC FUNCTIONAL GENETICS CENTRE, OXFORD, UK 3 DEPT. BIOMEDICAL SCIENCES, COLLEGE OF VETERINARY MEDICINE, IOWA STATE UNIVERSITY, USA 4 PFIZER ANIMAL HEALTH, KALAMAZOO, USA Nematodes of the genus Ascaris are gastrointestinal parasites of medical and veterinary importance; human parasite Ascaris lumbricoides infects ~1 billion people globally, and the pig parasite Ascaris suum causes significant animal welfare problems and has economic implications for farmers. Nicotinic acetylcholine receptors are important targets of the anthelmintic drugs levamisole and pyrantel, and more recently, the AADs. The large size of Ascaris has allowed studies of nematode nAChR pharmacology to be carried out using Ascaris muscle. In contrast to this, most molecular studies of nematode nAChRs have been carried out using C. elegans, which has the largest known nAChR gene family. Using a bioinformatics approach applied to the genomes of Brugia malayi and Trichinella spiralis, we have demonstrated that these parasites have very few nAChR genes (9 and 8 respectively). Certain genes encoding neuromuscular nAChR subunits are absent from these parasites (notably lev-1, lev-8 and acr-16) although other proposed components of the levamisole-sensitive neuromuscular nAChR (unc-38, unc-29 and unc-63) are remarkably well conserved throughout the Nematoda. Additionally, there is one subunit gene, acr-26, which is present in B. malayi, but is absent from C. elegans and other free-living nematode species examined. We have cloned cDNAs encoding UNC-29, UNC-38, and UNC-63 (partial) from A. suum and raised antibodies against these subunits. Ascaris UNC-29, UNC-38 and UNC-63 are expressed on body-wall muscle cells, and UNC-29 and UNC-38 clearly co-localise. When co-expressed in Xenopus oocytes, Ascaris UNC-29 and UNC38 form heteromeric channels gated by levamisole, acetylcholine and nicotine. Order of agonist potency is levamisole > acetylcholine > nicotine, which matches the described pharmacology of the native receptors present on Ascaris muscle cells. Secreted proteins of Trichinella spiralis modulate purinergic signalling in immune cells KLEONIKI GOUNARIS, SONJA KOCK, EMILY ELEFTHERIOU AND HOLLY C. AFFERSON. DEPARTMENT OF LIFE SCIENCES, DIVISION OF CELL AND MOLECULAR BIOLOGY, IMPERIAL COLLEGE LONDON, LONDON SW7 2AZ, UK Nucleotides accumulate at sites of inflammation and act as endogenous danger signals to the immune system. Extracellular nucleotides ligate to purinergic receptors and are involved in activation/deactivation of many different immune responses. We have shown that Trichinella spiralis secrete enzymes which can metabolise extracellular nucleotides and thus have the potential to alter host purinergic signalling and the ensuing immunological response. Murine bone marrow-derived dendritic cells (mBMDC), mast cells of a mucosal phenotype (mBMMC) and platelets were used to investigate the effect of T. spiralis secreted proteins on nucleotide-induced effector functions. We showed that ADP is responsible for inducing chemotaxis of both immature and mature mBMDC via activation of the P2Y12 purinergic receptor. ADP-induced chemotaxis was inhibited by T. spiralis secreted proteins, and we identified the secreted 5 ́-nucleotidase as the enzyme responsible. Adenosine, generated by the action of this enzyme, activates P1 receptors and inhibits chemotaxis not only towards nucleotides but also towards MIP-1α and MIP-3β. Adenine nucleotides induced mBMMC exocytosis, resulting in the release of mouse mast cell protease-1 (mMCP-1) and β -hexosaminidase, and this was inhibited by T. spiralis secreted proteins. Extracellular nucleotides were unable to directly stimulate cytokine production in mBMMC, but stimulated inhibitory pathways that led to the down-regulation of LPS-induced TNF-α, a process that was again inhibited by T. spiralis enzymes. It is well established that ADP causes platelet aggregation via activation of the P2Y1 and P2Y12 receptors. T. spiralis inhibits ADP-induced platelet aggregation and P2Y receptor activation via the action of the secreted 5 ́nucleotidase. Overall, our data suggest that nucleotide-metabolising enzymes secreted by T. spiralis can counterbalance inflammatory stimuli and potentially downregulate inflammatory responses. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~SESSION 5S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~SESSION 5 Worm-Induced Modulation of Lung Immunity Alan L. Scott. Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD A number of important helminth parasites have incorporated a short-term residence in the lungs as an obligate phase of their life cycle. While the significance of the lung phase to parasite development is not clear, this short-term exposure is thought to have a lasting impact on the immunobiology of the lung. We have exploited the transient pulmonary phase of Nippostrongylus brasiliensis (Nb) to study the innate immune responses as well as the long-term consequences of this infection on the immunological status of the lungs. Nb induces a rapid and robust Th2-dominated innate response in the lungs resulting in eosinophil infiltration, goblet cell hyperplasia and alternate activation of the lung macrophage population. While eosinophil levels and goblet cell hyperplasia are transient, there are marked Nb-induced, T cell-dependent changes to the lung that persist long after the adult worms are expelled. Weeks after infection, Th2 and Th1 cytokines are constitutively elevated, lung macrophages are maintained as alternatively activated cells with distinct morphological, surface and functional phenotypes and there is a significant reduction in airway responsiveness and inflammation to subsequent allergen challenge. The importance of IL-4/IL-13-mediated signaling to the induction and maintenance of the altered lung environment was studied in STAT6-/animals. In the absence of STAT6, Nb induces a robust Th17 response indicating that STAT6 signaling in the lungs is a key component of the mechanism that regulates Th17 immunity and pathology in the lungs. In addition, Nb infection of STAT6deficient animals also reveals a complex pattern of regulation of Arg1, Fizz1 and Ym1 in the lungs. The results demonstrate that even a transient exposure to a helminth parasite can affect significant and protracted changes in the immunological environment of the lung and that these complex molecular and cellular changes are likely to play a role in modulating subsequent responses to antigen challenge. miRNAs in Brugia malayi L. A. McREYNOLDS, C. B. POOLE, D. SPIRO, E. A. McREYNOLDS and P. DAVIS1, DIVISION OF RNA BIOLOGY AND DIVISION OF PARASITOLOGY, NEW ENGLAND BIOLABS, IPSWICH, MA, 2 THE INSTITUTE FOR GENOME RESEARCH, ROCKVILLE, MD, DEPARTMENT OF MOLECULAR BIOLOGY AND BIOCHEMISTRY, UNIVERSITY OF CALIFORNIA IRVINE, IRVINE, CA. MicroRNAs and other small RNAs play a major regulatory roll in eukaryotic cells. We are characterizing the endogenous small RNAs and the enzymes that generate them in Brugia malayi. We have identified Dicer, Drosha and RNA dependent RNA polymerase, which are involved in the cleavage and propagation of small RNAs. Comparison of the RNAse III domains of Dicer and Drosha suggests that these enzymes have an ancient origin that predates the divergence of plants from animals. Though the processing enzymes, Drosha and Dicer, are conserved there is no evidence in B. malayi of a homolgue for SID-1, which is the protein involved in spreading dsRNA between cells in C. elegans. This could have implications for the use of exogenous dsRNA for RNAi in filarial parasites. MicroRNAs and other small RNAs in B. malayi were identified using both bioinformatics and small RNA cloning. Of the 30 miRNAs so far identified the majority have close homologues in C. elegans, however, three are not in miRBase and appear to be B. malayi specific. Two members of the miR 100 family identified in B. malayi are more closely related to the vertebrate and insect miR-100 families then they are to the related miRNA in C. elegans. One microRNA identified in Brugia, miR-153, has no homologes in protostomes, that includes nematodes and insects but is found in deuterostomes, that includes chordates. It is not known how this miRNA was acquired by Brugia. In addition to miRNAs we have also cloned small RNAs that are coded by the Pao retrotransposon family. These RNAs may be required to activate the RNAi pathway and prevent transposon hopping which would cause genomic instability. Deep sequencing of small RNAs from Brugia will allow a detailed comparison of the miRNAs in a free living nematode, C. elegans, to those of a filarial parasite. Characterization of the diversity of miRNAs is an essential step in identification of the target mRNAs that these small RNAs regulate. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 6S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~ SESSION 6 Development of RNA interference in Nippostrongylus brasiliensis MURRAY E. SELKIRK, CHING-CHENG HUANG, DANIELLE SMYTH & GLYN BALL. DEPARTMENT OF LIFE SCIENCES, IMPERIAL COLLEGE LONDON, LONDON SW7 2AZ Exploitation of RNA interference (RNAi) has revolutionised work on Caenorhabditis elegans, and considerable success has been reported in the last few years with plant-parasitic nematodes. Somewhat surprisingly, it has proven difficult to transfer this technology to animal parasitic species, with a few reports of successful knockdown which appear to be gene specific. We are attempting to develop conditions for RNAi in the strongylid nematode Nippostrongylus brasiliensis. Although we initially reported suppressed expression of secreted acetylcholinesterase by soaking adult worms in double stranded RNA (dsRNA), RNAi should ideally be applied to larval stages in order to assess effects on infectivity and parasite development in vivo. Culture of N. brasiliensis on E. coli supports development from L1 to infective L3, however growth of larvae on E. coli HT115 expressing different target mRNAs gave no consistent reductions in transcript levels. Infective L3 do not feed or ingest dsRNA, but can be can be induced to do so by activation at 37C in a serum-independent manner. The susceptibility of activated L3 to RNAi via soaking and electroporation with dsRNA and siRNA was nevertheless still observed to be irreproducible. Use of mRNA encoding firefly luciferase identified uptake into larvae or adult worms as a major impediment. Defects in RNA uptake and/or spreading in parasitic nematodes have been suggested by others, and are consistent with the absence of sequences homologous to sid-1 and sid-2 in Haemonchus contortus and Brugia malayi, two species for which close to complete genome sequence is available. Development of a delivery system will therefore most likely be required to yield robust methods for RNAi in several animal parasitic species. RNA Metabolism and Molecular Adaptation in Ascaris Embryos RICHARD E. DAVIS, ADAM WALLACE, WEIZHI LIU, MAKEDONKA MITREVA AND GREG HANNON. DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR GENETICS, UNIVERSITY OF COLORADO SCHOOL OF MEDICINE, AURORA, CO. GENOME SEQUENCING CENTER, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MO. HHMI, COLD SPRING HARBOR LABORATORY, COLD SPRING HARBOR, NY. Embryos of the nematode Ascaris provide a powerful system for developmental and molecular studies. Unlike C. elegans, large amounts of synchronized populations of developing embryos can be obtained, enabling a variety of staged molecular and proteomic analyses. Molecular and biochemical tools developed in Ascaris embryos include in vitro RNA transcription, splicing, translation, and decay systems, as well as biolistic transfection of both DNA and RNA. Using these Ascaris embryo tools, we have examined the functional contribution of spliced leader trans-splicing to mRNA translation and decay. Both in vitro and in vivo studies demonstrate that spliced leader trans-splicing (the addition of a new cap and 22 nt sequence to the 5’ end of mRNAs) does not confer any measurable advantage to mRNA translation or stability when compared to non-trans-spliced mRNAs. The new trimethylguanosine (TMG) mRNA cap derived from trans-splicing alone does not efficiently support translation. Efficient translation of TMG capped mRNAs requires the accompanying spliced leader sequence added with the new cap to the 5’ end of the mRNA. We have identified sequences within the spliced leader that are necessary and sufficient for efficient translation of mRNAs with the TMG cap. Interestingly, sequences required for the efficient translation of TMG capped mRNAs, also inhibit mRNA decapping, an initiating event in mRNA decay. Efforts are underway to mechanistically determine how and through what proteins the spliced leader sequence influences these post-transcriptional processes. Many cap-interacting proteins in Ascaris have adapted to recognize and act on the atypical trans-spliced TMG cap present on the majority of nematode mRNAs. These cap-interacting proteins, such as the translation initiation factor eIF4E, are essential in nematodes, and as they have a unique ability to recognize the TMG cap compared to their mammalian hosts, are potential targets for rational drug design. Crystallographic and NMR approaches are underway to characterize the biochemical and biophysical basis for helminth eIF4E recognition of the TMG cap. Finally, we have initiated small RNA discovery and profiling during early Ascaris development using “deep sequencing”. These studies have identified novel Ascaris embryo miRNAs and other small RNAs that are not present in C. elegans. The expression profiles of these small RNAs have been characterized in fertilized embryos through L2 larvae. Several of these small RNAs are maternally contributed, whereas the transcription of others begins as early as the 1-2 cell stage, earlier than most nematode embryo transcription was thought to occur. Efforts are underway to characterize the transcription of these small RNAs, to identify the mRNA targets for these small RNAs, and to develop competent in vitro systems to examine mechanisms of mRNA repression that contribute to early nematode development. ABSTRACTS ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~SESSION 6S ~ SUNDAY 14 SEPTEMBER ~ DAY 2 ~SESSION 6 Cellular energy metabolism and ageing in Caenorhabditis elegans NEKTARIOS TAVERNARAKIS. INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY, FOUNDATION FOR RESEARCH AND TECHNOLOGY HELLAS, HERAKLION, CRETE, GREECE The biogenesis and function of mitochondria, the energy-generating organelles in eukaryotic cells, are primary longevity determinants. However, the molecular mechanisms regulating mitochondrial energy metabolism during ageing are poorly understood. Prohibitins are ubiquitous, evolutionarily conserved proteins, which form a ring-like, high molecular weight complex at the inner membrane of mitochondria. Prohibitin function has been implicated in carcinogenesis and replicative senescence. We have found that the mitochondrial prohibitin complex promotes longevity and tumor growth by moderating fat metabolism and energy production in the nematode Caenorhabditis elegans. Prohibitin deficiency shortens the lifespan of otherwise wild type animals. In sharp contrast, knockdown of prohibitin promotes longevity under dietary restriction and in diapause mutants. In addition, prohibitin deficiency extends the lifespan of animals with compromised mitochondrial function or fat metabolism and restores normal lifespan in mutants with lethal germline tumors. Depletion of prohibitin influences ATP levels, adipose tissue fat content and mitochondrial proliferation in a genetic background and age-specific manner. Together, these findings reveal a novel mechanism of regulating mitochondrial energy metabolism and fat utilization during diapause and ageing. Levamisole channel currents and resistance in adult Caenorhabditis elegans muscle A. P. ROBERTSON, H. QIAN, J. A. POWELL-COFFMAN, R. J. MARTIN DEPT BIOMEDICAL SCIENCES & DEPT OF GENETICS, DEVELOPMENT AND CELL BIOLOGY, IOWA STATE UNIVERSITY, AMES, IA, USA Nematode nicotinic acetylcholine receptors (nAChRs) are the site of action of the anthelmintic levamisole. We have used single-channel recording techniques to examine these receptors in the model nematode C. elegans. Although single-channel current recordings from these receptors have been possible using cultured embryonic muscle cells, in situ recordings from adult C. elegans have not yet been possible because the receptors are aggregated at the synapse and are inaccessible. In order to record channel currents in situ and to investigate the resistance at the channel level we exploited a lev-10 mutant. LEV-10 is a receptor associated protein in C. elegans which is required for postsynaptic clustering of the levamisole-selective receptor but not the nicotineselective receptor. Previous observations had suggested in lev-10 mutants that the levamisole receptors distribute extra-synaptically. We tested this hypothesis by observing single-channel currents in extra-synaptic muscle membrane patches from wild type and lev-10 mutants and found a population of 30 pS channels activated by acetylcholine and levamisole that increased in number in lev-10 knockouts. We were then able to examine at the single-channel level, the in vivo effects of removing either the LEV-8 subunit or the LEV-1 subunit using the double mutants lev-10;lev-8 and lev-10;lev-1. In lev-10;lev-8 mutants the mean closed time of the channels increased (control 133ms, lev10;lev-8 410ms) without significant change in channel conductance or mean open-time. In lev-10;lev-1 mutants the channel properties were unaltered but the number of functionally expressed channels was reduced. Our observations using the LEV-10 knockouts provide an explanation for levamisole resistance produced by LEV-8 and LEV-1 mutants at the single-channel level. They also allowed a direct comparison with levamisole activated channels from the parasitic nematode Ascaris suum which we have already described. The channels from C. elegans had shorter mean open-times, were not activated by nicotine, and did not have as big a conductance range. These differences may arise from differences in the subunit composition of the levamisole receptors between the two species. The project was supported by Grant Number R 01 AI 047194 from the national Institute of Allergy and Infectious Diseases to RJM ABSTRACTS ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 7S ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 7 MITR-1: a conserved novel nematode-specific protein required for mitochondrial respiration in C. elegans N.M. JOHNSON, R.S. KUMARASINGHE, T.S. SLOAN-GARDNER, J.-A. FRITZ, S.B. HETHERINGTON and C.A. BEHM. SCHOOL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, AUSTRALIAN NATIONAL UNIVERSITY, ACT 0200, AUSTRALIA. Mutations affecting components of C. elegans mitochondria have a variety of effects on the worm, from increased susceptibility to oxidative stress and developmental defects, to the extension of life span. We have identified a novel, nematode-specific nuclear gene required for mitochondrial function in C. elegans. mitr-1 (mitochondrial respiration defective) plays an essential role in mitochondrial respiration and energy production and has many of the hallmarks of the clk genes, including an increased life span following gene knockdown. mitr-1(RNAi) worms are small, slow growing, have small brood sizes and display a reduced rate of some rhythmic processes such as defecation and egg-laying, which manifests as a highly penetrant lethal egglaying defect (Egl). Those worms that do not die from Egl have significantly extended life spans in a manner independent of the insulin-like/DAF-2 signalling pathway. MITR-1::GFP is localised to mitochondria throughout the worm and is expressed at all development stages. Biochemical analysis reveals that MITR-1 is required for mitochondrial respiration: mitr-1(RNAi) worms have low oxygen consumption rates and low free-ATP levels. Consistent with this, these worms display a heightened sensitivity to hypoxic stress. Silencing of mitr-1 also results in expression of hsp-6::GFP, which is a reporter of mitochondrial stress and protein instability. In an attempt to better understand how MITR-1 contributes to mitochondrial respiration, we performed yeast twohybrid screening to identify protein interactors of MITR-1. A putative physical interaction between MITR-1 and components of the E1 subunit of the pyruvate dehydrogenase complex (PDC) was identified and confirmed by GST pull-down experiments. This implicates MITR-1 at the site of a critical metabolic reaction: the production of acetyl CoA from pyruvate. Homologues of mitr-1 have been identified in C. briggsae, Haemonchus contortus and Brugia malayi, which suggests that mitr-1 might play a similar role in other nematodes, including parasites. A hypodermally expressed Haem peroxidase is critical for viability and cuticle formation in C. elegans ANTONY P. PAGE, MELANIE C THEIN, GILLIAN STEPEK, ALAN D. WINTER AND GILLIAN MCCORMACK. INSTITUTE OF COMPARATIVE MEDICINE, VETERINARY FACULTY, UNIVERSITY OF GLASGOW, SCOTLAND. The nematode cuticle is a collagenous extracellular matrix that is repeatedly shed and re-synthesised throughout post-embryonic development via the moulting cycle. We study the enzymes involved in the synthesis, modification, processing and crosslinking of the cuticle components in the tractable model C. elegans. Using a combination of genetics and biochemistry we have identified an essential peroxidases called MLT-7 that in combination with the dual oxidase BLI-3 is responsible for cuticle cross-linking, maintenance of structural integrity and viability of C. elegans. Orthologues of these essential enzymes exist in the genomes of the human parasitic nematodes Brugia malayi and the sheep parasite, Haemonchus contortus We will discuss the individual and combined functions of these enzymes with respect to cuticle formation in nematodes. ABSTRACTS ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~SESSION 7S ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~SESSION 7 Molecular mechanisms of drug resistance in Haemonchus contortus P. MÄSER. INSTITUTE OF CELL BIOLOGY, UNIVERSITY OF BERN, SWITZERLAND The nematode Haemonchus contortus is a gastro-intestinal parasite of ruminants. Infections are controlled primarily with three classes of anthelmintics: the benzimidazoles, imidazothiazoles, and macrocyclic lactones. However, the genetic versatility of H. contortus combined with the large numbers of produced offspring promote the emergence of drug resistance and recently, H. contortus field isolates were described that are crossresistant to all current anthelmintics. Such mutants present an existential threat to sheep farms. We are investigating the molecular mechanisms of drug resistance in H. contortus in order to (i) device tests for early recognition of resistance alleles in the gene pool of the parasites and (ii) learn more about the mode of action of novel anthelmintics. Of particular interest are the amino-acetonitrile derivatives (AAD), a promising class of compounds with strong activity against nematodes and good tolerability in mammals. Based on the molecular characterization of worms selected for AAD-resistance, the targets appear to be nicotinic acetylcholine receptors of the DEG-3 subfamily which only occurs in nematodes. This provides a possible explanation for the selective action of the AADs. Anti-wolbachial chemotherapy of onchocerciasis – a macrofilaricide at last? ACHIM HOERAUF*, SABINE SPECHT, ALEX DEBRAH, ROLF FIMMERS, SABINE MAND, KEN PFARR, OHENE ADJEI, DIETRICH W. BÜTTNER INSTITUTE OF MEDICAL MICROBIOLOGY, IMMUNOLOGY AND PARASITOLOGY, UNIVERSITY BONN MEDICAL CENTER, BONN, GERMANY. KUMASI CENTRE FOR COLLABORATIVE RESEARCH IN TROPICAL MEDICINE (KCCR), KUMASI, GHANA. INSTITUTE FOR MEDICAL BIOMETRY, INFORMATICS, AND EPIDEMIOLOGY, UNIVERSITY BONN MEDICAL CENTER, BONN, GERMANY. BERNHARD NOCHT INSTITUTE FOR TROPICAL MEDICINE,HAMBURG, GERMANY The treatment of human onchocerciasis has long been hampered by the fact that ivermectin, the only drug that is in use, is mainly microfilaricidal and only shows moderate sterilising activity on the level of female worms and practically no macrofilaricidal effect. Our group has earlier introduced the concept of chemotherapy by antibiotics that target the Wolbachia endosymbionts, with doxycycline as the best usable drug, but also rifampicin showing effects in animal models. While in lymphatic filariasis, we had previously demonstrated that doxycycline given for 4 or 6 weeks had a strong macrofilaricidal effect (> 90%) and also ameliorates lymphatic pathology, whereas in onchocerciasis the effect seemed to be a long-term sterilising only. However, in a placebo-controlled study, doxycycline for 4 or 6 weeks at a daily dose of 200 mg clearly demonstrated a macrofilaricidal effect of up to 70%. A further open study also showed a macrofilaricidal effect when doxycycline was given at the lower dose of 100 mg/d for 5 weeks. The reason for our inability to detect a macrofilaricidal effect of doxycycline in earlier studies seems to be that this effect occurs only after a comparatively long observation period (18-20 months after doxycycline). The chemotherapy with doxycycline is currently limited to individual treatment, e.g. in patients presenting to outpatient clinics. However, first results from Cameroon suggest that provided there is sufficient training it is also possible to administer doxycycline within the activities of the so-called community-directed intervention (ComDT). In addition, new data from animal studies and humans suggest that combination with rifampicin may augment treatment efficacy or shorten total treatment time. We are collaborating in a large consortium to exploit the Wolbachia genome for the detection of new anti-Wolbachia drugs against filariasis using different new approaches such as bio-informatics and aptamer technology (SELEX). ABSTRACTS ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 8S ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 8 Filarial Wolbachia lipoprotein stimulates innate and adaptive inflammatory responses through TLR2 and TLR6 and induces disease manifestations of lymphatic filariasis and river blindness MARK J TAYLOR*, R. STUART LANGLEY, JOSEPH D. TURNER, KELLY JOHNSTON, LOUISE FORD, KATRIN DAEHNEL, ERIC PEARLMAN, BO WU AND BARTON SLATKO FILARIASIS RESEARCH LABORATORY, LIVERPOOL SCHOOL OF TROPICAL MEDICINE, PEMBROKE PLACE, LIVERPOOL, L3 5QA UK, DEPT. OF OPHTHALMOLOGY, CASE WESTERN RESERVE UNIVERSITY, CLEVELAND, OHIO, USA, NEW ENGLAND BIOLABS, IPSWICH, MA, USA The filarial nematodes that cause lymphatic filariasis and river blindness are host to a symbiotic bacterium, Wolbachia, which contributes to the inflammatory pathogenesis of filarial disease. Using siRNA knockdown of constitutive TLR1 or TLR6 expression in HEK-TLR2 cells and TLR1 and TLR6 mice we determined that extracts of Brugia malayi containing Wolbachia (BMFE), but not those derived from antibiotic treated nematodes (BMFEtet), induce inflammation through recognition by TLR2/TLR6 and to a marginal extent TLR2/TLR1 heterodimers. Removal of lipids or proteins from BMFE eliminated inflammatory activity. We used the TLR recognition profile to focus bioinformatic searches of wBm for putative lipoproteins. We identified the lipoprotein biosynthesis-encoding genes: Ltg, (prolipoprotein diacylglyceryl transferase) and Lspa, (lipoprotein signal peptidase II), but an absence of Lnt, (apolipoprotein aminoacyl transferase), the enzyme responsible for tri-acylation of the N-terminus amide group in most bacteria. Thus diacylation of Wolbachia lipoproteins occurs but further acylation is prevented through lack of Lnt, accounting for the predominant recognition by TLR2/6. Searches of three predictive lipoproteins databases revealed from 3-11 potential lipoproteins from wBm. Two proteins, peptidoglycan-associated lipoprotein (PAL) and VirB6, type IV secretion system protein were predicted by all three programs and Wolbachia PAL (WoLP1) was selected for further characterization. Antibodies raised to WoLP1 stained the surface of bacteria and detected a single 18kDa band in BMFE. Synthetic 20-mer peptides of the N-terminus of WoLP1, which were either diacylated or triacylated at the N-terminal cystiene residue, showed that the diacylated peptide had a near identical TLR2/TLR6 and TLR2/TLR1 receptor usage compared with native Wolbachia-containing BMFE. In contrast triacylated WoLP1 displayed no dependency on TLR6 and partial dose-dependency on TLR1. Injection of diacylated WoLP1 into mice resulted in systemic TNFα production and neutrophil mediated keratitis in a murine model of river blindness, which was significantly reduced in TLR6 but not TLR1 mice. BMFE and WoLP1 synergise with sub-inflammatory levels LPS and activate monocytes to induce up-regulation of podoplanin on lymphatic endothelial cells. WoLP molecules are also responsible for driving enhanced dendritic cell (DC) maturation and induction of DC cytokines, IL-12/IL-23 p40 monomer/monodimers, IL-12p70 and IL-23p40/p19 heterodimers and TNFα . DC primed with BMFE generated a mixed Th1/Th2 CD4+ T cell profile, whereas DC primed with BMFEtet polarised to only to a Th2 profile. Mice inoculated with BMFE generated IgG1 and IgG2c antibody responses. TLR2 or MyD88 mice generated similar levels of IgG1 but failed to produce IgG2c responses suggesting Wolbachia lipoproteins drive IFNγ dependent antibody switching to filarial antigens. Together our results support a role for Wolbachia lipoproteins as the TLR2/TLR6 ligands associated with the activation of innate and adaptive inflammatory immune responses associated with the pathogenesis of filarial disease. ABSTRACTS ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 8 ~S ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 8 ~ Atypical modulation of calcium currents by a schistosome calcium channel beta subunit VICENTA SALVADOR-RECATALA AND ROBERT M. GREENBERG*. DEPARTMENT OF PATHOBIOLOGY, UNIVERSITY OF PENNSYLVANIA SCHOOL OF VETERINARY MEDICINE, PHILADELPHIA, PA. USA. Voltage-gated Ca (Cav) channels are members of the voltage-gated ion channel superfamily that underlies electrical excitability in cells. Cav channels couple membrane depolarization to entry of extracellular Ca and represent likely targets for new anthelmintics, as well as candidate targets for praziquantel, the current drug of choice against schistosomiasis. Cav channels consist of a main pore-forming and voltage-sensing α1 subunit and auxiliary subunits such as β and α2/δ. The function of Cav channels greatly depends on functional coupling to the cytoplasmic accessory β subunit, which promotes surface expression of the α1 subunit, and also modulates several gating and kinetic parameters of Ca currents. Thus, when co-expressed with α1 subunits, Cav channel β subunits typically increase currents and modulate other biophysical aspects of the channel. We have previously shown that schistosomes and other platyhelminths express two β subunit subtypes: a conventional β subunit (SmCavβ) and a variant β subunit (SmCavβvar) that has unusual functional properties and appears to be involved in the action of the antischistosomal drug praziquantel. We have recently been using whole-cell patch clamp to characterize the functional modulation of the conventional SmCavβ subunit on the human Cav2.3 α1 subunit stably expressed in HEK-293 cells. SmCavβ dramatically increased Cav2.3 currents, slowed macroscopic inactivation and shifted the steady state inactivation in the hyperpolarizing direction, effects similar to those found for mammalian Cavβ subunits. Interestingly, currents produced by the Cav2.3/ SmCavβ complex run down to about 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration, while currents produced by Cav2.3 subunits alone or with mammalian Cavβ2a or schistosome SmCavβvar were stable over the same time frame. The kinetics of inactivation and steady-state properties were the same before and after rundown, indicating that rundown is not the result of the loss of interaction between Cav2.3 and SmCavβ. Recordings using Ba as the charge carrier run down at the same rate, indicating that regulation of rundown does not distinguish between Ca and other divalent cations. Interestingly, rundown does not occur in the absence of Mg-ATP or Na. Deletion analysis indicates that the molecular substrate for this β subunit-dependent rundown is within the highly acidic N-terminal domain of SmCavβ, a domain not found in other β subunits. We are currently exploring in greater detail the cellular factors that regulate this unusual modulatory effect and the role of this atypical domain in signal transduction. Identification of new drug leads targeting redox biochemistry for the control of schistosomiasis D. L. WILLIAMS, A. A. SAYED, C. L. CASS, G. RAI, D. J. MALONEY, W. LEA, A. JADHAV, A. SIMEONOV, C. J. THOMAS, J. INGLESE, C. P. AUSTIN. DEPARTMENT OF BIOLOGICAL SCIENCES, ILLINOIS STATE UNIVERSITY, NORMAL, ILLINOIS, USA, NIH CHEMICAL GENOMICS CENTER, NATIONAL HUMAN GENOME RESEARCH INSTITUTE, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND, USA. Treatment for schistosomiasis depends almost exclusively on praziquantel. Tens of millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. Phosphinic amides and oxadiazole 2-oxides identified from a quantitative high-throughput screen were shown to be inhibitors of a parasite enzyme, thioredoxin glutathione reductase (TGR), with activities in the low micromolar to low nanomolar range. Incubation of parasites with these compounds led to rapid inhibition of TGR activity and parasite death. The activity of the oxadiazole 2-oxides was associated with a donation of nitric oxide. Treatment of experimental schistosome infections with 4-phenyl-1,2,5-oxadiazole-3-carbonitrile-2-oxide led to dramatic reductions in worm burdens from treatments against multiple parasite stages. Highly significant reductions in egg-associated pathologies also resulted. In addition, this compound was active against the three major schistosome species infecting humans. These protective effects exhibited by this compound exceed benchmark activity criteria set by the WHO for lead compound development for schistosomiasis. We also discuss the development of these lead structures for potency and definition of mechanism. ABSTRACTS ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 8S ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 8 Probing the helminth host/parasite interface with phage-displayed antibodies CHARLES B. SHOEMAKER, JORGE SEPULVEDA, JACQUE TREMBLAY, DAVID MAASS, JEFF BETHONY, ALEX LOUKAS, PETER HOTEZ AND PATRICK J. SKELLY. DEPT. OF BIOMEDICAL SCIENCES, TUFTS CUMMINGS SCHOOL OF VETERINARY MEDICINE, NORTH GRAFTON, MA 01536. VICTORIA UNIVERSITY, WELLINGTON, NEW ZEALAND. DEPT. MICROBIOLOGY AND TROPICAL MEDICINE, GEORGE WASHINGTON UNIVERSITY, WASHINGTON DC 20037. DIVISION OF INFECTIOUS DISEASES AND IMMUNOLOGY, QUEENSLAND INSTITUTE OF MEDICAL RESEARCH, BRISBANE, AUSTRALIA Helminth parasites largely interact with hosts through their exposed surfaces, and these surfaces are the primary sites where the parasite must defend itself against immune recognition and effector action. It is also this surface that is the target of the few successful examples of metazoan parasite vaccines (e.g. Boophilus microplus, Haemonchus contortus, Taenia ovis and Echinococcus granulosus). Hosts of helminth parasites mount widely variable antibody responses against antigens exposed at the host/parasite interface, and these differences often strongly correlate with differences in the ability of the hosts to reject infection. We are using phage-displayed antibody methods to identify the surface antigens that are selectively recognized by antibodies from helminth resistant hosts. We hypothesize that exposed epitopes on antigens selectively recognized by resistant hosts are excellent targets of protective immunity. Using display methods, we found that mucosal antibodies from sheep that have been protectively immunized to gastrointestinal strongylid parasites by multiple truncated infections almost exclusively recognize only two antigens present on the L3 stage cuticle. One antigen, the glycan CarLA, displays remarkable intraspecific variation in which different anti-CarLA clonal Abs recognize different worm subsets, suggesting a novel immune evasion strategy possibly involving carbohydrate remodeling. The second antigen, the glycoprotein P6, turns over or sheds rapidly at the L3 surface in some strongyle species. Interestingly, P6 is the predominant hookworm L3 surface antigen recognized by dogs and hamsters successfully immunized by truncated infections or irradiated larvae. In schistosomiasis models, Fisher rats reject S. mansoni infections after about four weeks as compared to mice that are fully susceptible. The resistant rats mount a significantly more robust antibody response against the schistosome tegument than mice. We identified a panel of phage displayed antibodies from immune rats that recognize epitopes at the host-interactive surface of living schistosomes. The recombinant antibodies recognize both protein and nonprotein antigens. In two cases, we identified the protein targets as Sm-TSP-2 and Sm29, which are among the small subset of schistosome proteins found to be exposed on the tegument by proteomic studies. We find that both antigens are selectively recognized by schistosome immune Fisher rats as compared to susceptible mice. Both proteins are also selectively recognized by a small subset of individuals in Brazil that appear potently resistant to schistosome infection as compared to the general population. Identification and characterization of helminth host-interactive surface antigens selectively recognized by immune animals should lead to new insight into host/parasite interaction and could identify novel and promising vaccine antigens. ABSTRACTS ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 9S ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 9 Functional characterization of the schistosome surface Z. FAGHARI, G. KRAUTZ-PETERSON, D. NDEGWA, R. BHARDWAJ, P.J. SKELLY. DEPARTMENT OF BIOMEDICAL SCIENCES, DIVISION OF INFECTIOUS DISEASES, CUMMINGS SCHOOL OF VETERINARY MEDICINE, TUFTS UNIVERSITY, NORTH GRAFTON, MA, USA The tegument of intravascular schistosomes is a syncytium that is bounded on the exterior by a double lipid bilayer. This surface constitutes a major site of interaction with the host. In recent years proteomic analysis has been used to catalog the protein composition of the tegumental surface bilayers. The precise functions played by many of the molecules found in the tegumental surface membranes, and how they avoid being targeted by effective host immunity, are largely unknown. We are using RNA interference (RNAi) to suppress the expression of genes whose products are found in the host-exposed tegumental membranes. In this manner, we have begun a functional characterization of the schistosome surface. For example, we have identified a cDNA potentially encoding a 304 amino acid protein (SmAQP1) that is expressed in the tegumental membranes and has sequence similarity to members of the aquaporin protein family. Treatment of schistosomula with short interfering RNAs (siRNAs) targeting SmAQP1 results in potent (>90%) gene suppression 48 hours later but leads to no overt phenotype in parasites cultured for up to 14 days in rich medium. However, when these parasites are placed in hypotonic solution they resist swelling, unlike their control counterparts which rapidly double in volume. These experiments reveal a heretofore unrecognized role of the tegument in controlling water movement into the parasites and thus in osmoregulation. In a second example, treatment of parasites with siRNAs targeting either the alpha or the beta subunit of schistosome tegumental NaKATPase (SNaK1) leads to decreased parasite viability in vitro. In addition, significantly fewer parasites (~10%) are recovered from mice infected with schistosomula whose SNaK1 alpha and beta genes have been suppressed by siRNA treatment compared with controls, and recovered parasites exhibit a stunted appearance. In agreement with the work of other groups on the proposed role of tegumental molecules in signal transduction, our preliminary evidence suggests that SNaK1 in the schistosome tegument can act to transduce signals in response to the endogenous steroid hormone, ouabain. Finally, we are examining the role of the tegument in immune modulation by silencing the expression of genes whose tegumental products we hypothesize can act to diminish host immune signaling through DAMPs (Danger Associated Molecular Patterns). These experiments are designed to generate a comprehensive understanding of the role of the schistosome tegumental surface in promoting parasite survival through immune modulation, signaling and by controlling the movement of metabolites into and out of the tegument. Hsp90 and the biology of parasitism EILEEN DEVANEY, VICKI GILLAN, KIRSTY MAITLAND AND NIK A.I.I. NIK HIM, PARASITOLOGY GROUP, DIVISION OF VETERINARY INFECTION & IMMUNITY, UNIVERSITY OF GLASGOW, GLASGOW The molecular architecture of Hsp90 is highly conserved throughout evolution. However, C. elegans Hsp90 is unique amongst eukaryotes because of its inability to bind to the Hsp90 inhibitor Geldanamcyin (GA). In contrast Hsp90 from the parasitic nematode Brugia pahangi specifically binds to GA. We have investigated the GA-binding characteristics of Hsp90 from an additional 23 species of nematode. The results demonstrate that Hsp90 from both free-living and parasitic species belonging to Clade V are non-binding while Hsp90 from T. spiralis (Clade I) and from Clade III nematodes, including other filarial worms and ascarids, can bind to GA. Thus the life history of the species may determine whether or not Hsp90 binds to GA; species that have a freeliving larval stage in the soil do not bind GA, while those species which are obligate parasites (Trichinella and the filarial worms), or which are enclosed within a protective egg shell while in the environment (Ascarids), possess an Hsp90 that has retained GA binding. Our results support the concept of the adaptive evolution of Hsp90. GA is synthesized by a soil-dwelling Streptomyces species and, as Hsp90 is essential in all eukaryotes, it would clearly be disadvantageous for worms sharing the same ecological niche to possess a GA-sensitive Hsp90. Current efforts are focused on understanding the molecular basis of GA sensitivity. ABSTRACTS ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 9S ~ MONDAY 15 SEPTEMBER ~ DAY 3 ~ SESSION 9 Transcriptomic analyses of schistosome biology: what we have learned and where we are headed. K.F. HOFFMANN. INSTITUTE OF BIOLOGICAL, ENVIRONMENTAL & RURAL SCIENCES, ABERYSTWYTH UNIVERSITY, EDWARD LLWYD BUILDING, ABERYSTWYTH, SY23 3DA, UK Unquestionably, DNA microarrays have positively contributed to a variety of research programmes studying helminth and nematode organismal biology. Developing and utilizing these tools in our laboratory has allowed us to ask fundamental questions regarding schistosome conjugal biology and sexual maturation. Over the past several years we have elucidated a transcriptional basis underlying gender in adult S. mansoni and S. japonicum worms, have illustrated that adult worm gene expression in both sexes is dependent upon dioecious interactions and have demonstrated that strain-specific transcription can be detected in adult S. japonicum worms originating from different Chinese provinces. Collectively, these high-throughput transcriptional studies logically led to more hypothesis-directed investigations, which culminated in the characterization of new drug targets (schistosome tyrosinases) involved with the egg laying machinery and novel vaccine candidates (schistosome venom allergen like molecules) associated with cercarial penetration and schistosome development. In addition to continuing these focused investigations on gene products identified from the study of adult schistosome conjugal biology, we have recently applied DNA microarrays to the specific analysis of cercariae dioecy. Interestingly, these studies have demonstrated that morphologically identical, but chromosomally distinct, cercariae do express divergent gene repertoires. Subsets of these differentially expressed genes are: 1) characteristic of ones previously described in functionally-distinct dimorphic adults, 2) likely instrumental in explaining male-biased sex ratios commonly observed during definitive host infection and 3) possibly associated with processes involved in maintaining long-term female genomic stability. These results suggest that the short-lived cercarial life stage is not only transcriptionally active, but more importantly, is starting to establish gender-associated patterns of gene expression critical for labour division in the adult. Finally, we will present our current transcriptional understanding of schistosome development as inferred from an intensive DNA microarray analysis of the parasite’s lifecycle. We will demonstrate how this data is currently being linked to SchistoGeneDB and will discuss how future developments can dramatically improve community access. We will additionally discuss how interrogation of this lifecycle dataset is contributing to our continued efforts in identifying urgently needed novel drug and vaccine candidates. Use of the Caenorhabditis elegans model system to identify the receptors in parasitic helminths modulating serotonin-stimulated paralysis R.KOMUNIECKI, R. HOBSON, G. HARRIS, V.HAPIAK, AND P.R. KOMUNIECKI. DEPARTMENT OF BIOLOGICAL SCIENCES, THE UNIVERSITY OF TOLEDO, TOLEDO, OH Almost all anthelminthics act as agonists to stimulate inhibitory inputs into locomotion and/or pharyngeal pumping. Similarly, exogenous serotonin (5-HT) and dopamine paralyze nematodes from all clades, suggesting that serotonergic/dopaminergic signaling includes potential targets for anthelminthic development. Using the well characterized C. elegans model system, we and others have identified 16 putative biogenic amine receptors in C. elegans and characterized their pharmacology and coupling after heterologous expression in a variety of systems. Using this approach, four C. elegans 5-HT G-protein coupled receptors and at least one 5-HT-gated channel have been identified. Surprisingly, null mutations in the genes encoding each of these receptors reduce 5-HT dependent paralysis, and ser-4;mod-1 null animals are completely resistant. Based on promoter:gfp translational fusions, both ser-4 and mod-1 appear to be expressed in interneurons, and not in motorneurons or muscle, suggesting that 5-HT acts at the level of the animals decision to move and rather than its ability to move. Genes encoding 14 of these 16 putative biogenic amine receptors have been tentatively identified in the Ascaris suum genome. Based on work from C. elegans, we are currently heterologously expressing the A. suum and Brugia malayi ser-4/mod-1 homologues, and attempting to rescue C. elegans null mutants with receptors from the parasites to confirm the predicted roles of these receptors. In addition, we are focusing on the identification of the signaling pathways and ion channels downstream from these receptors. Certainly, many differences between C. elegans and the parasites are to be anticipated, but C. elegans has proven to be a useful tool for identifying core signaling pathways. Indeed, this approach has been used to identify molecular targets for most anthelminthics and should have even increased utility in the future. This work is supported by NIH AI 0145147 and the Julius Jacobson Professorship in Biomedical