7^th International Workshop on the Molecular Biology and Genetics of the Lepidoptera

By Marian Goldsmith

We dedicate this year’s Lepidoptera Workshop to the memory of Michael Wells, who died in May. Mike’s passing was a profound loss to our community. He was a long time supporter and contributor to the Lepidoptera Workshops, going back to its founding days in the late 1980s. His lab created and hosted the meeting’s website, giving us wider international exposure and facilitating registration. The research areas that he opened up in insect biochemistry and physiology provided a foundation for many of the projects reported at the meeting over the years. He was always available with constructive advice and support, both scientific and professional, and mentored many junior and senior scientists, not only in the lively discussion sessions at the meeting but also in private conversation. At the opening of this year’s Workshop people commented that one of his greatest joys was in educating school children and the general public about science using Manduca sexta, his research organism, as a model. To recognize Mike’s dedication to the spirit of science and dialogue is especially fitting in the context of the Orthodox Academy of Crete, where we regularly hold the Lepidoptera Workshops.

Abstracts are listed in alphabetical order by the last name of the first author.

A. Abd-Alla^{1, 2}, F-X. Jousset¹, F. Cousserans¹ and M. Bergoin¹

¹UMR 1231 BIVI, Univ. Montpellier 2, 34095 Montpellier, France, ²Entomology Unit FAO/IAEA Agriculture and Biotechnology Laboratory, A-2444 Seibersdorf, Austria.

Correspondence: bergoin@ensam.inra.fr

The cap gene of JcDNV occupies the 5’ half of one strand of its ambisense genome and is under control of the P9 promoter. The 4 structural polypeptides of the virion are translated from a single unspliced 2.6 kb mRNA by translation initiation at the 1st, 2nd, 3rd and 4th AUG codons according to a "leaky scanning" mechanism. We previously reported the production of JcDNV virus-like particles using different recombinant baculoviruses (Croizier et al., 2000 J. Gen.Virol, 81, 1605–1613). However, the role of each polypetide in virus assembly to produce infectious virions has not been established. We report here the effect of punctual mutations deleting one or more capsid polypeptides on virus production in cell culture and larvae. Six constructs were generated from pBRJ, a plasmid encompassing an infectious viral sequence, by site-directed mutagenesis of the 5 in-frame ATG’s at positions 555 (pJm1), 1386 (pJm2), 1521 (pJm3), 1668 (pJm4), and 1674 (pJm5) of the cap ORF. Other plasmids pJm2+3 and pJm4+5 contained the double ATG2+ATG3 and ATG4+ATG5 mutations, respectively while pJm336–7 contained a double TAA mutation upstream of ATG4. With the exception of pJm4+5, all these constructs generated mutant virions (VJm) when transfected to Ld 652 cells. However, infectivity tests performed by injecting virions produced in Ld 652 cells to 3rd instar Spodoptera littoralis larvae revealed significant differences among them: VJm2, VJm3, VJm4 and VJm5 virions lacking VP2 and VP3 respectively but containing VP4 appeared to be as infectious as wt virions. In contrast, Vjm1, VJm2+3, and VJm336–7 virions lacking VP1, VP2+VP3, VP1+VP2 +VP3 in their capsid respectively, were not infectious for S.littoralis larvae. Finally, mutations were performed in two regions assumed to be critical: the N-terminal “VP1up” sequence containing a phospholipase A2 activity and a Lysine-Arginine-reach region close to the N-terminal sequence of VP2. Mutation of the PLA2 active site drastically reduced infectivity of mutant virions while mutation in VP2 N-terminal region completely abolished infectivity. As demonstrated by dot-blot hybridization and PCR, all the mutant virions contained DNA. These results will be discussed in the light of confocal observations of Ld 652 cells infected with different mutants.

H. Abe¹, T. Fujii², K. Mita³, M. Ajimura³, T. Shimada²

¹Department of Biological Production, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan; ²Department of Agricultural and Environmental Biology, Graduate school of Agricultual and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; ³Laboratory of Insect Genome, National Institute of Agrobiological Science, Tsukuba 305-8634, Japan.

Correspondence: kmita@nias.affrc.go.jp

In the silkworm Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which the autosomal fragment containing the dominant marker genes were translocated onto the W chromosome. In this study, we analyzed the W chromosomal regions of the two sex-limited yellow cocoon strains (T(W;2)Y-Abe and -Chu types) and the newly obtained chromosomal fragment from the Zebra-W strain (T(W;3)Ze chromosome) at the molecular level. Initially, we obtained the W chromosome variant from the T(W;3)Ze chromosome by X-ray irradiation and obtained one zebra male larva having the chromosome fragment of T(W;3)Ze (W(B-YL-YS)Ze chromosome). The normal W chromosomes of the strains in Japan contained the 12 RAPD markers (W-Kabuki, W-Kamikaze, W-Sasuke, W-Musashi, W-Sakura, BMC1-Kabuki, W-Rikishi, W-Yukemuri-L, W-Yukemuri-S, W-Bomsai, W-Samurai and W-Mikan), the W(B-YL-YS)Ze chromosome contained three (W-Yukemuri-L, W-Yukemuri-S and W-Bonsai), the T(W;2)Y-Chu chromosome contained six (W-Rikishi, W-Yukemuri-L, W-Yukemuri-S, W-Bonsai, W-Samurai and W-Mikan), and the T(W;2)Y-Abe chromosome contained only one (W-Rikishi). Using prepared sucking stomachs of the p50 strain having the normal W chromosome and the sex-limited yellow cocoon strain (T(W;2)Y-Abe type), a single sex heterochromatin body was detected in nuclei of the p50 female moth, while no sex heterochromatin body was detected in the sex-limited yellow cocoon strain (T(W;2)Y-Abe). Therefore, we conclude that the regions of the W chromosome of the T(W;2)Y-Abe containing 11 of the 12 W-specific RAPD markers were deleted, leaving the extremely limited region containing W-Rikishi RAPD marker, which is sufficient to determine femaleness.

M. Ajimura¹, K. Sahara², H. Abe³, T. Tamura⁴, T. Shimada⁵, K. Mita¹

¹Insect Genome Research Unit, Nat'l Institute of Agrobiol. Sciences, Tsukuba, 305-8634, Japan; ²Div. of Applied Bioscience, Grad. School of Agric., Hokkaido University. Sapporo, 060-8589, Japan; ³Faculty of Agric., Tokyo University of Agric. and Tech., Tokyo, 183-8509, Japan; ⁴Transgenic Silkworm Res. Center, Nat'l Institute of Agrobiol. Sciences, Tsukuba, 305-8634, Japan; ⁵Dept. of Agric. and Environ. Biology, University of Tokyo, Tokyo 113-8657, Japan.

Correspondence: majimu@nias.affrc.go.jp,

Correspondence: kmita@nias.affrc.go.jp

The silkworm (Bombyx mori) has 27 pairs of autosomes and 2 sex-chromosomes (Z and W). A putative sex-determining gene (Fem) is located on the W chromosome and autonomously determines the femaleness in each cell during embryogenesis. According to the previous studies on the genome structure of W, most part of the W chromosome is heterochromatic region consisting of different types of transposons and very little euchromatic region has been isolated so far. Furthermore, it is difficult to construct a genetic map of the W chromosome because no recombination is carried out in female meiosis. For such reasons the isolation of Fem has not been succeeded yet. If Fem located in the W chromosome, one unique sequence must be present as the Fem gene at least. Based on the hypothesis, we carried out a cloning of Fem by a genomic subtraction method (RDA). By the extensive screening, a 380 bp DNA fragment (18G08) showing W-specific characteristics was obtained. We isolated W-BACs covering the region and analyzed the whole sequences. The sequencing of the BACs showed that the region contains an euchromatic region expanding about 50 kbp and 250 kbp of transposon-rich part. The region includes two genes (z1 and z20) having zinc-finger motifs, C3H1- and C2H2-types. Further analysis of the genes revealed that the Bombyx genome has 6 copies of z1 and z20 homologs; 2 copies in the chromosome XXV and 4 copies dispersed along the W chromosome. In each case, both z1 and z20 homologs are present as a pair mostly in tail-to-tail orientation. Sequence comparison of these repeats revealed the four z1-z20 copies in the W chromosome were generated from a autosomal gene by repeated duplications. Results of PCR and FISH experiments with several W deletion/traslocation chromosomes indicated z1-z20 could be mapped within a putative Fem gene (about 10 % of the W chromosome). An analysis of expression pattern of the z1 and Z20 genes during embryogenesis showed that no transcripts of z1 and z20 was detected at an early stage of embryonic development. The transcription was observed before sex-determination (at the first to second days after fertilization). On the other hand, transcriptions of the autosomal zinc-finger motif genes are observed constitutively through embryogenesis. Interestingly, we found that initiation site of the mRNA for both z1 and z20 genes in the W were located within transposons which were present outside of euchromatic region. This suggests that the transcription of the z1 and z20 are caused either by transposon sequence or by W-specific structure at hetero-euchromation boundary. These observations suggest both z1 and z20 genes in the W are candidates for Fem gene. We have constructed transgenic silkworm for z1 and z20 genes and are currently analyzing their phenotypes whether these genes are involved in sex-determination process. Dr. Nagaraju’s group also isolated the identical gene (z1) independently by differential display and will talk on their results in this session.

M. Altstein, A. Hariton, M. Davidovitch, and O. Ben-Aziz.

Department of Entomology, The Volcani Center, ARO, Bet Dagan, 50250, Israel.

Correspondence: vinnie2@agri.gov.il

The PK/PBAN family is a multifunctional family of peptides that plays a major role in the physiology of insects. The family comprises of peptides such as PBAN, melanization and reddish coloration hormone (MRCH), pyrokinins, myotropins, pheromonotropin (Pss-PT) and diapause hormone (DH) - which have been found to control a variety of functions such as: stimulation of sex pheromone biosynthesis in adult female moths, cuticular melanization in moth larvae, contraction of the locust oviduct, myotropic activity of the cockroach and locust guts, egg diapause in the silkworm, and acceleration of pupariation in flesh-fly larvae. Despite the intensive studies of the bioactivity of this family, very little is known about the molecular and cellular basis that underlies the functional diversity of the PK/PBAN family. In the past few years our studies focused on a detailed characterization of the structure activity relationship (SAR) of the PK/PBAN family, on characterization of the PK/PBAN receptor and on the development of a novel strategy for the generation of PK/PBAN antagonists and their employment in studying the mode of action of the PK/PBAN peptides. Our results on those topics and their implementation for the development of a novel approach for rational design of insecticide prototypes will be presented.

Barat-Houari M.¹, Hilliou F.², Jousset F.-X.¹, Sofer L.², Deleury E.², Rocher J.¹, Ravallec M.¹, Galibert, L.¹, Feyereisen R.², Fournier P.¹ and Volkoff A-N.¹

¹UMR 1231 Biologie Intégrative et Virologie des Insectes. INRA - Université de Montpellier II. Place Eugène Bataillon, Case Courrier 101, 34095 Montpellier Cédex, France; ²UMR 1112 R.O.S.E. INRA - Université de Nice-Sophia Antipolis, Laboratoire de Génomique Fonctionnelle des Insectes, 400 route des Chappes, BP 167, 06 903 Sophia Antipolis cedex, France.

Correspondence: volkoff@ensam.inra.fr

To succeed in their development, parasites must render their host suitable and circumvent the host immune defence. One strategy developed by some endoparasitic wasps of the ichneumonoid family is the association with symbiotic prokaryotes of the polydnavirus family. Polydnaviruses are maintained as proviruses integrated in the wasp genome. Virus particles that contain the typical polydnaviral segmented genome are produced in the wasp ovaries and then injected by the female wasp in the lepidopteran host during oviposition. In the lepidopteran host, infection of several tissues by polydnaviruses results in physiological alterations, including immune suppression, which are required for pre-imaginal development of the wasp. In order to improve our knowledge on host-parasite interactions involving polydnaviruses, we have developed a transcriptomic approach that has enabled us to start analysing the interactions between the parasitoid Hyposoter didymator, an ichneumonid wasp associated with the polydnavirus HdIV, and its lepidopteran host Spodoptera frugiperda. A microarray with 1751 cDNAs was used to analyse the gene expression profile of S. frugiperda hemocytes and fat bodies collected from last instar larvae, 24 hours after injection of filter-purified HdIV. Our results showed that approximately 6% of the 1751 arrayed host genes varied significantly in response to HdIV injection, indicating that factors associated with a parasitic wasp do affect the host transcriptome. HdIV injection mainly causes down-regulation of cellular genes (76% of the genes that vary are down-regulated). Interestingly, several of these genes have been previously shown to be involved in Lepidoptera innate immunity. For example, levels of transcripts related to calreticulin (−2.5), prophenoloxidase-activating enzyme (−2.2), immulectin-2 (−2.0), actin-binding proteins (−1.8 to −2.2) and a novel lepidopteran scavenger receptor (−3.0) are decreased in hemocytes of injected caterpillars. Thus different components of the host immune response (non-self recognition, humoral and cellular responses) seem to be affected and modulated 24 hours after HdIV injection. Conversely, we found an increased rate of transcription for a galactose-binding lectin (+5.5), a c-type lectin (+1.7) similar to a molecule encoded by bracoviruses, and surprisingly, for the prophenoloxidase subunits (+4.9 and +5.9). The role of these genes in S. frugiperda physiology and/or the reason their rate of transcription vary in response to HdIV injection will need to be further investigated. This novel microarray approach will allow identifying new targets for the virulence factors derived from parasitic wasps and their associated polydnaviruses and will certainly provide new insights on the strategies employed by parasites to manipulate their host physiology.

Simon W. Baxter¹, Nicola Chamberlain², Riccardo Papa³, Sean J. Humphray⁴, Richard H. ffrench-Constant², W. Owen McMillan³ and Chris D. Jiggins¹

¹Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, West Mains Road, EH9 3JT, Edinburgh, U. K.; ²Department of Biology and Biochemistry, University of Bath, South Building, Claverton Down, BA2 7AY, Bath, U. K.; ³Department of Biology, University of Puerto Rico, P.O. Box 23360, San Juan, PR 00931; ⁴The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hixton, CB10 1SA, Cambridge, U. K.

Correspondence: sbaxter@staffmail.ed.ac.uk

Identifying a gene responsible for a trait of interest within Lepidoptera is a multi-step process. Commonly, this involves performing crosses to enable trait segregation, using progeny to create linkage maps, identifying the genome region responsible for variation and then genetic mapping of candidate genes to test for associations. If candidate genes tested do not map to the chromosome or locus of interest, sequencing genomic BAC clones spanning the genome region is required. Here we present a molecular method for identifying DNA markers tightly linked to a quantitative trait, which can be used to screen BAC libraries, using wing colour variation in Heliconius butterflies as a model. Heliconius melpomene and H. erato are co-mimics that share the same warningly coloured wing patterns where they cohabit throughout Central and South America. Approximately 30 races of wing colour variants have been characterised and identifying the colour determining genes will be of value to developmental and evolutionary biology, and population genetics. We have used AFLP methods to identify the genetic locus of a strong visual trait in these sympatric species – red wing patches. Two dominant loci on the same chromosome of H. melpomene have previously been found to control red wing colouration: “B” is a large red spot on the forewing and “Dennis” (D) includes ray patterns on the hind wing and red colour on the proximal forewing. Crosses were performed between B/D heterozygotes (BbDd x BbDd) then AFLPs were performed on two progeny bulks; i) homozygotes lacking the B spot (bbD-) or ii) lacking the Dennis pattern (B-dd). When AFLP analysis was performed, it was expected that both bulks would share identical banding patterns, except in areas tightly linked to the B or D loci. AFLP analysis was performed using fluorescently labelled primers on a 3730 DNA Analyzer (Applied Biosystems). From approximately 300 primer combinations, nine bands were identified and confirmed linked the B or D locus. AFLPs were sequenced and used to create genotyping assays for additional broods, which found B and D to be tightly linked. Multiple of the AFLP markers were within 1 centimorgan (~180kb) of this region, and were used as probes to screen a H. melpomene BAC library. A similar strategy has been used to identify an AFLP band close to the Dry locus in H. erato, which controls the same phenotype as B and D in H. melpomene. Mapping a gene close to the H. erato AFLP in H. melpomene, has shown homologous genetic regions are responsible for similar red wing phenotypes in these two species. Performing AFLP analysis on bulks of progeny, selected for the presence or absence of a quantitative trait, could be applied to other lepidopteran species.

G.T. Behere, D. Russell and P. Batterham, W. T. Tay

ARC Special Research Centre for Environmental Stress and Adaptation Research (CESAR), Department of Genetics, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Victoria 3010, Australia.

Correspondence: p.batterham@unimelb.edu.au

The use of mitochondrial DNA Cytochrome Oxidase I (mtDNA COI) gene in species identification has gained popularity in recent years, with its effectiveness in identifying cryptic and new species demonstrated in birds and Lepidoptera. The noctuid moths of the genus Helicoverpa include two of the most devastating agricultural pest species: H. armigera of the old world, and H. zea found exclusively in the north and south American continents. Both of these species are polyphagous targeting >150 crop species. Phylogenies of H. armigera and H. zea have to-date been constructed based on coding nuclear DNA sequences and morphological characters, but a mtDNA phylogeny of H. zea and H. armigera has been lacking. Differentiating H. zea and H. armigera based on morphological characters relies almost exclusively on characters of the male genitalia, although accurate identification has remained problematic due to over-lapping ranges in character measurements. H. zea and H. armigera are presently recognised as two separate species despite successful bi-directional, ‘inter-specific’, mating experiments that gave rise to viable offspring, and the trapping of H. zea males when using H. armigera sex pheromones in the North American continent. Using a 511 base pair sequence of a partial mtDNA COI gene, we analysed the phylogenetic relationships amongst 228 H. armigera individuals sampled from China, Australia, Africa, India and Pakistan, plus 14 H. zea from North America, H. punctigera from Australia and H. assulta from India, using Heliothis virescens as an outgroup. Our mtDNA COI phylogeny of Helicoverpa species indicates that H. punctigera is ancestral to H. assulta which is in turn ancestral to H. armigera and H. zea. Furthermore, the long branch-length of H. zea from the H. armigera clade suggests a recent bottleneck event in H. zea’s separation from H. armigera. H. zea and H. armigera show an intermediate level of nucleotide diversity, lying between expected values for intra-specific and inter-specific sequence comparisons, possibly suggesting rapid nucleotide divergence in H. zea due to selection pressures imposed on movement by agricultural practices.

P. Beldade^1,2, S. Rudd³, J.D. Gruber¹, A.D. Long¹

¹Department of Ecology and Evolutionary Biology, University of California at Irvine, USA; ²Institute of Biology of the University of Leiden; The Netherlands; ³Turku Centre for Biotechnology, Turku, Finland.

Correspondence: pbeldade@biology.leidenuniv.nl

Bicyclus anynana (Nymphalidae, Satyrinae) has been established as a laboratory organism in the study of the evolution and development of wing patterns. I will describe an Expression Sequence Tag (EST) project for B. anynana that has identified the largest publicly available collection to date of expressed genes for any butterfly. By targeting cDNAs from developing wings at those stages when pattern is specified, we biased gene discovery towards genes potentially involved in pattern formation. Assembly of 9,903 ESTs from a subtracted library allowed us to identify 4,251 genes of which 2,461 were annotated based on blast analyses against relevant gene collections. Gene prediction software identified 2,202 peptides, of which 215 longer than 100 amino acids had no homology to any known proteins and thus potentially represent novel or highly diverged butterfly genes. We combined gene and Single Nucleotide Polymorphism (SNPs) identification by constructing cDNA libraries from pools of outbred individuals, and by sequencing clones from the 3′ end to maximize alignment depth. Alignments of multi-member contigs allowed us to identify over 14,000 SNPs, with 316 genes having at least one high confidence double-hit SNP. We furthermore identified 320 microsatellites in transcribed genes that could potentially be used as markers. These resources are being used to develop genomic tools for B. anynana and will be invaluable for exploring the potential of this and butterflies in as models in ecological, evolutionary, and developmental genetics.

B. Breugelmans¹, G. Simonet¹, S. Van de Velde², S. Van Soest¹, G. Smagghe², J. Vanden Broeck¹

¹Laboratory of Developmental Physiology, Genomics and Proteomics, Department of Animal Physiology and Neurobiology, Zoological Institute, K.U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. ²Laboratory of Agrozoology, Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.

Correspondence: Bert.Breugelmans@bio.kuleuven.be

Pacifastins are canonical serine protease inhibitors which appear to be restricted to Arthropods. Members of this family were originally purified from a Crustacean and have been identified in two Orthopteran locust species and in a Hymenopteran wasp. In silico data mining predicted the existence of more pacifastin-like members in several other insects such as the malaria mosquito (Anopheles gambiae) and the silkworm (Bombyx mori). Every member of this family is derived from a precursor protein. Except for one, all pacifastin precursors contain multiple inhibitor domains, which are usually separated from each other by dibasic cleaving sites. Using the available genome and EST databases, we have cloned the first lepidopteran pacifastin precursor from fat body of the silkworm (Bombyx mori). The precursor, named BMPP-1, codes for 13 pacifastin-like inhibitory domains and constitutes by far the largest pacifastin-like precursor in Arthropods. This brings the total of pacifastin-related peptides up to 45. Although numerous pacifastin-like peptides have been identified in several insect orders, only for a limited number of locust peptides the inhibitory activity towards serine proteases has been analysed. As a consequence, for the majority of the pacifastin-related peptides the (inhibitory) activity remains as yet to be studied. Therefore, in order to further unveil the biochemical and functional properties of this inhibitory family, we have optimised the recombinant production of both locust and Bombyx pacifastin-related inhibitors. This allowed us to test the inhibiting capacities of the recombinant peptides towards mammalian proteases, as well as towards endogenous proteases in crude tissue extracts (haemolymph, fat body and gut) of Spodoptera littoralis, Bombyx mori, Schistocerca gregaria and Locusta migratoria. Preliminary results of these in vitro tests show that (i) in addition to bovine (chymo)trypsin insect gut and hemolymph proteases are inhibited, (ii) not all inhibitors have the same potency, (iii) most inhibitors have a specificity for either trypsin- or chymotrypsin-like proteases, conforming to the P1-residue and (iv) there seems to be species selectivity but only for trypsin-like pacifastin inhibitors. Furthermore, the effect of recombinant peptides on a cell line of lepidopteran midgut cells has been analysed. Initial results show that one particular locust peptide has a distinct inhibitory effect on cell proliferation.

Clark, R.¹, Brown, S.² Heckel, D.³, Jiggins, C. D.⁴, Collins, S.⁵ and Vogler, A. P¹.

¹ Entomology Department, The Natural History Museum, SW7 5BD, London, UK and Department of Life Science, Imperial College, SW7 2AZ, London, UK. ² Dept. Genetics, University of Melbourne, Parkville 3010, Victoria, Australia. ³ Max Planck Institute of Chemical Ecology, D-07745 Jena, Germany ⁴ Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, EH9 3JT, Edinburgh, UK. ⁵ African Butterfly Research Institute (ABRI), Nairobi, Kenya.

Correspondence: rebecca.clark@ic.ac.uk

Papilio dardanus has been an iconic example of selection at work in the classical evolutionary and genetics literature due to the extensive polymorphism and Batesian mimicry found in the females of this species. This phenotypic diversity is controlled by a single autosomal genetic locus, termed H, of which ten distinct alleles have so far been identified through Mendelian crossing experiments. Despite extensive debate as to the nature of H (using supergene, single regulatory gene or combinatorial hypotheses) the locus has remained enigmatic. As a single locus within a single species (and therefore expressed on a relatively uniform genetic background) controlling extensive phenotypic diversity (some of which has known adaptive value) the H locus system is ideal for evolutionary developmental studies. The current assumption that major developmental genes identified in model organisms as acting during patterning through all stages of development also specify the pattern on the wing can be addressed through the identification of H and the mechanism by which alleles at this locus affect the phenotype across the wing. Molecular genetics approaches to the identification of H have resulted in the delimitation of the H genome region, through the production of flanking AFLP markers, and the mapping of a major regulatory gene to this region. This candidate gene co-segregates with the H phenotype in all female offspring (from crosses involving several colour morphs) including those known to be recombinants between the AFLP markers and H. With the identification of such a strong candidate for H population level studies are being used to strengthen the evidence supporting the association between the genotype at the candidate locus and the H phenotype.

N. Chamberlain¹, S. Baxter², C. Jiggins² and R.H. ffrench-Constant¹

¹Center for Evolutionary and Conservation Biology, Tremough, University of Exeter, UK; ²Institute of Evolutionary Biology, University of Edinburgh, UK.

Correspondence: nc230@bath.ac.uk

We are studying two species as models for both Batesian mimicry and Mullerian mimicry. In the Eastern Tiger swallowtail of North America, which is a Batesian mimic of the Pipevine swallowtail, we are studying the biochemical basis of melanism in the melanic female mimics. We have defined the biochemical reactions that lead to the abnormal melanism of the female and highlighted the role of both dopa-decarboxylase (DDC) and BAS (the homolog of the fly gene ebony). The role of the rate of scale development in this phenomenon will also be discussed. More recently, we have been working with C. Jiggins on Mullerian mimicry in Heliconius melpomene. In this species, we are carrying out a chromosomal walk to clone the gene, Yb, which is responsible for the yellow band on the hind wing. We will describe progress in the BAC walk and look at any candidate genes in the region. The Yb locus is interesting as it seems to represent a ‘super-gene’ controlling all of the pattern in a different species H. numata (M. Joron, submitted).

O. Chortyk, J. Friz, C. Thompson, P. Kumar, C. Tice, B. Vertin, R. Palli, M. Kumar, A. Meyer, T. Meteyer, H. Smith, D. Cress, B. Li, and R. Hormann. RHormann@Rheogene.com

RheoGene, Inc., 2650 Eisenhower Ave., Norristown, Pennsylvania, 19403, U.S.A.

Drug-inducible regulation of multiple genes independently, simultaneously, and silently with respect to host metabolism is a highly desirable capability in gene therapy and tissue engineering. Insect-derived ecdysone receptor (EcR)-based gene switches are an intriguing option due to anticipated fundamental orthogonality to transcription events related to mammalian homeostasis. The essential components of a highly orthogonal EcR dual channel gene switch and even a potential three-channel gene switch using ligands representing two and three different chemotypes, respectively, have been previously described (Kumar MB et al. 2002. Proceedings of the National Academy of Sciences USA 99:14710-14715; Mohan BK et al. 2004. Journal of Biological Chemistry, in press) Orthogonal actuation of multiple genes with ligands selected from a common chemotype, rather than several unrelated chemotypes, could provide advantages in simplifying pharmacokinetics and pharmacodynamics, and could also potentially ameliorate possible undesired pleiotropic effects. Toward the general goal of orthogonal multiplex gene-switch systems and the specific goal of systems using a single chemotype, a designed library of 246 ligands, including diacylhydrazines and ecdysteroids, was screened against a panel of 68 wild-type and LBD-mutant EcRs in a transcriptional activation assay. EcRs were fused to the GAL4 DNA binding domain in conjunction with a chimeric RXR fused to a VP16 activation domain. Luciferase was employed as a reporter gene, and the vectors were transfected into 3T3 cells. EC50 values for each receptor-ligand pair were obtained and multiple two-, three- and four-channel orthogonalities were observed. We report the influence of chemotype, ligand library subpopulation, and design principles of both mutants and ligands in their contribution to the number and robustness of the resultant orthogonal switch systems. Diacylhydrazine intra-chemotype specificity of selected duplex switches is described using CoMFA methodology employing designed training sets and diversity-selected test sets. Receptor-independent QSAR is considered in view of a diacylhydrazine-docked EcR homology model.

Derek Collinge^1–2, Karl Gordon¹, Carolyn Behm², Steve Whyard³.

¹CSIRO Entomology Canberra, Australia; ²The Australian National University Canberra, Australia; ³University of Manitoba, Canada.

Correspondence: derek.collinge@csiro.au

Stable transformation is an essential tool for molecular biologists working on non-model organisms. The ability to introduce and express genes of choice in an organism provides a means to investigate important molecular questions such as gene function, biochemical pathway analysis, reporter gene studies and developmental processes. My PhD studies have focused on the transformation of the pest Helicoverpa armigera with the reporter gene EGFP (enhanced green fluorescence protein). There are essentially two parts to transformation, 1) DNA delivery and 2) target gene integration. In insects the latter is commonly mediated by the use of transposons gene movement. I have used the class II transposon piggyBac to facilitate the movement of EGFP into the genome of H. armigera eggs as a proof of integration. Biolistics DNA delivery used microscopic gold particle coated with DNA of choice which are accelerated at high velocity into cells. In the past biolistics has been used to transform many kinds of plant tissue and has had mixed success transforming Drosophila eggs. Extensive attempts to adapt biolistics to transform H. armigera eggs proved fruitless with too many technical hurdles to over come. However, recent attempts using microinjection DNA delivery have been far more encouraging with a high frequency of transient EGFP expression and the generation of two putative EGFP stably transformed H. armigera lines. The development of an effective microinjection technique has also allowed us to look at the roll of RNAi in H. armigera with the view to using RNAi to knockdown the expression of essential/specific genes which in turn will form the basis for the development of a genetic control mechanism. By co-injecting an EGFP construct and either siRNA or dsRNA against EGFP, I have observed a significant reduction in the frequency and level of EGFP expression in eggs injected with dsRNA, and to a lesser extent with siRNA’s. This indicates conservation of the RNAi pathway in H. armigera and will allow me to begin testing phenotypic effects of silencing new gene targets. For RNAi to be developed as an effective pest control mechanism, the parameters of RNAi in specific pests must be well understood. In specific the systemic nature or RNAi within H. armigera must be analysed. For RNAi to be most effective, the silencing signal must be able to spread through all cells in the organism. One gene identified in C. elegans known as SID-1 plays a roll in mediating the systemic spread of the RNAi signal which may involve the cell-cell movement of siRNA’s. Not all organisms contain a SID-1, for example no SID-1 has been identified in Drosophila and as a result systemic silencing is absent. Recently I have identified 2 different SID-1-like genes in H. armigera and will soon be undertaking detailed spatial and functional studies once the two variants have been fully cloned. The presence of SID-1-like genes in H. armigera strongly suggest the presence of systemic RNAi in this organism and supports further studies into the use of RNAi as a pest control mechanism.

d’Alençon¹ E., Audant² P., Bernard-Samain³ S., Bidegainberry² V., Brehélin⁴ M., Brun-Barale² A., Cousserans¹ C., Duvic⁴ B., Escoubas⁴ J-M., Feyereisen² R., Fournier¹ Ph., Gagneur¹ C., Gordon⁵ K., Gimenez¹ S., Heckel⁶ D., Hotelier⁷ Th., Hilliou² F., Mita⁸ K., Negre⁷ V., Sabourault² C., Suraporn² S., Volkoff¹ N., Weissenbach³ J.

Correspondence: abrun@antibes.inra.fr,

Correspondence: heckel@ice.mpg.de

¹Biologie Intégrative & Virologie des Insectes, INRA-UMII, Université Montpellier II, 34095 Montpellier Cedex 5, France; ²Réponse Organismes aux Stress Environnementaux, INRA / Univ-Nice, 400 rte Chappes, BP167, 06903 Sophia-Antipolis Cedex, France; ³Génoscope, Centre National de Séquençage, 2 rue Gaston Crémieux CP5706, 91057 Evry Cedex, France; ⁴Ecologie Microb. Insectes & Interactions Hôtes Pathogènes, INRA-UMII, Université Montpellier II, 34095 Montpellier Cedex 5, France; ⁵CSIRO Entomology, GPO Box 1700, Canberra ACT 2601, Australia; ⁶Max Planck Inst. für Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany; ⁷Unité Informatique Centre, AgroM-INRA, 2 place Viala, 34060 Montpellier Cedex 02, France; ⁸Insect Genome Lab., National Inst. of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.

Species of the genus Spodoptera are pests which cause severe economic damage on a variety of crop plants in all continents. Several of these species are used as model for studies on pesticide resistance and host-pathogens interactions. New developments in biology and pathology of these insects now rely upon a functional genomics approach. For this purpose, we (groups 1–2 & 7, see above) established sets of cDNA from tissues which were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction, i.e. hemocytes, midgut and fat body. A total of 29,325 ESTs sequences were obtained, which were clustered into non-redundant sets (2294 clusters and 6103 singletons) and integrated into a database. SPODOBASE (Negre et al., 2006, BMC Bioinformatics, submitted. http://bioweb.ensam.inra.fr/Spodobase/) is constructed in such a way that ESTs from other Spodoptera species can also be added. User can retrieve information using text searches, pre-formatted queries, query assistant or BLAST searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary. The whole set of ESTs (From part of this set of data a first Spodoptera microarray was printed and used to study host-parasitoid interaction at the transcriptomic level, see communication by Volkoff et al., this meeting) was compared with databases and this revealed that 15 % of the S. frugiperda ESTs - after checking that these correspond to potentially coding regions - do not have a match in BLAST searches against neither NCBI-nr nor B. mori EST databases. We also found that only 48 % of the contigs and 30 % of the singletons show a significant similarity with a Uniprot entry. This opens a research towards the identification of potential Lepidoptera or even Spodoptera specific genes. Phylogeny among Lepidoptera is still poorly known and a synteny programme will allow us to develop a comparison between gene order, gene orientation, intergenic distance and sequence similarities among different species. For this purpose a consortium of 6 laboratories launched a collaboration towards the sequencing of 60 selected genomic regions for both Spodoptera frugiperda and Helicoverpa armigera, to be then compared with the same regions of the B. mori genome. The regions to be sequenced were selected on the basis of the presence of genes representing different major biological functions of the insect, like immune response, olfaction, phylogenic markers, targets of insecticide resistance, P450 and GST families. BAC libraries were constructed for both species and various sequences were chosen as probes against the high-density filters. These probes were obtained either from the ESTs collections or from already sequenced cDNAs. More than 320 genes have now been identified among the first 28 BACs already sequenced representing 1.9 Mb of S. frugiperda and 1.5 Mb of H. armigera genomes. Present results also show presence of a high density of sequences displaying similarities with known repeated elements (retrotransposons with or without LTR, DNA transposons of several types). They were slightly more abundant in S. frugiperda than in H. armigera (237 vs. 124), and their relative frequency also varies between the two species. BAC sequencing is in progress at the French Genoscope National Center of Sequencing and results will be discussed.

De Simone Anna Maria, Sorrentino Angela, Di Cara Francesca, Lino Polito and Digilio F. Anna

Institute of Genetics and Biophisics "A. Buzzati-Traverso", Via P. Castellino 111, 80131 Naples, Italy.

Correspondence: digilio@igb.cnr.it

The legume pod borer Maruca vitrata (Fabricius) (syn. M. testulalis) (Lepidoptera: pyralidae), is a pest of legume crops such as cowpea, pigeonpea, and common bean from the tropics to the temperate zone all over the world. Larvae damage flower buds, flowers and immature pods, causing severe economical damage to several crops. One of the most important host is cowpea (Vigna unguiculata) which is a highly important grain legume crop grown in semi-arid and dry savannah areas of the tropics. Cowpea provides a cheap source of nutritious food and is equally important for improving soil fertility, soil conservation and the sustainability of various cropping systems. Although the basic biology of M. vitrata has been studied extensively, no molecular studies have so far been carried out on this species. In order to obtain more information on this insect, we are developing tools for a molecular approach. Specifically, as a first molecular characterization of developmental genes in M. vitrata and with the future prospect of generating sterile mutations in this species, we decided to characterize the sex-determining genes, starting from the major determinants of the fruit fly sex-determining genes and using the knowledge gained in Drosophila melanogaster. Within the highly diversified Class Insecta, in spite of the existence of a variety of sex-determining mechanisms, the final regulatory genes as well as doublesex (dsx), intersex (ix) and transformer 2 (tra2) are well conserved, and their have been identified in many species other than Drosophila: Ceratitis capitata, Megaselia scalaris, Anopheles gambiae, Musca domestica and in the silkworm Bombyx mori. Based on this consideration, we started our molecular approach searching for dsx, ix and tra2 homologues in M. vitrata, by using D. melanogaster and B. mori genes as heterologous probes. Until now these studies allowed us to identify the M. vitrata ix gene homologue and several other genes that we can use to perform a comparative study between Maruca vitrata and other lepidoptera characterized to date and in broad terms between Diptera and Lepidoptera. The comparative study of these two systems (Lepidoptera and Diptera) will provide both basic knowledge and data that can be applied to develop strategies focused on biological control of this pest.

J-M Drezen, A. Bezier, J. Lesobre, E. Huguet and C. Dupuy

Institut de Recherche sur la Biologie de l’Insecte CNRS, Tours, France.

Correspondence: drezen@univ-tours.fr

Hundreds of thousand species of parasitoid wasps develop during their larval stage within the body of other insects while the adults are free. In particular 17500 species of wasps, phylogeneticaly related -the microgastoids- are obligatorily associated with viruses of the bracovirus genus. Permanently integrated into the wasp chromosomes, bracoviruses are thought to originate from the same virus integrated in the genome of the ancestor of the microgastroids that lived approx. 70 millions years ago. Bracoviruses are essential for parasitism success. Mature virions are produced in the wasp ovaries and injected by female into the host lepidopteran larvae, along with the wasp’s eggs. The virus particles enter host cells where viral genes are expressed, causing several alterations to the host physiology which are beneficial for the development of the wasp progeny, comprising disruption of the immune defenses, retarded growth and inhibition of metamorphosis. Sequencing of a bracovirus genome have allowed the characterization of several gene families encoding potential factors involved in parasitism success. Interestingly some of the genes recently identified contain conserved protein domains found in virulence factors from other parasites or pathogens that are known to be involved in the suppression of the immune response of mammals. This suggests a convergent evolution between parasitoid wasps, bacterial pathogens, and parasitic nematodes (filariae) to target pathways of the host cellular response conserved in vertebrates and invertebrates. The characterization of genes conserved in the virus genomes of different parasitoid species will also be an essential tool to understand the role played by bracoviruses in the radiation of the parasitoid wasp families, highly diversified.

I. Eleftherianos¹, P. J. Millichap¹, G. Felföldi^1,2, F. Gökcen³, N. Waterfield¹, D. J. Clarke¹, R. H. ffrench-Constant⁴, S. E. Reynolds¹

¹Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; ²Department of Biochemistry, Eötvos Lörand University, Pazmany setany 1/C, Budapest, H-1117, Hungary; ³Institut für Allgemeine und Spezielle Zoologie, Justus-Liebig-Universität, Stephanstr. 24, 35390, Giessen, Germany; ⁴School of Biosciences, University of Exeter, Cornwall, UK.

Correspondence: bssie@bath.ac.uk

Photorhabdus is a highly virulent pathogen that produces a range of lethal toxins to kill its insect host. Here we show that Photorhabdus is recognised by the immune system of its host Manduca sexta, as indicated by a rapid increase in the levels of mRNAs encoding three different inducible microbial recognition proteins, Hemolin, Immulectin-2 and Peptidoglycan Recognition Protein in the fat-body. RNAi knock-down of any one of these genes markedly decreased the ability of the insects to withstand infection when exposed to Photorhabdus. RNAi against Immulectin-2 caused the greatest reduction in host resistance to infection. The decreased resistance to infection was associated with reduced haemolymph phenoloxidase activity. We also show that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by Photorhabdus. Induction of this protective effect is associated with up-regulation of both microbial recognition protein genes and anti-bacterial effector genes. RNAi knock-down of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. We present evidence that the protection elicited by previous exposure to E. coli is due to the presence of factors within the haemolymph plasma that inhibit the growth of Photorhabdus. We have also used RNAi to investigate the function of recognition protein genes expressed in M. sexta haemocytes. RNAi of Hemolin expression was associated with reduced ability of haemocytes to aggregate, form nodules and phagocytose the invading bacteria. A different approach to investigate pathogen-host interactions is to identify genes that enable Photorhabdus to persist and multiply within the insect. In order to discover such persistence genes we screened cosmids from a fully sequenced Photorhabdus genome and identified a kdp operon that encodes the protein subunits of a bacterial K⁺ ion transporter and the two-component regulator that governs their expression. Expression of these Photorhabdus genes allows E. coli to persist within phagocytic hemocytes; disruption of any one of the operon’s genes prevents persistence. Since expression of the two-component regulator genes alone is sufficient for persistence, we conjectured that the distinctive feature of Photorhabdus kdp is the sensor-regulator pair, which can also regulate expression of the E. coli kdp genes to the detriment of the host insect. We confirmed this hypothesis by showing that whereas E. coli kdp genes are not expressed after phagocytosis, the kdp genes of Photorhabdus are strongly expressed within M. sexta phagocytes. Finally we show that Photorhabdus produces a hydroxystilbene antibiotic compound that also acts as an inhibitor of phenoloxidase, an important component of the insect immune system. These results show not only that Photorhabdus is recognised by the M. sexta immune system but also that the insect’s immune system plays an active, but ultimately ineffective, role in countering infection.

M. Elias¹, M. Joron¹, K. Willmott², V. Kaiser¹, K. L. Silva-Brandão³, A.V.L. Freitas³, C. Arias Mejía⁴, L.M. Gomez Pineres⁵, A.V.Z. Brower⁶ and C. Jiggins¹

¹ Institute of Evolutionary Biology, University of Edinburgh, Scotland, UK. ² Florida Museum of Natural History, University of Florida, Gainesville, USA. ³ Instituto de Biologia, Universidade Estadual de Campinas, São Paulo, Brazil. ⁴ Universidad de los Andes, Bogotá, Colombia. ⁵ Universidad de Medellín, Medellín, Colombia. ⁶ University of Tennessee, USA.

Correspondence: marianne.elias@ed.ac.uk

Species-level phylogenetic hypotheses can be used to explore patterns of divergence and speciation. Warningly-colored mimetic butterflies are interesting models, because their wing patterns are under strong selection, and might be involved in speciation. Here we present an almost complete phylogenetic hypothesis for the neotropical mimetic butterfly genus Napeogenes (20 species), based on mitochondrial genes (entire cytochrome oxydase I and II) and nuclear genes (tektin, 715bp; and elongation factor 1α, 1028bp). Whenever possible, we included several subspecies of the same species (with different wing patterns). The results showed good congruence between the different genetic regions. In most cases individuals of the same species clustered together, supporting the current taxonomy. However, N. larina otaxes and N. larina aethra appeared to be two sister species rather than two subspecies. A combined evidence topology is presented based on a Bayesian analysis of all the genes regions. Using the phylogentic hypothesis we investigate whether changes in wing pattern were associated with speciation, and whether speciation was dominantly sympatric or allopatric. Using the phylogenetic hypothesis we explore patterns of diversification, and we investigate whether changes in altitudinal range and in wing pattern were associated with speciation.

J.-M. Escoubas¹, P.-A. Girard¹, N. Volkoff², Y. Boublik³, F. Cousserans², E. D’Alençon², K. Mita⁴, P. Taillez¹, M. Brehélin¹.

¹Écologie Microbienne des Insectes et Interactions Hôte-Pathogène, Institut National de Recherche Agronomique, Université de Montpellier II, CC54, 2 place E. Bataillon, 34095 Montpellier, France;²Biologie Intégrative et Virologie des Insectes, Institut National de Recherche Agronomique, Université de Montpellier II, CC101, 2 place E. Bataillon, 34095 Montpellier, France;³Plateforme Protéines Recombinantes, C.R.B.M., Centre National de la Recherche Scientifique, 34293 Montpellier, France; Laboratoty of Insect Genome, National Institut of Agrobiological Sciences, Tsukuba, 305-8634, Japan.

Correspondence: jmescoubas@univ-montp2.fr

A new family of putative antimicrobial proteins, characterized by imperfectly conserved tandem repeats (up to 11) of cystein-stabilized alpha beta motifs (CS-αβ), a structural scaffold characteristic of invertebrate defensins and scorpion toxins, was identified through functional genomic approaches in the lepidopteran species Spodoptera frugiperda. Orthologs were also found in ESTs or genomic databases from other lepidopteran species (Bombyx mori, Galleria mellonella, Papillo dardanus), but not in other insect orders or other invertebrate or metazoans, thus suggesting that this protein family is specific to the lepidopteran insects. In S. frugiperda as well as in B. mori, more than one transcript can be related to this protein family. In silico analysis of B. mori genome (the sole lepidopteran genome available to date) suggests that the three mRNA encoding X-tox proteins that were characterized in this insect are generated through a mechanism of alternative splicing. This mechanism thus allows the production of different X-tox proteins containing five or six CS-αβ motifs. S. frugiperda X-tox genomic organization is currently under investigation. The role of these proteins in lepidopteran immune response was investigated by transcriptional analyses with one of the S. frugiperda cDNAs. Results indicate that the corresponding gene, spod-11-tox, is expressed mainly in larval fat body and hemocytes and that transcription is enhanced in both immune tissues upon bacterial challenge. Phylogenetic analyses show that CS-αβ motifs-containing proteins are divided into three clusters. The first, considered as “ancestral” group, comprises molecules from different invertebrate taxa (odonate insect, arachnids and mollusks) and even a fungus molecule whereas the second group includes defensins isolated from neopteran insects. Finally, the third cluster contains all the CS-αβ motifs stem from lepidopteran X-tox proteins. According to this phylogenetic analysis and to the position of lepidoptera in insect evolution (one of the latest order in insect speciation), we propose that X-tox proteins evolved from insect defensins to generate a new family of antimicrobial proteins. The main feature of this family is the multi-domain organisation who leads to molecular diversity generated through a mechanism of alternative splicing. The full characterization of this new protein family (gene organization and expression, protein structure and mechanism of action) in several lepidopteran species should contribute to solve the puzzle of the evolutionary history of arthropod defensins.

G. Felföldi^1,2, I. Eleftherianos¹, R. H. ffrench-Constant³, I. Venekei², S. E. Reynolds¹

¹Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; ²Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117, Hungary; ³School of Biosciences, University of Exeter, Cornwall, UK.

Correspondence: bssie@bath.ac.uk

Photorhabdus luminescens lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect. Here they secrete factors, which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes. One of the secreted proteins is an rtx-like zinc metalloprotease PrtA. Using a proteomic approach to determine the natural substrates of PrtA in host haemolymph, we identified a number of Manduca sexta haemolymph proteins that are selectively cleaved when plasma was incubated with purified PrtA. One of the PrtA protein targets in M. sexta haemolymph included the Serine Protease Homologue Protein 3 (SPH-3). We found that SPH-3 is not transcribed in the fat-body or haemocytes of naive M. sexta, but only when insects are exposed to bacteria, including Photorhabdus. We used RNA interference (RNAi)-mediated inhibition of expression through injection of double-stranded RNA of SPH-3 into M. sexta caterpillars and we achieved reduced transcription of SPH-3 in both fat-body and haemocyte tissues. Knock-down of SPH-3 dramatically reduced the ability of insects to resist infection to a standard dose of Photorhabdus, as measured by the rate at which infected insects die. RNAi of SPH-3 was also associated with decreased levels of phenoloxidase (PO) activity in haemolymph cell-free plasma from insects infected with either a non-pathogenic strain of E. coli or Photorhabdus. The reduction in PO activity was also reflected by a reduction in the number of melanotic nodules present in tissues of dissected larvae. Finally, we found that RNAi of SPH-3 did not affect the transcription of pattern recognition protein genes (Hemolin, Immulectin-2, Peptidoglycan recognition protein, Pattern recognition Serine Proteinase, β-glucan recognition protein-1, β-glucan recognition protein-2), but down-regulated the transcription of certain antibacterial effector genes (Attacin, Cecropin, Moricin). Our findings strongly suggest that SPH-3 serves specific immune functions and plays a distinctive role in M. sexta immune pathways.

H. M. Fischer¹, C. W. Wheat², U. Wittstock³, D. G. Heckel¹, H. Vogel¹

¹Entomology Dept., MPI for Chemical Ecology, D-07745 Jena, Germany; ² Dept. of Biological and Environmental Science, University of Helsinki, FI-00014 Helsinki, Finland; ³Inst. für Pharmazeutische Biologie, Technische Universität Braunschweig, D-38106 Braunschweig, Germany.

Correspondence: hfischer@ice.mpg.de

A metabolic diversion of a plant chemical defense caused by an enzyme (Nitrile Specifier Protein, NSP) enables a majority of Pieridae species to feed on Brassicaceous plants. The NSP is a newly recruited detoxifying protein in this family. It shows a specific repeat structure and has no sequence homology to any protein with a known function. We are trying to unravel the molecular mechanisms leading to the evolution of this protein and the subsequent adaptation to a plant defense system. The nucleotide sequence is known for two model species. By generating cDNA libraries of related species and amplifying intron and upstream regions, species and allelic differences are being located. Although NSP shows no sequence homology to any protein of known function, single domains show similarities to the so-called major allergen proteins, found in the gut lumen of many insect species. By establishing heterologous expression systems for both proteins we are trying to investigate the mode of action of NSP and the function of MA.

D. Freitak, D. Heckel, H. Vogel

Department of Entomology, Max Planck Institute for Chemical Ecology, Hans Knöll Str. 8, Jena D07745, Germany.

Correspondence: dfreitak@ice.mpg.de

One way to sense the pathogen load in the environment is through the food. lepidopteran larvae are consuming vast amount of food during their relatively short lifespan, this leads to the intake of large amount of possibly harmful microorganisms. Many pathogens are using digestive system as a route to enter the host. We hypothesize that this should have lead to the evolution of pathogen recognition capacities of the digestive system and the mounting of an effective immune response after recognition. In my experiments I raised T. ni larvae on sterile artificial diet and on artificial diet contaminated with bacteria. I collected the hemolymph and midguts of last instar larvae and measured the antibacterial activity and protein expression of the hemolymph. Furthermore, to estimate the status of the immune system, I measured the general lysosomatic and phenoloxidase activity. Larvae grown on the bacteria-contaminated diet showed higher steady state lytic activities and arylphorin concentration in their hemolymph. Contrary to this finding, phenoloxidase activity was higher in the individuals grown on the diet without bacteria. The type of diet also showed an effect on the growth and pupation of larvae. Larvae who were fed on diet contaminated with bacteria needed more time to reach the pupal state and had a lower pupal weight. This kind of effect is mediated via digestive system. I used Real-Time PCR to check the differential expression level of different immune response related genes and found out that several genes connected to synthesis of antibacterial proteins are up regulated in the midgut of the larvae grown on bacterial diet.

T. Fujii¹, H. Abe², K. Mita³, S. Katsuma¹, T. Shimada¹

¹Department of Agricultural and Environmental Biology, Graduate school of Agricultual and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; ²Department of Biological Production, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan; ³Laboratory of Insect Genome, National Institute of Agrobiological Science, Tsukuba 305-8634, Japan.

Correspondence: tsuguru2002@yahoo.co.jp

The silkworm, Bombyx mori, has a sex-determination system (ZW female: ZZ male) in which a W chromosome epistatically determines femaleness. Therefore, the putative Fem (female determinant) gene is assumed to be localized on the W chromosome. So far, 12 W-specific RAPD markers (W-RAPD) have been identified in the silkworm strains maintained in Japan. However, because recombination is restricted to the male in B. mori, genetic mapping of these W-RAPD markers and Fem gene to the W chromosome is impossible. On the other hand, the relative positions of the W-RAPD markers on the W chromosome can be determined using deletion mapping. In deletion-mapping of W-specific RAPD markers, we used X-ray irradiation to break the translocation-carrying W chromosome (W^Ze). We succeeded in isolating a fragment of the W chromosome (Ze^W) having three of 12 W-Specific RAPD markers. It was revealed that the Ze^W fragment does not contain Fem gene because it was transmitted to males. Unexpectedly, we found that the Z chromosome was also broken into a large fragment (Z_L) having the +^sch (1–21.5) and a small fragment (Z_S) having the +^od (1–46.9) by X-ray irradiation. Moreover, it was revealed that a new chromosomal fragment (Ze^WZ_S) was generated by a fusion event between the Ze^W and the Z_S fragments. We analyzed the genetic behavior of the Z_L fragment and the Ze^WZ_S fragment during female (2A : Z_L Ze^WZ_S/W) and male (2A : Z/Z_L Ze^WZ_S) meiosis using phenotypic markers. It was observed that the Z_L fragment and the W or the Z chromosomes separate without fail. On the other hand, non-disjunction between the Ze^WZ_S fragment and the W chromosome and also between the Ze^WZ_S fragment and the Z chromosome occurred. Furthermore, the males (2A : Z/Z_L) resulting from non-disjunction between the Ze^WZ_S fragment and the W chromosome or the Z chromosome had a phenotypic defect. Namely, Z/Z_L male moths could not flap their wings due to indirect flight muscle dystrophy while they could walk and copulate. It was confirmed that the Z_L fragment did not cause the flapless phenotype because Z/Z_L Ze^WZ_S male could flap their wings vigorously. These results suggest that there is haploinsufficient gene(s) which are involved in indirect flight muscle development on the Z_S region of the Z chromosome. In B. mori, absence of dosage compensation is suggested because 12 of 15 genes on the Z chromosome expressed more abundant mRNA in males than in females. In contrast, mRNA amounts of three of 15 genes on the Z chromosome were equivalent in males and females, or female biased (Koike et al., 2003). Therefore, if there are haploinsufficient gene(s) on the Z_S region of the Z chromosome, the expression of the gene(s) may be up-regulated in some stage of female (Z/W) development.

R. Futahashi, H. Fujiwara

Dept. of Integrated Biosciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan.

Correspondence: kk37524@mail.ecc.u-tokyo.ac.jp

Like the adult wings, the larvae of butterflies and moths are unique in their colouring, shape, and patterns. Because butterflies and moths spend most of their lives as soft-bodied larvae, larval body patterns of them are conspicuous traits for natural selection. However, the molecular bases of insect larval pattern formation are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its colour pattern dramatically during the fourth ecdysis. In the juvenile instars of P. xuthus, the larva have brownish-black integuments with white markings, which is inferred to mimic a bird dropping. On the other hand, the fifth (last) instar has a green camouflage colour, which is believed to allow the larger larvae to avoid predators better than the bird-dropping pattern. A comparison of the penultimate and final instar of P. xuthus should be useful in elucidating the regulation of larval pattern formation. In the present study, we aimed to identify the genes involved in the stage-specific larval mimicry markings of P. xuthus, and the hormonal control of their expression. We first compared the mRNA expression of epidermis between the third and fourth molts of P. xuthus using cDNA subtraction method. After analyzing 2,072 clones from two subtractive libraries, we obtained 31 and 64 candidate genes for final- or penultimate-instar-specific genes, respectively. The expression pattern of each gene during the second, third and fourth molts was examined by RT-PCR and whole-mount in situ hybridization. Among final-instar-specific genes (Fsg), Fsg02 and Fsg20 were expressed at the presumptive green region only during the fourth molt, suggesting that Fsg02 and Fsg20 were correlated with green coloration in the final instar. Among penultimate-instar-specific genes (Psg), we identified 10 cuticle genes expressing specifically in tubercle structures during the third molt. These genes were inferred to be involved in the formation of the unique exoskeletal structure observed in the juvenile instar. We next investigated the hormonal effect on the larval patterning. We found that the addition of juvenile hormone analogue to the 4th instar altered the fifth instar pattern into the juvenile-instar type, suggesting that juvenile hormone regulates the stage-specific larval pattern change. This study provides novel molecular markers and insights into the molecular mechanisms of the larval pattern formation and pattern change.

Annie Garel¹, Jérôme Briolay¹, Patrick Brouilly¹, Corinne Royer², Shun-ichi Sasanuma³, Motoe Sasanuma³, Céline Keime¹, Olivier Gandrillon¹, Gérard Chavancy², Kasuei Mita³, Pierre Couble¹

¹Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918 – 69622 Villeurbanne Cedex, France. ²Unité Nationale Séricicole, INRA, 25 Quai Jean Jacques Rousseau – 69350 La Mulatière, France. ³National Institute of Agrobiological Sciences, Owashi 1–2, Tsukuba, Ibaraki 305-8634, Japan.

Correspondence: Royer@cismsun.univ-lyon1.fr,

Correspondence: pierre.couble@univ-lyon1.fr

To identify functions that distinguish the posterior silk gland (PSG) and the median silk gland (MSG) of Bombyx mori, specialized in the production and secretion of fibroin and sericins respectively, serial analysis of gene expression (SAGE) profiles of PSG and MSG cells were analyzed and compared. 41311 and 22078 tags were extracted from respectively the fibroin and the sericin secreting cells. The construction of a B. mori reference tag collection from published resources and from a set of 38000 Bombyx EST sequenced from the 3’ side, helped us to identify a mRNA to more than 60% of the different tags whose incidence was found at more than one exemplary in the two SAGE libraries. Strikingly, a series of highly or mid abundant tags was identified in the MSG tag collection, that did not show up in the PSG SAGE library. cDNA corresponding to 19 of these MSG-specific tags were full-length sequenced. Most of these cDNA corresponded to mRNA encoding protéiens of unknown functions. Four of them were found highly abundant and coding virtually secreted proteins. Current efforts aim at identifying the role of these proteins in the secretion and spinning of silk. The analysis of these novel transcripts allow to suggest that middle silk gland cells realize more diversified functions than that already documented, of synthesis and secretion of sericins, the water soluble components of silk.

M. Geber, I. Faye, and O. Terenius

Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.

Correspondence: ingrid.faye@genetics.su.se

Hemolin, an immune protein belonging to Immunoglobulin gene superfamily is so far only demonstrated to exist in lepidopteran insects. Hemolin is upregulated by viral as well as bacterial infections and in addition, by ecdysone. Several of the putative regulatory elements responding to these inducers are conserved in the promoter region of the Hemolin genes in Hyalophora cecropia and Manduca sexta. One of the conserved upstream elements falls under the consensus of mammalian interferon regulatory factor elements IRF-E and its involvement in the regulation of Hemolin by viruses and the signalling pathways involved remains to be investigeted. From early experiments with H. cecropia it is clear that its NFκB homolog, CIF is not activated during viral challenge. Moreover, it has been shown that partial dsRNA silencing of the Hemolin gene in the Chinese Oak silk moth, Anthereae pernyi increases the progress of an infection by ApNPV in the pupal stage. Recently, we decided to look at the co-evolution of Hemolin and baculoviruses, specific for the different lepidopteran species used. Apart from the six hemolin genes that were already cloned, the longest exon (IV) was cloned from dried specimens of three additional species. From baculoviruses earlier isolated from these lepidopterans, the polyhedrin genes were available in databases and used in the phylogenetic comparisons. The general evolutionary relationship between lepidopteran species was based on the gene for elongation factor 1a, also available in the databases. This study is still in progress and data achieved so far will be presented and discussed.

M. Goldsmith¹, D. Proestou¹, D. Carter¹, E. Nicholson¹, C. Wu², and H. Zhang²

¹Biological Sciences Department, University of Rhode Island, Kingston, RI, USA; ²Department of Soil & Crop Sciences, Texas A&M University, College Station, TX, USA.

Correspondence: mki101@uri.edu

We constructed large insert BAC libraries in the vector, pECBAC1, for Manduca sexta (Ms), Heliconius erato (He), and Heliothis virescens (Hv). Two libraries were generated for each species using partial digests of pupal DNA with BamHI or EcoRI. Based on haploid genome size (M. sexta, 500 Mb, H. erato, 395 MB, and H. virescens 400 Mb, J.S. Johnston, personal communication), mean clone insert size (150–175kb), and number of clones (19.2–21.5 X10³), we estimated the genome coverage per library to be in the range of 6–9-fold. For an independent estimate of genome coverage, we screened high density BAC filters with putative single copy genes in the form of cloned cDNA or PCR-amplified genomic DNA, using the number of hits as a measure of genome redundancy. We used the non-radioactive Amersham ECL Direct system to label probes and detect hybridization by chemiluminescence with X-ray film. Advantages of this method include simple, rapid probe labeling and filter washing protocols coupled with the ability to rehybridize filters without stripping, although we found some signal carry over despite waiting the prescribed period of time before reusing filters. Our primary considerations for probe design and general findings were: 1) Probe length. Although we were able to detect positive signals on filters hybridized with probes as short as 350–400 bp, probes of 1 kb or greater gave more consistent signal-to-noise ratios. 2) Use of well-conserved sequences. To increase the information obtained from our validation studies, we asked colleagues for probes concerning key areas of research using lepidopteran models. These included genes whose products are involved in olfaction (MsOR1, MsOR3), nerve axon growth and guidance (MsNos128, MsEph, MsFasII, MsPlexA), hormone action (MsBroad, MsE75, HvPTTH), wing patterning (Hewg, Heptc, HeCi), Bt toxin action (HvAPN 120, Hvcad), and ribosome structure (HvRpS4, HeRpS5, HeRpS9, HeRpL3, HeRpL10). We reasoned that, considering the paucity of genomic data for lepidopteran species, identifying BACs for well-conserved genes would be of value for future comparative genomics studies. 3) Avoidance of conserved domains. The lack of large-scale genome sequence data for these species made it difficult to predict whether genes for related proteins containing conserved domains were present which might generate duplicate hybridization signals. This problem first arose with a probe for MsBroad containing two zinc fingers which generated twice the number of hits than average. 4) Attention to the presence of introns. Some probes amplified from genomic DNA contained introns, based on greater sequence length than predicted from cDNAs. These often gave an unexpectedly high number of positive signals, indicating the presence of a repeated element. Thus, we tried to avoid probes containing introns unless they were shown to be “clean” in this respect. 5) Amplification of cDNA-containing plasmid inserts with insert-specific primers. Initially we used universal sequencing primers to amplify inserts in plasmid vectors. In some cases the presence of short vector-specific sequences remaining on the PCR products cross-hybridized extensively with the pECBAC1 vector, generating high filter backgrounds. We subsequently switched to primers within the insert sequence. Of interest is that we usually obtained sufficient background on the filters to determine the address of positive clones despite having no known vector sequence in the probe. So far we have tested each library with 6–11 probes, of which 63–85% percent gave well-defined signals. By this approach library coverage averaged as expected for the M. sexta libraries but less than the values based on BAC library DNA content for the H. erato and H. virescens libraries. Possible explanations for the discrepancy will be discussed. Supported by NSF Grant IBN-0208388 to MRG, CW, and HZ. We thank L. Riddiford, F. Gould, and O. McMillan for generous gifts of insects, and A. Nighorn, H. Robertson, L. Riddiford, X. Zhou, P. Copenhaver O. McMillan, S. Gill, L. Gahan, F. Gould, R. Palli, and D. Knipple for probes.

K.P. Gopinathan, R. Parthasarathy and S. Dhawan

Microbiology & Cell Biology Department, Indian Institute of Science, Bangalore 560 012, India.

Correspondence: kpg@mcbl.iisc.ernet.in

Bombyx mori has long been used as a lepidopteran model system for basic studies on gene expression and its regulation. Recently the entire genome of B. mori comprising of about 500 million base pairs has been sequenced. However, the developmental studies at the molecular and genetic levels on this economically important organism has lagged considerably behind Drosophila, which has served as the paradigm for development. We have been exploiting B. mori as a model organism for development for the past several years. There are substantial differences in the developmental programmes between Drosophila and Bombyx. Although the overall programming of appendage development in both the organisms follow common principles, the timings of operation of the patterning mechanisms appear to be significantly different. The silk proteins are produced in the silk glands of B. mori, a pair of tubular structures arising from the labial segment and extending in length all the way up to the caudal region. The silk glands are functionally divided into three distinct compartments, the anterior (ASG), middle (MSG) and posterior (PSG) silk glands. PSG synthesizes the silk fibre proteins and MSG synthesizes the silk glue proteins, the sericins. We have identified the operation of the canonical Wnt signaling pathway in the subcompartment specification within the MSG to result in the territorial regulation of the different sericin proteins. On the other hand, the PSG specification is through the Ubx signaling pathways. The silk glands are often considered as modified salivary glands because of their labial origin, but unlike B. mori, Drosphila lacks the silk glands. B. mori additionally has a pair of salivary glands originating from the mandibular segment. However, there are no subcompartments within the salivary glands. Although there are similarities between the two sets of glands, there are also substantial differences between them.

K. Gordon, G. Colebatch, P.M. Campbell, I. Horne, and P.D. East.

CSIRO Entomology, Canberra, ACT, Australia.

Correspondence: karlg@ento.csiro.au

The lepidopteran insect, Helicoverpa armigera, or cotton bollworm, is a major pest of agriculture worldwide. It is characterised by a wide host plant range and the ability to develop resistance to many control agents. The larval midgut plays a central role in both interactions, being responsible for both digestion and defence against key environmental stresses -e.g. host plant defence chemicals or chemical pesticidal agents. It is the primary target of biological control agents like Bt, now widely used in transgenic crops for pest control. We are undertaking a functional genomics study of the midgut in this insect in order to understand the molecular basis of digestion, detoxification and resistance. Furthermore, this insect's ability to become resistant to chemical pesticides, coupled with the recent detection of genes for Bt-resistance, means an ongoing need for research into novel biological methods for its control. We have undertaken an EST project to identify genes important for midgut function, growth and development and gene/enzyme pathways involved in insect-plant and insect-pathogen interactions. Sequence analysis of midgut cDNA libraries now covers ~5,000 ESTs each from young (2nd and early 3rd instar) and from 5^th instar larvae. Clustering yielded a unigene set of ~3,900 contigs. Searches against databases such as UniProt (EBI) and model organism proteomes yielded confident Gene Ontology annotation for ~55% of the contigs. These include numerous genes associated with key midgut functions: digestive hydrolases; nutrient and ion transport and pH maintenance; a range of detoxification enzymes and genes forming the peritrophic membrane or involved in midgut cell proliferation, differentiation and adhesion. Many have not previously been identified in lepidopteran systems. Additionally, comparison with silkworm genes identified likely orphan lepidopteran genes. Some of the main groups we have analysed to date are: Proteases: Over 80 proteases have been identified. These are predominantly trypsins, with some chymotrypsins and a number of diverged proteases that may be inactive. A variety of amino- and carboxy-peptidases have also been identified. Lipases: Over 20 lipases have been identified. These include a number of diverged lipases that may be inactive.Detoxification: A number of representatives from each of the major classes of detoxification enzymes have been obtained. These include: 20 cytochrome P450 mono-oxygenases (Cyp450s), 6 glutathione-S-transferases (GSTs) and 28 carboxylesterases. Peritrophic membrane components: Three different types of proteins have been identified to date. These are several types of intestinal mucin, that have highly glycosylated mucin-like domains interspersed with cysteine-motif chitin-binding domains (CBDs); these appear to be unique to lepidoptera and appear likely to be an adaptation to their diet rich in plant matter that generates significant (oxidative) stress for the gut epithelium. A further type of peritrophin carries only CBDs; shorter versions of this are found in other arthropods. The third type contains a chitin deacetylase domain (CDA), either alone or with a CBD. These are widespread in arthropods. Immobilised enzymes appear to represent a further class of PM proteins. Microarrays are being used for expression profiling studies in the midgut and have identified numerous genes whose expression shows spatial variation. Expression of genes encoding PM components has been further studied using RT-PCR, whole-mount and in-situ hybridization experiments. Generation of this defence structure is ongoing along the midgut.

Identification of genes that may underlie Bt resistance in Helicoverpa, e.g. from families such as the candidate receptor aminopeptidases, provides a basis for development of strategies for mapping candidate resistance genes. Finally, the cDNA sequences are being used to screen BACs for sequencing by INRA (in collaboration with R. Feyereisen).

T.M. Hughes and J.M. Marcus

Department of Biology, Western Kentucky University, Bowling Green, KY USA.

Correspondence: morrit@wku.edu

The butterfly genus Basilarchia contains several well-known examples of mimicry, even though the majority of species in this genus do not mimic other butterflies. The orange and black viceroy butterfly (Basilarchia archippus) mimics the distasteful monarch butterfly (Danaus plexippus) through much of its range, but the viceroy subspecies found in Florida (B. archippus floridensis) mimics the queen butterfly (Danaus gilippus berenice) instead. Closely related to the viceroy is the red-spotted purple butterfly (B. arthemis astyanax), which is bright blue in color and thought to mimic the distasteful pipevine swallowtail butterfly (Battus philenor). Competing hypotheses for the origin of mimicry in this genus either suggest that mimicry evolved only once followed by a series of model switching events, or that mimicry has arisen independently at least twice in this genus. We tested these hypotheses by generating a molecular phylogeny based on DNA sequences for three genes (cytochrome oxidase I, cytochrome oxidase II, and wingless) obtained from representatives of each of the butterflies in the genus Basilarchia, plus representative outgroups. Our results support the hypothesis of two independent origins of mimicry in the genus Basilarchia, but they also suggest that the viceroy is much more distantly related to the red-spotted purple than had previously been suspected.

E. Huguet¹, C. Serbielle¹, V. Douris², G. Lalmanach³, K. Iatrou², J-M Drezen¹

¹Institut de Recherche sur la Biologie de l’Insecte, UMR CNRS 6035, Faculté des Sciences et Techniques, Parc de Grandmont, 37200, Tours, France; ²Institute of Biology, National Centre for Scientific Research “Demokritos”, P.O. Box 60228, Aghia Paraskevi 15310 Athens, Greece; ³INSERM U 618 « Protéases et Vectorisation Pulmonaires », Université François Rabelais, Faculté de Médecine, 8 ter, Boulevard Tonnellé, 37032 Tours cedex, France.

Correspondence: elisabeth.huguet@univ-tours.fr

Parasitoid wasps have developed amazing strategies to parasitize their insect hosts. One of the most effective involves the domestication of polydnaviruses (PDV). Viral DNA is integrated into the genome of the wasp and is transmitted vertically via wasp chromosomes. Virus excision and replication occurs only in wasp ovaries resulting in the production of segmented double stranded DNA circles which are injected in the host during wasp oviposition. Expression of viral genes inside the insect host alters its immune response and developmental program resulting in the emergence of adult parasitoid wasps. PDVs are divided in two genera, ichnoviruses and bracoviruses, which are associated with tens of thousands of species of parasitoid wasps belonging to ichneumonid or braconid families. Cotesia congregata is a braconid wasp that injects a bracovirus (Cotesia congregata Bracovirus, CcBV) during parasitism of the tobacco hornworm caterpillar, Manduca sexta. Complete sequencing of the CcBV genome has revealed the presence of numerous genes possibly involved in host deregulation. Here we describe the characterization of a CcBV multigene family encoding three proteins with homology to members of the cystatin superfamily. Cystatins are tightly binding reversible inhibitors of cysteine proteases. Cystatins have been described in numerous organisms and are often involved in host-pathogen interactions either acting as host defense proteins or as virulence factors. In filarial nematodes, for example, they account for a major portion of the immunosuppressive activity of secreted filarial proteins and are considered as major pathogenicity factors. Several lines of evidence suggest that viral cystatins could also play an important role in lepidopteran host immune suppression. Viral cystatins are expressed at an early stage and reach very high levels of expression during parasitism. Furthermore, the purification of a recombinant form of one of the CcBV cystatins, cystatin1, revealed that this viral cystatin is functional having potent inhibitory activity towards the cysteine proteases papain, human cathepsins L and B and Sarcophaga cathepsin B in assays in vitro. The identification of the targets of the viral cystatins in the host caterpillar should now help us to deduce the host physiological processes affected by viral cystatins.

Masatoshi Iga, Takayuki Sekimoto, Mohamed Elmogy, Masafumi Iwami and Sho Sakurai

Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan.

Correspondence: ssakurai@kenroku.kanazawa-u.ac.jp

Anterior silk glands (ASGs) of the silk worm Bombyx mori undergo programmed cell death (PCD) in prepupal period in response to the large increase in hemolymph ecdysteroid titer. ASGs loose the sensitivity to JH from day 3 (V3) to 5 of the fifth instar and acquire their responsiveness to 20-hydroxyecdysone (20E) from V5 to V6, the day of gut purge. Then the PCD sequence begins 2 days after gut purge (G2) at and after which 20E is not required for completing PCD. We examined the expression profiles of 11 transcription factor genes in the fifth instar. Developmental profiles of expressions of early genes (two isoforms of each of EcR, usp, E74, E75 and three isoforms of BR-C), an early-late gene (BHR-3) and βFTZ-F1 indicated that these genes were divided into two groups. Genes of one group are EcR-B1, usp-1, E74B and βFTZ-F1, which were maximally expressed from V4 to 6, indicating that those genes could be involved in the acquisition of competence to respond to 20E. The other group genes, that were up-regulated after gut purge, are EcR-A, usp-2, E74A, E75A, E75B and BHR3, showing a possible involvement of these genes in PCD execution. Three BR-C isoforms (Z1, Z2 and Z4) were induced in a stepwise manner; first increase from V5 to V6 followed by the second increase from G1 to G2. These expression profiles are different from those found in Drosophila salivary glands during the period from the time shortly before puparium formation to pupation. G0 ASGs undergo PCD when cultured with 1 μM 20E. During the first 24 h of the culture, EcR-A, usp-2 and E75A were up-regulated in 1 h while E74A, BRH-3 and three BR-C isoforms were at 8 h. By contrast, E74B did not respond to 20E and usp-1 was rather suppressed by 20E. Interestingly, Co-existence of CHX, a translation inhibitor, with 20E enhanced greatly the 20E-induced expressions of EcR-B1, usp-1, E74B and E75B, although these genes were immediate response genes. This indicates the presence of suppressing factors of those genes. 20E-concentration responses for gene expression revealed that the optimum 20E concentrations for individual gene expressions coincided with the hemolymph ecdysteroid titer at the time when individual genes were up-regulated in vivo. Accordingly, the differential gene expressions of those transcription factor genes are brought about by the changes in ecdysteroid titer. In an in vitro culture of G0 ASGs with 1 μM 20E, an addition of α-amanitin, a transcription inhibitor, from 0 h of culture completely inhibited PCD while that at 8 h allowed ASGs to undergo PCD with a slight delay, showing that gene expression required for PCD is completed by 8 h of 20E stimulation. By contrast, when ASGs were transferred differentially to a hormone-free medium, 20E was revealed to be required until 42 h of the culture for competing PCD. These results suggest that PCD of ASGs is governed by two different molecular mechanisms, one associated with de novo gene expression and the other mediated by nongenomic action of 20E. The non-genomic action was analyzed enzymatically by assessing activities of protein kinase C (PKC) and caspase 3-like protease and pharmacologically using various inhibitors and membrane-permeable second messenger analogues. PCD of ASGs proceeds in the order of cell shrinkage, nuclear condensation, DNA fragmentation, nuclear fragmentation and apoptotic body formation. We found that 0.2 mM CHX inhibited protein synthesis but not induced PCD. When ASGs were exposed to CHX and 20E, nuclear condensation, DNA fragmentation and nuclear fragmentation occurred, an indication that those responses do not depend on 20E-induced gene expression. In the nongenomic action of 20E, 20E acted through a calcium-PKC-caspase 3-like protease pathway for inducing DNA fragmentation and nuclear fragmentation, while PKC and caspase 3-like protease activation were not involved in inducing nuclear condensation. Although Ca²⁺ signal was prerequisite for nuclear condensation, calcium ionophore was capable of mimicking 20E only after 18 h preincubation of ASGs with 20E, indicating the cross talk of genomic and nongenomic action of 20E for completing the PCD.

E. Jacquin-Joly, C. Merlin, S. Malpel, J. Pelletier, I. Brigaud, M-C. François, M. Maïbèche

UMR « Insect Physiology: Signalization and Communication », INRA Versailles, Route de Saint-Cyr, F-78026 Versailles Cedex, France.

Correspondence: jacquin@versailles.inra.fr

In insects, among them economically important agricultural pests and disease vectors, olfaction plays a vital role in all aspects of their life. In particular, chemical signals underlie behaviors that are critical for mate (via sexual pheromones) or host recognition and selection (via allelochemicals). A better understanding of the molecular basis of this chemical communication could result in identifying new targets for future strategies to reduce their negative effects. Focusing on the major cotton pest, the noctuid moth Spodoptera littoralis, we set up an EST approach to dissect the olfactory reception mechanisms in the antennae. Indeed, recognition and selection of specific odorants by the antennae relay on the combinatorial expression and intervention of a multitude of different actors, most of which remain to be discovered, such as olfactory receptors or ion channels. Indeed, only few olfactory receptors are known to date in insects and they appear as new G-protein coupled receptor families of extremely divergent genes. Our EST strategy consisted in the elaboration of a cDNA library constructed from RNA extracted from 12000 S. littoralis male antennae, including a normalization step (Invitrogen, CA, USA) to enrich the library in rare transcripts, favouring the discovery of novel genes. Two thousands clones from the library were randomly sequenced from their 5’ end and sequences were analysed and compared to public databases using bioinformatics. This led to the identification of a panel of molecular elements potentially involved in the peripheral olfactory steps in S. littoralis. A repertoire of putative olfactory genes was established, including odorant-binding proteins, olfactory receptors, degrading enzymes, ion channels. In particular, different elements involved in the sex pheromone detection pathway were discovered, such as pheromone-binding proteins and putative pheromone receptors. In addition, numerous modulating elements (hormone receptors, clock genes) were discovered, suggesting that the olfactory response may be modulated by endogenous/exogenous factors at the peripheral level (hormonal statute, circadian clock). In collaboration with the French National Centre of Sequencing (Génoscope, Evry), high-throughput sequencing of the antennae library is under way to complete the antennal olfactory scheme in S. littoralis.

D.L. Jarvis, J.J. Aumiller, C. Geisler, J. Hensley, J.R. Hollister, and X. Shi.

Department of Molecular Biology, University of Wyoming, Laramie, WY, USA.

Correspondence: dljarvis@uwyo.edu

As eukaryotic organisms, insects have the enzymatic machinery needed to N-glycosylate their proteins. Insect protein N-glycosylation pathways generally appear to be intermediate in complexity relative to lower eukaryotes, such as yeasts, and higher eukaryotes, such as mammals. More specifically, insects generally perform the same early steps of protein N‐glycosylation, which include N‐glycan assembly, transfer, and trimming, as lower and higher eucaryotes. But, they lack some of the functions required for N‐glycan elongation. Thus, the major processed N‐glycans produced by insects are relatively simple paucimannose structures (Man₃GlcNAc₂‐R) with or without core fucose residues. An important implication of these observations is that recombinant N‐glycoproteins produced in insect-based systems might not have structurally authentic glycans. In fact, recombinant N‐glycoproteins produced using baculovirus-insect cell systems typically lack the complex, terminally sialylated glycans found on many native mammalian glycoproteins. The insect-derived products typically have paucimannose N-glycans at the sites occupied by complex, terminally sialylated N-glycans in the native mammalian products. In recent years, we have been addressing this problem by using metabolic engineering methods to extend the protein N‐glycosylation pathway of lepidopteran insect cell lines. These efforts have yielded transgenic insect cell lines that encode and express mammalian glycosyltransferases and enzymes involved in CMP-sialic acid biosynthesis. Relative to the parental lines, these new cell lines can still serve as hosts for baculovirus expression vectors and produce similar levels of recombinant glycoproteins. Unlike the parental insect cell lines, however, the transgenic lines can produce recombinant glycoproteins with complex, terminally sialylated N‐glycans. This presentation w