Eighth International Conference on the Juvenile Hormones

s are listed in alphabetical order by the last name of the senior author Characterization of a novel peptide with allatotropic activity in the fall armyworm, Spodoptera frugiperda M. Abdel-latief, M. Meyering-Vos and K.H. Hoffmann Department of Animal Ecology I, University of Bayreuth, 95440 Bayreuth, Germany [email protected] The biosynthesis of juvenile hormone (JH) by the corpora allata (CA) can be either stimulated or inhibited by neuropeptides termed allatotropin (AT) or allatostatin (AS), respectively. To date, only one AT has been identified. It was first isolated from heads of pharate adults of Manduca sexta (Manse-AT; GFKNVEMMTARGF-NH2). The gene encoding Manse-AT has been cloned from various lepidopterans, including S. frugiperda. Here we report the identification of a second peptide with allatotropic activity from the brain of S. frugiperda by molecular techniques. A cDNA that encodes 53 amino acids included one copy of the RVRG NPISCF-OH peptide. This peptide strongly stimulated the synthesis and release of JH in vitro by the CA of female adult S. frugiperda and was code-named Spofr-AT 2. The stimulation was dose-dependent with an app. EC 50 of ca. 10 M. CA that were activated with Spofr-AT 2 could be inhibited by the addition of Manse-AS to the incubation medium. Northern blotting and RT-PCR analyses revealed that the preprohormone is expressed in the brain, midgut, and ovary in a developmental-specific manner. Whole-mount in situ hybridisation confirmed the gene expression in various tissues of adult females. Supported by the DFG (Ho 631/15-4). Juvenile hormone accelerates ovarian development and does not affect age polyethism in the primitively eusocial wasp, Ropalidia marginata M. Agrahari1 and R. Gadagkar1,2 Indian Institute of Science, Centre for Ecological Sciences, Bangalore 560 012, India Jawaharlal Nehru Centre for Advanced Scientific Research, Evolutionary and Organismal Biology Unit, Jakkur, Bangalore 560 064, India [email protected] Juvenile hormone modulates post-imaginal reproductive division of labor in primitively eusocial species and promotes the production of queens (e.g., Polistes) while it modulates age polyethism and promotes the production of foragers in highly eusocial species (e.g., the honey bee). Ropalidia marginata is a primitively eusocial wasp that shows both post-imaginal regulation of reproductive division of labor as well as age polyethism. Hence, R. marginata is a particularly interesting model system to study the effect of juvenile hormone. We demonstrate here that a single, topical application of 100 μg of juvenile hormone-III per female wasp accelerates ovarian development of wasps held in isolation. Similar application to wasps released back on to their natal nests has no effect on their rate of behavioral development as witnessed from the age of first performance of feed larva, build, bring pulp and bring food. We conclude therefore that in R. marginata, juvenile hormone has retained its function of modulating reproductive division of labor and has not acquired the function of modulating age polyethism. Uncovering Juvenile Hormone’s Mode of Action in Ips pini (Say) J.C. Bearfield, S. Young, G. J. Blomquist and C. Tittiger Department of Biochemistry, University of Nevada, Reno, NV 89557-0014, USA [email protected] In the pine engraver beetle, Ips pini, pheromone components are produced de novo via the mevalonate pathway. Feeding and JH III treatment induce an increase in pheromone biosynthetic gene expression in male midgut cells, especially 3-hydroxy-3methylglutaryl-CoA reductase (HMG-R) and synthase (HMG-S). Microarray analysis has identified several other mevalonate pathway genes, as well as a few unknowns, that appear to be JH-responsive. 2 JH-8 2004. Eighth International Conference on the Juvenile Hormones. 17pp. Journal of Insect Science, 4:28, Available online: insectscience.org/4.28 Further expression analyses using real-time and semi-quantitative RT-PCR prepared from midguts at various time points (within the first 8 hours) after JH treatment have revealed some that are rapid, primary JHresponders, with transcripts visible after only 1 hour. The promoter regions of JH-responsive genes are needed to search for putative JH response elements. Therefore, an I. pini genomic library was screened using JH-responsive clones in order to isolate their 5’ flanking regions. Electrophoretic mobility shift assays using nuclear extracts prepared from Sf21 cells treated with either hormone or carrier alone were performed to identify regions of JHinduced protein binding. Also, important regions for induction are being mapped using transcriptional assays with luciferase reporter constructs. The isolation of JH-inducible genes and their regulatory sequences will provide vital information about the mechanism of JH action in the Coleoptera. Unique structural features of moth farnesyl diphosphate synthase: implications for the biosynthesis of homologous juvenile hormones C. Béliveau1, S. Sen2, P. Rehse3 and M. Cusson1 Laurentian Forestry Centre, NRCan-CFS, 1055 du PEPS, Sainte-Foy, QC, G1V 4C7, Canada Dept. of Chemistry, Indiana University-Purdue Universit, Indianapolis, Indiana, 46202, USA RIKEN Harima Institute, Mikazuki, Sayo, Hyogo, 6795148, Japan [email protected] Although the vast majority of insects produce only one form of juvenile hormone (JH III), the Lepidoptera produce a total of five JHs, including four structural homologs that feature one or more ethyl side chains. In a key step of JH biosynthesis, three isoprene units undergo a head-totail condensation catalysed by farnesyl diphosphate synthase (FPPS), generating the C 15 farnesyl diphosphate – the backbone of JH – or one of its C 16-18 structural homologs. We cloned and sequenced FPPS cDNAs from two species of moths. A comparison of these sequences with those of all other known FPPSs – cloned from various organisms ranging from Drosophila melanogaster to Homo sapiens – revealed several amino acid substitutions within the catalytic cavity. Molecular modeling of moth FPPS suggests that some of these substitutions could provide the extra space needed for binding the bulkier ethyl-substituted substrates/products. In assays aimed at assessing the substrate specificity of the recombinant enzymes, we observed that moth FPPS displays a relative activity twice as high as that of Drosophila’s in the presence of homogeranyl-PP and [H]-isopentenyl-PP, suggesting that the moth enzyme shows preference for homologous substrates Juvenile hormone regulation of bark beetle monoterpenoid pheromone biosynthesis G.J. Blomquist, C.I. Keeling, A. Gilg-Young, J. Bearfield, M.D. Ginzel, S. Young and C. Tittiger Department of Biochemistry, University of Nevada, Reno, NV, 89557-0014, USA [email protected] Juvenile hormone (JH) III regulates de novo aggregation pheromone production in bark beetles. Feeding on host phloem or application of JH III induces the production of ipsdienol in male Ips pini and frontalin in male Dendroctonus jeffreyi in a dose and time related manner. The incorporation of [C]mevalonolactone into ipdienol and frontalin demonstrated that these pheromone components were produced via the mevalonate pathway. JH III induction of 3hydroxy3-methylglutaryl-CoA reductase, a key regulatory enzyme in the mevalonate pathway, was localized to midgut tissue by in situ hybridization. Studies with [C]acetate demonstrated that midgut tissue from male I. pini produced ipsdienol and from male D. jeffreyi produced frontalin. This work led to a paradigm shift in which it is now recognized that most, but not all, monoterpenoid pheromone components from bark beetles are produced de novo. It also provided the background to current studies using microarrays to examine JH III regulation of mevalonate pathway enzymes in midgut tissue, the characterization and JH III regulation of geranyl diphosphate synthase, the discovery of putative JH III regulatory elements and a novel regulatory motif in I. confuses. The effect of juvenile hormone on the PBAN-receptor and pheromone production in the moth Helicoverpa armigera: An aging hormone in adult females? R. Bober, T. Zacharova and A. Rafaeli Institute for Technology & Storage of Agricultural Products, ARO, Volcani Center, P.O. Box 6, Bet Dagan, Israel [email protected] The present study was designed to elucidate the effect of Juvenile Hormone (JH) on PBAN activation of pheromone production in vitro and on specific binding to a PBAN-receptor (PBANR) in pheromone glands of H. armigera females. PBAN can activate pheromone biosynthesis only in pheromone glands of newly emerged adult females, after which levels increase with age and reach maximum levels in 3 and 4 day old female glands, thereafter the pheromonotropic response decreases. Female pharate pupae do not respond to PBAN but, in the presence of JH II or its analog, fenoxycarb (FX), PBAN can induce a response in pharate females. In addition, a photoaffinity-biotin-labeled PBAN-analog, specifically binds to a 50 kD putative PBAN membrane receptor in adult females. This binding is absent in pharate females but can be induced after JH II or FX treatments to levels of newly emerged females, thereby providing evidence that JH up-regulates the PBAN-R in immature females. In contrast, in 3-4 day-old mature adult females, FX causes down-regulation of both pheromone production and PBAN-R binding to levels present in older females. Taken together these data indicate that juvenile hormone treatment shifts the normal age-dependent response to PBAN. 3 JH-8 2004. Eighth International Conference on the Juvenile Hormones. 17pp. Journal of Insect Science, 4:28, Available online: insectscience.org/4.28 Effect of Precocene II on Fatty Acid Metabolism in the Pea Aphid, Acyrthosiphon pisum Under Cold Stress Zhaorigetu Chen1, Robin D. Madden2, & Jack W. Dillwith2. 1Section of Integrative Biology, University of Texas at Austin, TX 78712 2Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078, USA [email protected] Pea aphid, Acyrthosiphon pisum, accumulate high levels of myristic acid as triglycerides at 10°C. To investigate whether juvenile hormone (JH) mediates fatty acid metabolism in the aphids, the aphids from 25°C colony were treated with precocene II (a JH antagonist) at 0.5 and 2 μg/aphid, respectively, then reared at 10°C for 16 days. An increase in fatty acid content was observed in the aphids treated with 0.5 μg, whereas no change was observed in 2 μg treatment. When the aphids were retreated with same amount of precocene II 24 h after 1st treatment, the fatty acid content in the aphids did not change in either treatment. A further experiment was conducted to investigate the reproductive fecundity in the aphids treated with 2 μg precocene II. The nymphs per aphid in the treated aphids decreased significantly at either 25°C or 10°C compared to that in the control aphids. However, the nymphs per day in treated aphids at 10°C is similar to that in the control aphids. These results imply that JH may mediate fatty acid metabolism and affect the reproduction in the pea aphids reared at low temperatures. Molecular characterization of the lipophorin receptor of Blattella germanica L. Ciudad, M.D. Piulachs and X. Belles Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona (CSIC), Jordi Girona 18, 08034 Barcelona, Spain [email protected] We have cloned and molecularly characterized a Blattella germanica lipophorin receptor (BgLpR) cDNA. It has a length of 2595 bp encoding a 865-residue protein with a predicted molecular mass of 96.8 kDa. BgLpR is a member of the low density lipoprotein receptor family with five domains: ligand domain, epidermal growth factor precursor, putative O-linked sugar, transmembrane domains, and cytoplasmic tail with the internalization signal FDNPVY. In adult females, BgLpR is expressed in the fat body and the ovary during the 7 days of the first gonadotrophic cycle. In the ovary, mRNA levels are high during the first 5 days and suddenly decrease on days 6 and 7. In the fat body, mRNA levels are high at adult emergence, decrease on days 1 and 2, increase from day 3 to day 5, and slightly decrease on days 6 and 7. The pattern in the ovary corresponds to the dynamics of lipophorin incorporation into the ooplasm during the vitellogenic cycle. That in the fat body may be related with a lipid scavenger activity. The pattern difference in the fat body and in the ovary suggests that the regulatory mechanisms in these two organs are different. The possible role of JH in BgLpR regulation is discussed. Expression and localization of a putative farnesoic acid omethyltransferase (FAMeT) in Drosophila melanogaster L. Dayton1, S.S. Tobe2 and W.G. Bendena1 1 Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6, Canada 2 Department of Zoology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada [email protected] Farnesoic acid o-methyl transferase (FAMeT) is an important enzyme in the pathway leading to juvenile hormone biosynthesis. Juvenile hormone participates in the development and reproduction of most insects. Database searches based on sequence identity with crustacean FAMeT has revealed a putative gene product in Drosophila melanogaster. This gene encodes a protein with 41% sequence identity to Metapenaeus ensis. It is thought that FAMeT may play a rate limiting role in juvenile hormone biosynthesis in D. melanogaster; however, no experimental evidence has been reported. In order to elucidate the putative Drosophila FAMeT orthologue’s role in juvenile hormone biosynthesis, we have investigated protein distribution, activity and in vivo transcription in D. melanogaster. This has been examined by 1) protein expression to test FAMeT’s activity in vitro and 2) by fusion of the putative FAMeT gene promoter to green fluorescent protein (GFP) within a p-element vector. Germ-line transformations of this construct will be carried out to provide localization data. Up-regulation of JH levels in Lacanobia oleracea larvae attacked by the parasitoid wasp Eulophus pennicornis, is associated with reduction in host JH esterase activity J.P. Edwards1, H.A. Bell1, G.C. Marris1, A. Kirkbride-Smith1, G. Bryning1 and M. Cusson2 Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK Laurentian Forestry Centre, Sainte-Foy, Quebec, G1V 4C7, Canada [email protected] The ectoparasitoid wasp Eulophus pennicornis attacks 5 th and 6th instar larvae of its host Lacanobia oleracea. We measured the activity of juvenile hormone esterase (JHE) daily in unparasitized and parasitized penultimate (5) and final (6) instar host larvae. In unparasitized larvae, JHE activity was low throughout the penultimate larval stadium, but was markedly higher in final instar larvae. In parasitized penultimate instar larvae, JHE levels were similar to those in unparasitized insects. However, in parasitized final instar larvae, JHE activity was markedly reduced when compared to the levels in unparasitized larvae. In the light of these differences, we measured 4 JH-8 2004. Eighth International Conference on the Juvenile Hormones. 17pp. Journal of Insect Science, 4:28, Available online: insectscience.org/4.28 the titres of juvenile hormones in parasitized and unparasitized larvae, on days 2 and 5 of the final larval stadium. Whereas JH levels were virtually absent in unparasitized larvae, substantial quantities of JH (~100-fold higher) were detected in parasitized larvae of the equivalent ages. We have also investigated the JH present in larval and adult stages of the parasitoid. In both parasitoid larvae and in adult wasps, we found high titres of JH III, but little JH II, and no JH I. The making of a yolky egg: It takes more than juvenile hormone and reserves