Hormones (19th Ecdysone) Internaonal Workshop, 2013

July 21 to 26 University of Minnesota Minneapolis USA

Workshop website: hp://www.cbs.umn.edu/gcd/insect-hormones-ecdysone-workshop Thank you to our sponsors:

U of M sponsors:

John and Margaret Ordway Chair Department of Developmental Biology Genetics, Cell Biology and Development

Local Organizers: Mary Muwahid Michael B. O’Connor MaryJane Shimell International Insect Hormone (19th Ecdysone) Workshop 2013 July 22-26, University of Minnesota Minneapolis, Minnesota, USA

Monday, July 22, 2013

Johnson Great Room at McNamara Center, 200 Oak Street S.E., Minneapolis Campus

8:00 - 8:45 am Registration and Breakfast (outside Johnson Room in Memorial Hall)

8:50 - 9:00 am Welcome: Mike O’Connor, University of Minnesota

Session I: Nuclear hormone receptors (Chair: Ron Hill)

9:00 - 9:25 Ron Hill, CSIRO, Australia. Mutagenesis of Recombinant Ecdysone Receptor Ligand Binding Regions

9:30 - 9:55 Masako Asahina, Biology Center, U. of South Bohemia, Czech Republic Sumoylated NHR-25/Ftz F1/NR5A regulates cell fate in C. elegans

10:-00 - 10:25 Guy Smagghe, Ghent University, Belgium Cell based reporter assay for identifying EcR agonist/antagonists

10:30 - 11:00 Coffee break

Session II: Hormone Regulation of Metabolism (Chair: Jason Tennessen)

11:00 - 11:25 Jason Tennessen, University of Utah, USA Drosophila larval development uses aerobic glycolysis to support rapid growth

11:30 - 11:55 Arpan Gosh, University of Minnesota, USA Activin signaling regulates sugar homeostasis and pH balance in Drosophila

12:00 - 12:25 Hui Bai, Brown University, USA Juvenile hormone interacts with Insulin signaling to control lipid metabolism

12:30 - 2:00 Lunch Coffman Union

2:00 - 3:30 Poster Session I – Memorial Hall, McNamara Center (poster stands will be up until Tuesday evening)

Session III Neuroendocrine signaling (Chair: Ryuske Niwa)

3:30-3:55 Ryuske Niwa, University of Tsukuba, Japan Regulation of ecdysteroid biosynthesis through serotonin-producing neurons

4:00 - 4:25 Qiuxiang Ou University of Alberta, Canada Drosophila neurotropin Spatzle 5 is required for ecdysone synthesis

4:30 - 4:55 Shi-Hong Gu Museum of Natural Science, Taiwan Role of Bombyx AMPK in PTTH-stimulated Ecdysteroidogenesis

5:00 - 5:25 Masatoshi Iga, University of Toyko, Japan PDF mediated regulation of ecdysone biosynthesis in the PG of Bombyx

5:30 - 5:55 Yuya Ohhara, University of Shizuoka, Japan Autocrine monoaminerigic signaling triggers ecdysone biosynthesis in Drosophila

Dinner on the town

Tuesday, July 23, 2013

8:00 – 8:30 am Breakfast: Comstock Dinning Hall or outside meeting room

Session IV: Hormones and Trafficking events (Chair: Andrew Anders)

8:30 - 8:55 Andrew Andres, University of Las Vegas, USA Using E23 as genetic tool to block tissue-specific responses to 20-hydroxyecdysone in Drosophila.

9:00 - 9:25 Samantha Hindle, UCSF, USA ABC Transporters Regulate CNS Chemoprotection through Ecdysone signaling at the Blood Brain Barrier

9:30 - 9:55 Rosa Barrio, CIC bioGUNE, Spain Steroid synthesis and Growth control

10:00 - 10:30 Coffee Break

Session V: Developmental timing (Chair: Jim Truman)

10:30 - 10:55 Jim Truman, HHMI Janelia Farms, USA The role of molt timers in determining instar length in Manduca Sexta

11:00 - 11:25 Hitoshi Ueda, Okayama University, Japan Control of developmental timing by fat body transcription factors in Drosophila

11:30 - 11:55 Yuichiro Suzuki, Wellesley College, USA Ventral veins lacking interacts with JH and Ecdysone to influence the timing of metamorphosis

12:00 - 12:25 Kim Rewitz, University of Copenhagen, Denmark Feedback circuits shape the metamorphosis-inducing steroid pulse

12:30 - 2:00 Lunch

2:00 - 3:00 Poster Session II

Session VI: Hormonal control of body size and organ growth/remodeling (Chair: Sheng Li)

3:00 - 3:25 Sheng Li, Chinese Academy of Sciences, China Fat Body remodeling and it hormonal regulation in Drosophila

Tuesday, July 23, 2013 continued

3:30 - 3:55 Naoki Okamoto, RIKEN center for Developmental biology, Japan A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth

4:00 - 4:25 Rewatee Gokhale, Michigan State University, USA Ecdysone signaling Mediates intra-organ growth coordination in Drosophila

4:30 - 4:55 Wu-Min Deng, Florida State University, USA Tissue repair through cell competition and compensatory cellular hypertrophy in postmitotic epithelia

5:00 - 5:30 Takashi Koyama, Instituto Gulbenkian de Ciencia, Portugal Nutrition regulates body size through FoxO-Ultraspiracle mediated ecdysone synthesis

Light Rail trip to Mall of America for Dinner and shopping?

Wednesday, July 24

8:00 - 8:30 Breakfast: Comstock Dinning Hall or outside meeting room

Session VII: Hormones and Behavior-Nongenomic 20-E responses (Chair: Naoki Yamanaka)

8:30 - 8:55 Naoki Yamanaka, University of Minnesota, US PTTH coordinates physiological and behavioral transitions during Drosophila development

9:00 - 9:25 Xiao-Fan Zhao, Shandong University, China Regulation of cyclin-dependent kinase 10 phosphoryation by steroid hormone 20-E through GPCR signaling

9:30 - 9:55 Hiroshi Ishimoto, University of Iowa, USA A novel action of the steroid hormone ecdysone on behavioral plasticity in adult Drosophila

10:00 - 10:30 Break

10:30 - 10:55 Hiroko Sano, Kurume University, Japan Analysis of the peripheral tissue-derived peptides, CCHamide-1 and 2 and dRYamide 1 and 2 in Drosophila

11:00 - 11:25 Taketoshi Kiya, Kanazawa University, Japan Identification of Hr38 as a conserved neural activity-induced gene in insect brains

11:30 - 12:15 Box Lunches Great Hall

12:15 Board bus for Harriet Island

12:30 - 1:30 Transportation to Harriet Island in St. Paul Lorenz Bus Service will be arriving at 12:15 and will depart McNamara at 12:30

1:30 - 2:00 pm Board the Betsey Northup Harriet Island, St. Paul

2:00 - 5:00 Sail on the Betsey Northup

5:00 Transportation back to McNamara Center Lorenz Bus Service

Dinner on the town

Thursday, July, 25

8:00 - 8:30 Breakfast: Comstock Dinning Hall or outside meeting room

Session VIII JH signaling (Chair: Xavier Belles)

8:30 - 8:55 Xavier Belles, CSIC-UPF Barcelona, Spain A single miRNA drives hemimetabolan metamophosis to a right end

9:00 - 9:25 Tetsuro Shinoda, NIAS Tsukuba, Japan Hormonal regulation of Kruppel homolog 1 and its mechanisms or repressing metamorphosis in Bombyx

9:30 - 9:55 Takahiro Shiotsuki, NIAS Tsukuba, Japan The characteristics of JH epoxide hydrolase genes in

10:00- 10:30 Coffee break

Session IX JH signaling continued and JH-Ecdysone-interactions (Chair, Lynn Riddiford)

10:30 - 10:55 Lynn Riddiford, HHMI Janelia Farm USA Roles of JH receptors Methoprene-tolerant and Germ cell-expressed in Drosophila larval development and metamorphosis

11:00 - 11:25 David Martin, CSIC-UPF Barcelona, Spain Ecdysone meets juvenile hormone: nuclear receptors seven-up and FTZ-1 control juvenile hormone biosynthesis during hemimetabolan metamorphosis

11:30 - 11:55 Vincent Henrich, University of North Carolina at Greensboro, USA Transcriptional regulation of two Drosophila early puff genes in the larval salivary gland by 20-E and juvenile hormone: the role of Met and GCE

12:00 – 12:25 Aaron Bauman, HHMI Janelia Farms USA Subfunctionalization of duplicate juvenile hormone receptors in higher Diptera

12:30 - 2:30 Lunch Coffman Union

Session X Hormonal Control of Reproduction and Evolution (Chair: Thomas Flatt)

2:30 - 2:25 Thomas Flatt, University of Lausanne, Switzerland The contribution of Endocrine genes to Latitudinal Population Differentiation in Drosophila

2:30 - 2:55 David Dolezel, ENTU Biology Center, Czech Republic Juvenile hormone and circadian genes regulate reproductive diapause in Pyrrhocoris apterus

Thursday, July 25 continued

3:00 - 3:25 Mark Brown, University of Georgia, USA Ovary ecdyseroidogenic hormone: function and signaling in mosquitoes

3:30 - 3:55 S. Reddy Palli, University of Kentucky, USA Hormonal Regulation of Reproduction in the Red Flour Beetle, Tribolium castaneum

4:00 -5:00 Business Meeting: Planning the next workshop 2015?

5:00 -6:00 Reception for Alex Raikhel, Indoor Club, TCF Bank Stadium 2009 University Avenue S.E. Minneapolis

6:00 - 6:50 Karlson Lecture: Alex Raikhel The role of JH and 20-E in Mosquito reproduction

7:00 – 9:00 Banquet buffet, Indoor Club TDF Bank Stadium

Friday, July 26

8:00 - 8:30 Breakfast: Comstock Dinning Hall or outside meeting room

Session XI Ecdysone biosynthesis and regulation (Chair: Marek Jindra)

8:30 - 8:55 Marek Jindra, Biology Centre ASCR, Czech Republic Ecdysoneless -Why is it ecdysone-less?

9:00 - 9:25 Sora Enya, University of Tsukuba, Japan A novel Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis in the prothoracic gland

9:30 - 9:55 Hajime Ono, Kyoto University, Japan Characterization of 3-oxo steroids as intermediates in the Black Box of the ecdysone biosynthetic pathway

10:00 – 10:30 coffee break

Session XII Young Investigators (Chair, Arash Bashirullan)

10:30 - 10:55 Arash Bashirullah, University of Wisconsin, USA Forward Genetic Analysis of Ecdysone-Triggered responses During Metamorphosis

11:00 - 11:25 Viviane Callier, Arizona State University Oxygen affects the developmental physiology of growth and metamorphosis initiation in Drosophila

11:30 - 11:55 Megha, National Centre for Biological Sciences, India A role for the IP3 receptor in neuropeptide-producing cells of Drosophila during larval development

12:00 - 12:25 Michael Texada, HHMI Janelia Farms USA Mapping the Drosophila peptide-hormone system

Closing remarks

End of conference

Safe Travels

Abstracts of speakers

in order of presentation

In vitro Site-directed Mutagenesis of Recombinant Ecdysone Receptor Ligand Binding Regions

Matthew Pollard1, Bin Ren2, Lloyd Graham1, Victor Streltsov2, Linda Howell3, Ross Fernley2, Julian Grusovin2, George Lovrecz3, Louis Lu3, Tram Phan3, Pat Pilling2, Garry Hannan1,Thomas Peat2, Dave Winkler3 , Michael Lawrence4 and Ron Hill1

1CSIRO Division of , Food and Health Sciences, PO Box 52, North Ryde, NSW, 1670, Australia 2CSIRO Division of Materials Science and Engineering, 343 Royal Parade, Parkville, VIC 3052 Australia 3CSIRO Division of Materials Science and Engineering, Bag 10, Clayton South MDC 3169, Australia 4 Walter and Eliza Hall Institute, 1G Royal Parade, Parkville Victoria 3052, Australia & Department of Medical biology, University of Melbourne, Australia

We have cloned the EcR and USP subunits of ecdysone receptors from six insect pests spanning four orders: Lucilia cuprina, Myzus persicae, Bemisia tabaci, Nezara viridula, Helicoverpa armigera and Bovicola ovis. The C-terminal ligand binding regions of these EcR and USP nuclear receptor proteins have been co-expressed in insect cells employing a baculovirus system. Recombinant ecdysone receptor ligand binding domains (LBDs) exhibit ligand binding properties that largely reflect those of the holo-receptors in vivo. However, a nested series of D-domain truncations produced by in vitro site-directed mutagenesis has led to a family of recombinant proteins that demonstrates a significant influence of the linker domain on stability of the EcR-USP LBD heterodimer. Concomitant effects on hormone binding are also observed. During the course of this work a new conformation for the heterodimeric ecdysone receptor LBD has been observed. This distinctly different three-dimensional structure will be compared with the canonical ecdysteroid-bound LBD structure and possible implications for juvenile hormone binding considered.

Ashburner (1972) reported competition between N-ethylmaleimide and 20-hydroxyecdysone for a site on the then hypothetical ecdysone receptor in his model to explain hormone induced puffing of specific loci on Drosophila melanogaster polytene chromosomes. On the basis of X-ray structures of ecdysone receptor LBDs and molecular modelling we have suggested a possible basis for this competition at an atomic level (Hill et al., 2013). Amino acid replacements produced by in vitro site- directed mutagenesis now provide definitive verification of the precise site in the ligand binding pocket of the ecdysone receptor at which this competition occurs.

Ashburner M. 1972. N-ethylmaleimide inhibition of induction of gene activity by hormone ecdysone. FEBS Lett. 22:265—69 Hill R.J., Billas I.M., Bonneton F., Graham L.D. and Lawrence M.C. 2013. Ecdysone receptors: From the Ashburner model to structural biology. Ann. Rev. Entomol. 58: 251-271

Sumoylated NHR-25/Ftz-F1/NR5A regulates cell fate during C. elegans vulval development

Jordan D. Ward1, Nagagireesh Bojanala2, Teresita Bernal1, Kaveh Ashrafi3, Keith Yamamoto1 and Masako Asahina1,2,3 1Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA, USA; 2Biology Centre ASCR and University of South Bohemia, Ceske Budejovice, Czech Republic; 3Department of Physiology, UCSF, San Francisco, CA, USA.

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The exquisite cell and tissue specificity of gene regulation by a given TF appear to be achieved combinatorially, within multifactor regulatory complexes with distinct compositions and activities. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here we examine how a single regulatory input (sumoylation) differentially modulates the activity of the conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. NHR-25, the single Ftz-F1 homolog in C. elegans plays pleiotropic roles in molting, epithelial cell differentiation, cell fate decision, morphogenesis and fat metabolism. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on three lysines. Genetic studies revealed that loss of smo-1 phenocopied NHR-25 overexpression, with respect to maintenance of the 3º cell fate in vulval precursor cells (VPCs) during development. Furthermore, in vivo overexpression using NHR-25 alleles that could not be sumoylated and SUMO-NHR-25 fusions indicated that NHR-25 sumoylation is critical for maintaining 3º cell fate. SUMO also regulated formation of an NHR-25 accumulation gradient in VPCs. Using an NHR-25::GFP translational reporter, we discovered that NHR-25 levels were uniform across VPCs at the beginning of development, but as cells began dividing a smo-1-dependent NHR-25 gradient formed with highest levels in 1º fated VPCs, lower levels in 2º fated VPCs, and the lowest levels in 3º fated VPCs. Our findings support a model where the ratio of unsumoylated to sumoylated NHR-25 regulates 3º cell fate determination and maintenance during vulval development. Supported by GAAV IAA500960906, MODBIOLIN 7FP-REGPOT-2012-2013-1, MSM6007665801, GACR: 204/09/H058, Terry Fox Foundation (#700046), CIHR (#234765) and the NIH (CA20535).

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in (Bm5) and Diptera (S2) cell cultures, followed by modeling of ecdysteroid-EcR interactions and normal modes analysis

Moisés J. Zotti a,f, Ellen De Geyter a, Luc Swevers b, Antônio S.K. Braz c, Luis P.B. Scott c; Pierre Rougé d, Josep C. Toledano e, Anderson D. Grutzmacher f, Eder J. Lenardão g and Guy Smagghe a a Department of Crop Protection, Ghent University, Coupure links 653, 9000 Ghent, Belgium. b Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research “Demokritos, 153 10 Aghia Paraskevi, Athens, Greece. c Laboratory of Computational Biology and Bioinformatics, Federal University of ABC, Santo André, Brazil. d UMR UPS-IRD 152 Pharma-Dev, Faculté de Pharmacie, Université Paul Sabatier, 31062 Toulouse Cedex 9, France. e Department of Biological Organic Chemistry, CID-CSIC, E-08034 Barcelona, Spain. f Department of Crop Protection, FAEM, Federal University of Pelotas, P.O. box 354, CEP: 96010-900, Pelotas, RS, Brazil. g Laboratory of Clean Organic Synthesis, CCQFA, Federal University of Pelotas, Pelotas, RS, Brazil.

Abstract Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysteroid receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC50 = 3.3 µM) and Bm5 cells (EC50 = 5.3 µM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50 = 0.71 µM and 0.00089 µM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039 µM; in Bm5 cells this effect only became visible at much higher concentrations (IC50 = 18 µM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidences that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be more interesting for dipterans because the activity of dibenzoylhydrazines is dependent of an extension in the EcR-LBD binding pocket but so far present only in lepidopterans.

KEYWORDS: cell-based screening system; ecdysteroid agonist; ecdysteroid antagonist; molecular modeling; normal mode analysis, steroids

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Drosophila larval development uses aerobic glycolysis to support rapid growth

Jason M. Tennessen1,2, Geanette Lam2, Janelle Evans2, and Carl S. Thummel2. 1Department of Biology, Indiana University, Bloomington, IN, 47405 2Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112

Drosophila larval metabolism is exquisitely tuned to promote exponential growth in response to environmental nutrients. This growth phase is in contrast with embryonic metabolism, which is dependent on intrinsic energy reserves. Little is known, however, about how a developing animal transitions between the embryonic and larval metabolic states. We have determined that the onset of larval growth is preceded by an embryonic metabolic transition (EmbMT) that induces the coordinate expression of nearly every gene involved in glycolysis, the pentose phosphate pathway (PPP), and lactate production. Subsequent GC/MS-based metabolomic analysis has revealed that the larval metabolic program displays the central hallmarks of aerobic glycolysis, which is analogous to the Warburg effect in proliferating cancer cells and is ideally suited to convert dietary carbohydrates into biomass. We have also discovered that the EmbMT is dependent on the Drosophila ortholog of the Estrogen-Related Receptor (ERR) class of nuclear receptors. In dERR null mutants, nearly every gene that encodes an enzyme in glycolysis and the PPP, as well as Lactate Dehydrogenase/ImpL3 (Ldh/ImpL3), are not properly upregulated during the EmbMT. As a result, dERR null mutants die during larval development with a metabolomic profile indicative of a block in aerobic glycolysis. dERR appears to directly regulate these pathways, as there are predicted dERR binding sites in nearly all of the misregulated genes, and we have confirmed that dERR protein binds directly to the promoter of the phosphofructokinase gene.

Our results demonstrate for the first time that aerobic glycolysis can be utilized in the context of normal developmental growth and establish Drosophila larval development as a model for studying this metabolic state. We are now exploiting this system by characterizing key enzymes involved aerobic glycolysis. Toward this goal, we have determined that mutations in the ecdysone-regulated gene Ldh/ImpL3 significantly disrupt aerobic glycolysis and larval growth. Intriguingly, GC/MS-based analysis of Ldh mutants reveals an unexpected link between the production of lactate and the oncometabolite 2-hydroxyglutarate (2-HG). Although abnormal 2-HG production promotes the progression of glioblastomas, it has never been linked with normal developmental growth. Our findings suggest that 2-HG is more than an aberrant metabolite produced in cancer cells, but rather is part of a conserved metabolic program that contributes to normal larval development and aerobic glycolysis.

Systemic Activin signaling independently regulates sugar homeostasis and pH balance in Drosophila melanogaster.

Arpan C. Ghosh and Michael B. O’Connor GCD, University of Minnesota, Minneapolis, MN-55455

Ability to maintain physiological homeostasis is key for survival of all multi- cellular organisms in changing environmental conditions. However, mechanisms involved in achieving physiological homeostasis are poorly understood. We find that Drosophila Activin-like ligand Dawdle (Daw) is required for maintaining both sugar and pH homeostasis in the larvae. Canonical Smad signaling downstream of Daw regulates sugar homeostasis primarily by controlling insulin release from the larval insulin producing cells. Independent of insulin release, canonical Daw signaling also controls hemolymph pH balance by regulating accumulation of metabolic acids possibly by affecting mitochondrial metabolism. Consistent with deregulation of sugar and pH homeostasis we find that, compared to controls, daw mutant larvae are significantly more vulnerable to high sugar and acidic food conditions. Interestingly, release of Daw from multiple tissues can rescue the metabolic and sugar homeostasis defective phenotypes of daw mutants in a dose-sensitive manner. These results suggest a novel hormonal role of Daw in independently maintaining sugar and pH levels within ranges conducive for normal development and adult survival.

Abstract for 2013 Insect Hormones Workshop Minneapolis MN

Juvenile hormone interacts with insulin signaling to control lipid metabolism

Hua Bai and Marc Tatar Department of Ecology and Evolutionary Biology, Brown University, Providence, RI 02912

Juvenile hormones (JH) produced in corpora allata (CA) are involved in a variety of biological processes. Beside the role on female vitellogenesis, the effects of JH and its analog on lipid metabolism have also been studied in several insects. However, the underlying mechanism is poorly understood. Here we investigated the regulation of JH on lipid metabolism in female Drosophila. We found that short period application of JH analog (methoprene) increases the level of triglyceride (TAG), while CA ablated (CAKO) and putative JH receptor (Met) mutants have reduced TAG. Interestingly, we identified an insulin-like peptide (dilp6) whose expression can be directly induced by methoprene application in ex vivo fat body culture. This induction requires Met and Kr- h1, two key players involved in JH signaling. Consistently, overexpression of either Kr-h1 or dilp6 using within fat body promotes the accumulation of TAG. Furthermore, the expression of Kr-h1 and dilp6 are altered in responding to different nutrient conditions. These results suggest that JH may regulate lipid metabolism through a fat body expressed insulin-like peptide.

Adaptive regulation of ecdysteroid biosynthesis in the prothoracic gland through serotonin-producing neurons in the fruit Drosophila melanogaster

Ryusuke Niwa1,2, Yuko Shimada-Niwa1, and Yosuke Umei1

1) Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan 2) PRESTO, JST, Japan

During insect larval stages, ecdysteroids are synthesized in a special endocrine organ called the prothoracic gland (PG). Ecdysteroid biosynthesis in the PG is controlled in response to several external conditions, such as nutrition, temperature and photoperiod. Adaptive changes in ecdysteroid biosynthesis result in flexible alterations for developmental timing such as molting. However, it remains unclear how external information is transmitted to the PG to control ecdysteroid biosynthesis. To elucidate genes involved in controlling the adaptive regulation of ecdysteroid biosynthesis, we conducted a transgenic RNAi screen of the fruit fly Drosophila melanogaster using PG-specific GAL4 drivers. We found that knocking down a gene encoding an isoform of the serotonin receptors caused a decrease in ecdysteroid biosynthesis and delay in development. We also identified the serotonin-producing neurons directly innervating the PG. Genetic manipulations that inhibit projections into the PG correlated with a delay in molting. Dendrites of these serotonergic neurons extended toward the subesophageal ganglion, known as the feeding center of insects, implying that these serotonergic neurons may receive signals from food. Furthermore, the projection of the neurons into the PG was affected by nutrient conditions. We propose that environmental conditions are reflected in the timing of metamorphosis though neuronal control involving serotonin and its receptor.

Drosophila neurotrophin Spätzle5 is required for ecdysone synthesis Qiuxiang Ou, Brian Phelps, Kirst King-Jones Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada

Ecdysone is converted from cholesterol through a series of enzymatic steps in the prothoracic gland. However, signaling pathways controlling this process remain poorly characterized. To identify novel components required for ecdysone production in the prothoracic gland, we initially performed whole-genome microarray analysis of hand-dissected ring glands (which harbor the prothoracic gland) and compared the signal to microarrays of whole larvae samples, which allowed us to identify genes with specific expression in the ring gland. The 100 highest-scoring genes were then subjected to prothoracic gland-specific RNA interference (RNAi), which resulted in 25 genes that have likely novel roles in ecdysone synthesis. One of these hits, a gene called spätzle5, has homology to vertebrate neurotrophins, which are signaling molecules that have neuroprotective functions during brain development. RNAi knockdown of spätzle5 caused arrest of larval development, which can be rescued by ecdysone feeding, indicating that this neurotrophin is necessary for ecdysone synthesis. We also demonstrate that spätzle5 is required for the production of nitric oxide (NO) possibly through controlling the activity of nitric oxide synthase (NOS). Prothoracic gland-specific RNAi knockdown of NOS also results in L3 arrest, consistent with the idea that this neurotrophin recruits NO signaling to govern ecdysone synthesis. These data provide evidence that neurotrophins are involved in the regulation of steroid hormone synthesis.

Involvement of Phosphorylation of Adenosine 5'-monophosphate-activated Protein Kinase in PTTH-stimulated Ecdysteroidogenesis in Prothoracic Glands of the Silkworm Bombyx mori

Shi-Hong Gu*, Yun-Chin Hsieh, Pei-Ling Lin Department of Zoology, National Museum of Natural Science, Taichung, Taiwan 404, ROC

The involvement of the phosphorylation of adenosine 5’-monophosphate-activated protein kinase (AMPK) in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori was investigated. It was found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPKα phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70

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ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. We also investigated the relationship between Ca2+ and AMPK/TOR signaling. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, plays a role in PTTH stimulation of ecdysteroidogenesis.

* Corresponding author: Shi-Hong Gu, [email protected]

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Pigment dispersing factor mediated regulation of ecdysone biosynthesis in the prothoracic glands of Bombyx mori

Masatoshi Iga, Takayoshi Nakaoka, Yutaka Suzuki, Hiroshi Kataoka Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Japan

The insect steroid hormone, ecdysone is important for regulating insect growth and development. The ecdysone is predominantly synthesized in the prothoracic glands (PGs), and the synthesis is intricately regulated by several neuropeptides. To date, four prothoracicotropic and three prothoracicostatic peptides have been identified in Bombyx mori, whereas still remains the possibility that other factor(s) is involved in the regulation. Therefore, we performed next-generation sequencing RNAseq analysis by illumina genome analyzer in the PGs of B. mori and searched a novel regulatory mechanism of ecdysone biosynthesis by focusing on the receptors that are specifically expressed in the PGs. By this screening, we succeeded to pickup an orphan G-protein coupled receptor (GPCR), Bombyx neuropeptide GPCR gene-B2 (BNGR-B2). In addition, we succeeded to identify a ligand for the BNGR-B2, and it was pigment dispersing factor (PDF), the ortholog of pigment dispersing hormone in crustacean. The PDF stimulated ecdysone biosynthesis in the cultured PGs, and the synthesized amount of ecdysone and expression level of BNGR-B2 showed clear relationship. Thus, presence of PDF mediated regulatory mechanism of ecdysone biosynthesis was revealed. Furthermore, the signaling pathway of PDF was investigated by pharmacological analysis, phosphorylation state change of several components involved in the PTTH signaling and analysis of ecdysteroids by LC-MS/MS. In this presentation, we also discuss the difference and similarity between PDF and PTTH mediated regulation of ecdysone biosynthesis.

[This study was supported by the grant from Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry.]

Autocrine monoaminerigic signaling triggers ecdysone biosynthesis in Drosophila prothoracic gland Yuya Ohhara1, 2, Yuko Shimada-Niwa3, Yasunari Kayashima1, Yoshiki Hayashi2, Ryusuke Niwa3,4, Satoru Kobayashi2, and Kimiko Yamakawa-Kobayashi1 1 School of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Japan 2Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, Japan 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan 4PRESTO, JST, Japan

In Drosophila, the larval-prepupal transition is elicited by ecdysteroids, including ecdysone and its derivative 20-hydroxyecdysone. Ecdysone is produced in the endocrine organ, the prothoracic gland (PG). In the PG, ecdysone biosynthesis is under the control of several neuropeptides, such as the prothoracicotropic hormone (PTTH) and Drosophila insulin-like peptides (Dilps). These neuropeptides stimulate ecdysone biosynthesis through kinase-linked receptors. In contrast, it has long been proposed that a G-protein coupled receptor (GPCR) and its downstream signal transduction cascade are involved in ecdysone biosynthesis in the PG. However, the GPCR(s) and its ligand(s) remain to be identified. Here we report that a member of GPCR superfamily, 3-octopamine receptor (Oct3R), and its ligand, tyramine, stimulate ecdysone biosynthesis in an autocrine manner. Oct3R and tyramine biosynthetic enzyme, tyrosine decarboxylase-2 (Tdc2), were expressed in the PG. The PG-specific knockdown of Oct3R or Tdc2 caused a halt in larval-prepupal transition due to a loss of ecdysone biosynthesis. Furthermore, the PG-specific knockdown of Oct3R resulted in a loss of PTTH and Dilps signaling in the PG, and a halt in larval-prepupal transition of Oct3R knockdown was rescued by forced expression of PTTH and Dilps signaling components. Together, we conclude that tyramine and Oct3R-mediated autocrine signaling in the PG stimulates ecdysone biosynthesis through the regulation of PTTH and Dilps signaling pathways. Using E23 as genetic tool to block tissue-specific responses to 20-hydroxyecdysone in Drosophila.

Elana Paladino, Gregory King, Kathryn Lantz, and Andrew Andres School of Life Sciences, University of Nevada, Las Vegas

E23 is a Drosophila primary-response gene originally identified because it forms a classic early puff on the polytene chromosomes of the larval salivary gland in response to 20-hydroxyecdysone (20E). The E23 amino acid sequence reveals that it is structurally most similar to ABC transporters of the same group as the product of the white gene. These transporters hydrolyze ATP in order to pump molecules against their concentration gradients, but generally each transporter is either dedicated to export or import but not both. In order to understand the normal function of E23, we ectopically overexpressed it in a variety of 20E responsive tissues during different developmental stages. In all cases examined, ectopic expression of E23 blocks the hormone response in that tissue at that time. In the salivary gland alone, ectopic expression of E23 blocks glue-gene induction at mid-third instar, glue protein secretion at late-third istar, and gland histolysis at the end of prepupal development. When overexpressed in the target tissues responsible for molting and ecdysis, it also blocks second- and third-instar transitions. We therefore propose that ectopic expression of E23 can be used as a tool to block any 20E response even in those tissues that utilize different ecdysone receptors. These observations support a model in which the normal function of E23 is to pump 20E out of some cells to mediate tissue-specific responses to a broad systemic titer of the hormone.

ABC TRANSPORTERS REGULATE CNS CHEMOPROTECTION THROUGH ENDOGENOUS ECDYSONE SIGNALLING AT THE BBB. Samantha Hindle, Souvinh Orng, Michael DeSalvo, Elena Dolgikh, Hiroshi Ishimoto, Fahima Mayer, Toshihiro Kitamoto, Matt Jacobson, Roland Bainton Department of Anesthesia, UCSF

Maintenance of homeostasis in the central nervous system (CNS) requires tight regulation over the metabolites and toxins entering the brain space; this function is performed by the blood-brain barrier (BBB). Understanding how the BBB orchestrates this is fundamental for both disease prevention and efficient targeting of therapeutics into the brain. The major challenge in understanding the BBB is being able to analyze it within the context of intact animals and under physiological conditions. We have pioneered the use of Drosophila (Dm) BBB surface glia layers for this purpose. P- glycoprotein (Pgp), a broad-specificity ABC transporter, is well known for its role as a xenobiotic efflux transporter. However, its role in controlling the localization of endogenous molecules is unclear. BBB knock-down of Mdr65, the Drosophila homolog of Pgp, allowed us to investigate the endogenous substrate specificity of Mdr65 and the physiological effect on the animal. We revealed that Mdr65 is important for controlling ecdysone partitioning between the humoral and brain space. Manipulations of Mdr65 function also impacted upon ecdysone-regulated behaviors, including eclosion, sleep and longevity. Our findings that Mdr65 is required for both xenobiotic and endogenous molecule partitioning suggest a central role for Pgp-like transporters in communicating exogenous chemical threats through their effect on endogenous molecule partitioning.

STEROIDS SYNTHESIS AND GROWTH CONTROL

Ana Talamillo1, Leire Herboso1, Coralia Pérez1, David Martín2, James D. Sutherland1 and Rosa Barrio1

1. CIC bioGUNE, Bizkaia Technology Park, Derio, Spain 2. IBE-CSIC, Barcelona, Spain

The steroid hormone ecdysone is produced in the prothoracic gland (PG) of insects. There, cholesterol intake is needed as source for ecdysone synthesis. We have demonstrated that the small ubiquitin-related modifier SUMO is necessary for lipid uptake in the PG. SUMO is required for the expression of Ftz-f1, an orphan nuclear receptor, which is in turn necessary for the expression of some members of the Scavenger Receptor Class B type I (SR-BI) family. These receptors belong to the mammalian CD36 family, which participates in the selective uptake of High Density Lipoprotein cholesteryl ester and in the formation of microvillar channels in the mammalian adrenal gland. Our results show that the SR-BI Snmp1 is involved in the cholesterol uptake mediated by SUMO and Ftz-f1. Snmp1 overexpression is able to rescue the lipid capture in PGs silenced for SUMO or Ftzf1. Furthermore, the requirement of SUMO/Ftz-f1/SR-BIs for lipid capture is conserved in other tissues. The ecdysone produced in the PG has non-autonomous consequences on the growth of other larval tissues, such as the imaginal discs. Without ecdysone, imaginal discs do not reach the proper size due to a reduction in cell proliferation and cell size. These parameters are recovered when the hormone is exogenously administrated. We are investigating the factors implicated in cell growth triggered by ecdysone.

The role of molt timers in determining instar length in Manduca sexta.

J. W. Truman1, Y. Suzuki2, T. Koyama3, K. Hiruma4, L.M.Riddiford1 1Janelia Farm Research Campus, HHMI. Ashburn, VA, 2Department of Biology, Wellesley College, Wellesley, MA, 3Instituto Gulbenkian de Ciencia, Oeiras, Portugal, 4Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki, Japan

Manduca sexta larvae have served as a model for growth control in insects, most notably for the demonstration of critical weight, a threshold weight that the larva must surpass before it can enter metamorphosis on a normal schedule, and the inhibitory action of juvenile hormone (JH) on this checkpoint. We examined the effects of nutrition on allatectomized (CAX) larvae that lack JH to impose the critical weight checkpoint. Normal larvae respond to prolonged starvation at the start of the last larval stage, by extending their subsequent feeding period to insure that they start metamorphosis above critical weight. CAX larvae, by contrast, show no homeostatic adjustment to starvation but start metamorphosis four days after being given food, regardless of larval size or the state of development of their imaginal discs. By giving starved CAX larvae food pulses of various durations, we found that feeding for only 12 to 24 hours was sufficient to result in metamorphosis on day four, regardless of further feeding or body size. By manipulating diet composition, we found that protein was the critical macronutrient to initiate this timing. This constant period between the start of feeding and the onset of metamorphosis suggests that larvae possess a molt timer that establishes a minimal time to metamorphosis. Ligation experiments indicate that a portion of the timing occurs outside of the head, perhaps in the prothoracic glands. This positive system that promotes molting and the negative control via the critical weight checkpoint provide antagonistic pathways that evolution can modify to adapt growth to the ecological needs of different insects.

Control of developmental timing by transcription factors in fat body during metamorphosis in Drosophila melanogaster

Abdel-Rahman Sultan1, Kazutaka Akagi1, Haruka Nishida1, Moustafa Sarhan1, Takumi Nakayama1, Azusa Koie1, Hiroyasu Oish2 and Hitoshi Ueda1,2

1) The Graduate School of Natural Science and Technology, Okayama University, Japan. 2) Department of Biology, Faculty of Science, Okayama University, Japan

ABSTRACT

Living organisms sometimes determine their specific developmental events at precise timing. However, little is known about how these timing are decided at molecular level. Insect metamorphosis is complicated process and it takes for around 4 days in Drosophila melanogaster, but we do not know the mechanism to determine the period. Our study showed the importance of ecdysone-inducible transcription factors Blimp-1 and FTZ-F1 for the determination of prepupal period at the onset of metamorphosis. Recent our study using temporally specific knockdown or induction of these factors revealed that these factors play important roles to determine the pupal period. Furthermore, our results indicate a critical role of fat body for the determination of the developing timing by these factors. We will also show the mechanism to determine the developing timing during prepupal period by these factors.

The POU domain protein Ventral veins lacking interacts with juvenile hormone and ecdysteroid signaling to influence the timing of metamorphosis.

CeCe Cheng1, Amy Ko1, Leila Chaieb1, Takashi Koyama2, Christen Mirth2, Wendy Smith3 and Yuichiro Suzuki1

1 Department of Biological Sciences, Wellesley College, Wellesley, MA 02481, USA 2 Development, Evolution and the Environment Lab, Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal 3 Department of Biology, Northeastern University, 360 Huntington Avenue, 134 Mugar Building, Boston, MA 02115, USA

In insect metamorphosis, the endocrine changes associated with metamorphosis are fairly well characterized. However, the regulatory mechanisms underlying the timing of metamorphosis is poorly understood. We examined the function of a POU domain protein, Ventral veins lacking (Vvl), in regulating hormonal pathways in the red flour beetle, Tribolium castaneum. RNA interference-mediated silencing of vvl expression led to precocious metamorphosis but prevented molting and pupation, suggesting that vvl influence JH and ecdysteroid signaling. Ectopic application of JHIII or methoprene on vvl knockdown larvae rescued the timing of metamorphosis and led to a longer larval stage although molting was still impaired. The expression of krüppel-homolog 1 was downregulated in the absence of Vvl, but this expression was rescued by exogenous methoprene application, indicating that vvl might act upstream of JH signaling. Vvl was also found to interact with Ecdysone receptor and Ultraspiracle. A model for how Vvl interacts with JH and ecdysteroid signaling will be presented. Feedback circuits shape the metamorphosis–inducing ecdysone pulse in Drosophila

Morten E. Moeller1, E. Thomas Danielsen1, Rachel Herder2, Michael B. O’Connor2, Kim F. Rewitz1,*

1Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark. 2Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT Pulses of ecdysone drive molting and metamorphosis in insects by initiating genetic response programs determined by pulse frequency, amplitude and duration. Although extensive studies have characterized the genetic response to ecdysone in Drosophila, the mechanisms generating a regulatory pulse are incompletely defined. The two key parameters that determine pulse shape (amplitude and duration) are activation and repression/clearance of synthesis. We have shown that the ecdysone pulse triggering metamorphosis in Drosophila, is shaped by feedback circuits in the prothoracic gland (PG), the ecdysone producing tissue. In response to the brain neuropeptide prothoracicotropic hormone (PTTH), ecdysone amplifies its own synthesis by acting on the PG. This leads to the high-level production of ecdysone that triggers pupariation. Following pupariation, ecdysone inhibits the ecdysteroidogenic activity of the PG in coordination with increasing peripheral clearance. The positive and negative feedback circuits rely on a developmental switch in the expression of Broad isoforms in the PG. These Broad isoforms regulate the ecdysteroidogenic activity of the PG by transcriptionally activating or silencing genes involved in ecdysone biosynthesis. These results demonstrate an autonomous feedback circuitry in the PG that ensures a rapid, self-limiting response to PTTH, illustrating a switch-like mechanism useful for producing steroid oscillations.

Fat body remodeling and its hormonal regulation in Drosophila

Sheng Li ([email protected] )

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China

The Drosophila fat body is an organ analogous to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. It undergoes a distinct remodeling process, including autophagy, apoptosis, and cell dissociation, during the larval-pupal transition, which is mainly regulated by the steroid hormone (20-hydroxyecdysone, 20E) and the sesquiterpenoid hormone (juvenile hormone, JH). We discovered: (1) The JH receptors MET and GCE mediate JH action to prevent 20E-induced apoptosis and cell dissociation. (2) A balancing crosstalk occurs between 20E-indcued autophagy and apoptosis and this crosstalk is predominantly transduced by E93, a 20E primary-response gene. (3) Two 20E-activated matrix metalloproteinases, MMP1 and MMP2, coordinately promote cell dissociation. We assume that the Drosophila fat body provides an exceptional in vivo model to study tissue remodeling and its hormonal regulation.

1

A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila

Naoki Okamoto, Takashi Nishimura

Laboratory for Growth Control Signaling, RIKEN Center for Developmental Biology, Japan

Members of the insulin peptide family have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. Drosophila insulin-like peptides (Dilps) promote tissue growth through the single insulin-like receptor (InR). Despite the important role of Dilps in nutrient-dependent growth control, the molecular mechanism that regulates the activity of circulating Dilps is not well understood. Here, we report the function of a novel secreted decoy of InR (SDR) as a negative regulator of insulin signaling. SDR is predominantly expressed in glia and is secreted into the hemolymph. Larvae lacking SDR grow at a faster rate, thereby increasing adult body size. Conversely, overexpression of SDR reduces body growth non-cell-autonomously. SDR is structurally similar to the extracellular domain of InR and interacts with several Dilps in vitro. We further demonstrate that SDR is constantly secreted into the hemolymph independent of nutritional status and is essential for adjusting insulin signaling under adverse food conditions. We propose that Drosophila uses a secreted decoy to fine-tune systemic growth against fluctuations of circulating insulin levels. Ecdysone signaling mediates intra-organ growth coordination in Drosophila melanogaster

Rewatee Gokhale1, Chris Mirque2, Alexander Shingleton2 1Department of Biochemistry and Molecular Biology, 2Department of Zoology, Michigan State University, East Lansing 48823.

Regulation of final organ size is a complex developmental process and involves the integration of systemic and organ specific processes. Together, these processes enable the coordination of organ growth with body growth and the achievement of correct organ size. In holometabolous insects like Drosophila, final organ size depends on the growth of the precursor imaginal discs during development. At the end of larval development, larvae undergo metamorphosis to form pupae, an event which is regulated by the level of 20-hyrdoxyecdysone (20E) signaling. Previous work from our laboratory demonstrated that in Drosophila, perturbing the growth of one imaginal disc during development results in the growth retardation of other discs and an extension of developmental time. This growth coordination is thought to prevent overgrowth of the non growth-perturbed organs at the end of larval growth. Excitingly, this inter-organ coordination of growth can be disrupted by exogenous application of 20E, indicating that low levels of 20E in vivo are responsible for mediating growth coordination between organs.

What about growth coordination within an organ ? Here we test the hypothesis that 20E signaling is also involved in mediating growth coordination within an organ. We generated growth perturbed (GP) larvae in which the two compartments of the wing imaginal disc have different rates of growth. We find that there is tight growth coordination between differentially growing compartments and that this growth coordination can be disrupted by exogenous treatment with 20E. This indicates that limiting levels of 20E mediate growth coordination between tissues within an organ. We further investigate the mechanisms downstream of ecdysone signaling which are involved in growth coordination. Our results implicate the insulin signaling pathway in mediating growth coordination in the imaginal disc. We find that manipulating the level of insulin signaling in the imaginal discs of these GP larvae recapitulates the disruption of growth coordination that is observed upon ecdysone treatment. Our ongoing research is directed at elucdiating the role of insulin- and ecdysone-signaling, and their interaction, in coordinating growth among developmental compartments within organs.

Tissue repair through cell competition and compensatory cellular hypertrophy in postmitotic epithelia

Yoichiro Tamori and Wu-Min Deng*

Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4295, USA

In multicellular organisms, tissue integrity and organ size are finely maintained through removal of aberrant or damaged cells and compensatory proliferation. Little is known, however, about this homeostasis system in postmitotic tissues, where tissue-intrinsic genetic programs constrain cell division and new cells no longer arise from stem cells. Here we show that, in postmitotic Drosophila follicular epithelia, aberrant but viable cells are eliminated through cell competition, and the resulting loss of local tissue volume triggers sporadic cellular hypertrophy to repair the tissue. This "compensatory cellular hypertrophy" (CCH) is implemented by acceleration of the endocycle, a variant cell cycle composed of DNA synthesis and gap phases without mitosis, dependent on activation of the insulin/IGF (insulin-like growth factor)-like signaling pathway. These results reveal a remarkable homeostatic mechanism in postmitotic epithelia that ensures not only elimination of aberrant cells through cell competition but also proper organ-size control that involves compensatory cellular overgrowth induced by physical parameters.

Nutrition regulates body size through FoxO-Ultraspiracle mediated ecdysone synthesis Takashi Koyama1, Alexander W. Shingleton2, Christen K. Mirth1

1Instituto Gulbenkian de Ciência, Oeiras 2781-901, Portugal. 2Department of Zoology, Michigan State University, East Lansing, MI 48824, USA.

Nutrition regulates body size in most animals through the action of the insulin/Target of Rapamycin (TOR) pathway. In Drosophila, this pathway controls a key developmental checkpoint, critical weight, by regulating the synthesis of the steroid hormone ecdysone in the prothoracic glands (PGs). Critical weight sets the duration of the growth period thereby fixing maximum body size. Here we show that a major downstream component of insulin/TOR signaling, Forkhead Box class O (FoxO), directly interacts with a component of the ecdysone receptor complex, Ultraspiracle (Usp), to suppress ecdysone synthesis at critical weight. Knocking down both FoxO and Usp in the PGs causes premature critical weight transition, therefore miniature final body size. In contrast, overexpressing both of these genes delays critical weight transition and eclose into large body size adults. In addition, specifically disrupting the FoxO-Usp binding in the PGs changes the timing of ecdysone synthesis, the timing of critical weight, and final body size. This highlights a key mechanism through which nutrition controls body size. PTTH coordinates physiological and behavioral transitions during Drosophila development

Naoki Yamanaka1,*, Nuria M. Romero2,3,4,*, Francisco A. Martin2,3,4,*, Kim F. Rewitz5, Michael B. O’Connor1,† and Pierre Léopold2,3,4,†

1Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, USA

2University of Nice-Sophia Antipolis, 3CNRS, 4INSERM, Institute of Biology Valrose, Nice, France.

5Department of Biology, Cell and Neurobiology, University of Copenhagen, Copenhagen, Denmark

*,†These authors contributed equally to this work.

Prothoracicotropic hormone (PTTH) is a brain neuropeptide that regulates the timing of molting and metamorphosis in insects. PTTH activates its receptor Torso expressed in the prothoracic gland (PG), which transduces a downstream signal through the MAPK pathway to stimulate the production of the steroid hormone ecdysone. Ecdysone in turn regulates the expression of multiple genes in most larval tissues to coordinate all the physiological and morphological changes that accompany molting and metamorphosis. To date, all known functions of PTTH during insect development appear to be mediated indirectly through control of ecdysone production and release. By using Drosophila genetic tools to manipulate the function of Torso in different cell types, we have recently uncovered that PTTH acts on two light sensors, the Bolwig’s organ and the peripheral class IV dendritic arborization neurons, to regulate light avoidance. We find that PTTH concomitantly promotes steroidogenesis and light avoidance at the end of larval stage, thereby driving animals towards a darker environment to initiate the immobile maturation phase. Thus, PTTH controls the decisions of when and where animals undergo metamorphosis, optimizing conditions for adult development. Regulation of cyclin-dependent kinase 10 (CDK10) phosphorylation by steroid hormone 20-hydroxyecdysone for gene expression

Wen Liu, Mei-Juan Cai, Xiao-Fan Zhao

The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China

Steroid hormone 20E-hydroxyecdysone (20E) regulates gene expression via a genomic pathway by binding with its nuclear receptor EcR. However, the existence of 20E in a nongenomic pathway before the initiation of gene expression by a genomic pathway remains unclear. In this study, we report that 20E induces a Ser/Thr protein kinase cyclin-dependent kinase 10 (CDK10) quick phosphorylation in Lepidoprea Helicoverpa armigera via a nongenomic pathway. G-protein-coupled receptors (GPCRs) and Phospholipase C (PLC) inhibitors can repress 20E-induced CDK10 phosphorylation. The phosphorylated CDK10 increases its interaction with heat shock proteins such as Hsc70 and Hsp90 in the 20E transcription complex. CDK10 is localized in the nucleus via its KKRR motif. The nuclear location and the ATPase activity site of CDK10 are necessary for its function in regulating gene expression. CDK10 knockdown occurs when larvae are fed with Escherichia coli-expressing CDK10 dsRNA, which results in developmental delay by suppressing the 20E-induced gene expression. These data suggest that 20E regulates CDK10 phosphorylation and participates in the gene expression in a 20E genomic pathway via the GPCR-PLC nongenomic pathway. Keywords: CDK10, development, phosphorylation, 20E, nongenomic. Acknowledgments: This work was supported by grants from the National Natural Science Foundation of China (31230067) and the National Basic Research Program of China (973 Program, Grant no. 2012CB114101).

A novel action of the steroid hormone ecdysone on behavioral plasticity in adult Drosophila

Hiroshi Ishimoto1, Zhe Wang2, Chun-Fang Wu2,3, and Toshihiro Kitamoto3,4 1Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya City, Aichi 464-8602, Japan, 2Department of Biology, College of Liberal Arts and Sciences, 3Interdisciplinary Programs in Genetics and Neuroscience, 4Department of Anesthesia, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.

Ecdysone is the major steroid hormone in insects and plays vital roles in development as well as adult physiology and behavior. Although ecdysone signaling is mediated primarily by the nuclear ecdysone receptors (EcRs), several lines of evidence suggest that ecdysone can also execute its functions by modulating intracellular signaling cascades rapidly and independently of transcriptional regulation. Despite the potential significance of such rapid steroid actions, their biological roles and the underlying molecular mechanisms are not well understood, particularly with regard to their effects on behavioral regulation. DopEcR is a unique dual G-protein coupled receptor (GPCR) responding both to ecdysteroids and catecholamine dopamine. Here we found that Drosophila mutants for DopEcR are defective in courtship associative memory. Ecdysone is the key ligand for the DopEcR actions in the regulation of courtship memory: memory impairment caused by defective ecdysone synthesis could be rescued by acute feeding of 20-hydroxy-ecdysone (20E) in a DopEcR-dependent manner. Although dopamine is essential for courtship memory it does not seem to play a major role in this particular DopEcR-mediated process. We also identified genetic interactions between ecdysone/DopEcR signaling and the cAMP pathway mediated by rutabaga (Ca2+/CaM-dependent adenylyl cyclase) and dunce (cAMP-phosphodiestrase), suggesting that activation of ecdysone/DopEcR signaling leads to a rapid increase in intracellular cAMP levels to achieve steady courtship memory. Taken together, this study has demonstrated that a novel action of ecdysone that is mediated via GPCR-cAMP signaling plays important roles in behavioral plasticity in adult flies. Analysis of the peripheral tissue-derived peptides, CCHamide-1 and -2, and dRYamide-1 and -2, in Drosophila

Hiroko Sano1, Takanori Ida2, and Masayasu Kojima1 1Institute of Life Sciences, Kurume University, Kurume 839-0864, Japan, 2Interdisciplinary Research Organization, University of Miyazaki, Miyazaki 889-2192, Japan

In mammals, the gastrointestinal tract and adipose tissues are important sources of hormones that signal to the brain for the regulation of food intake and energy expenditure. We use a Drosophila model to examine the mechanisms of how diverse signals from peripheral tissues are integrated in the brain to control metabolism or behavior. Here we present the analysis of CCHamide (CCHa)-1, -2 and dRYamide-1, -2, which we have biochemically purified from whole fly homogenate. CCHa-1 and CCHa-2 were identified as ligands for GPCRs encoded by CG30106 and CG 14593, respectively. CCHa1 is mainly expressed in the gut as well as the brain, and CCHa2 is specifically expressed in the fat body. Receptors for these ligands are specifically expressed in the brain. We show that the expression of CCHa1 and CCHa2 is affected by nutritional condition, in that CCHa1 levels are elevated by feeding after starvation, while CCHa2 expression is decreased by starvation. These results suggest that the Gut-Brain and Fat cell-Brain connections generated by CCHa-1 and -2, respectively, are involved in nutrition-related systems such as metabolism and food intake. We similarly identified dRYamide-1 and -2, which are synthesized from the same precursor protein, as ligands for GPCR encoded by CG5811. dRYamide-1 and -2 are detected in the brain and the gut, while their receptor is expressed in the brain. We show that the expression of dRYamide-1 and -2 is up-regulated by starvation, suggesting that dRYamide-1 and -2 are also involved in nutrition-related systems. We are currently testing the functions of CCHa1 and -2, and dRYamide-1 and -2, and herein present the recent progress in our research. Identification of Hr38 as a conserved neural activity-induced gene in insect brains: its application as a marker for neural activity and implication for neural activity-dependent modification of ecdysone signaling in the brain.

Nozomi Fujita1, Hiroki Yamahana1, Yuka Nagata1, Takumi Nishiuchi3, Makoto Sato4, Masafumi Iwami1,2, Taketoshi Kiya1,2

1Division of Biological Sciences, 2Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan, 3Division of Functional Genomics, Advanced Science Research Center, 4Brain/Liver Interface Medicine Research Center, Kanazawa University, 13-1 Takara-machi Kanazawa, Ishikawa 920-8641, Japan

Many insects exhibit stereotypic instinct behavior, but the neural mechanisms of the behavior are not well understood due to difficulties in detecting neural activity in free-moving insects. As a powerful approach to detect behavior-related neural activity, immediate early genes (IEGs), whose expression is transiently and rapidly upregulated by neural activity, are widely used in the studies of various vertebrates. In insects, however, because no conserved IEGs have been identified, the advantages of this powerful approach have not been realized. In the present study, we identified Hr38 as a gene whose expression is transiently upregulated by female odor stimulation in the male silkworm , Bombyx mori, and found that Hr38 can be used to detect neural activity accompanying the sex pheromone-induced sexual behavior. Using consecutive sections, we constructed for the first time a comprehensive map of the neural activity pattern induced in response to pheromones in the insect brain. Similarly, we found that in the brains of the vinegar fly, Drosophila melanogaster, Hr38 can be used as a neural activity marker, and constructed a comprehensive neural activity map in response to female presentation. Also, in the honeybee Apis mellifera L., Hr38 was expressed in a neural activity-dependent manner in the brain. These results demonstrate that Hr38 is the first conserved neural activity marker gene to be identified among insects and will be useful for a wide variety of neuroethological studies. Since Hr38 is known to interact with Ultraspiracle and fine-tune the ecdysone signaling pathway, we also investigated the importance of ecdysone signaling in the higher brain function using honeybees. Expression of Hr38 was significantly increased by associative learning and administration of 20E and 3-epi-20E potentiated the long-term memory in a timing-dependent manner. These results suggest that ecdysone signaling pathway is modified in a neural activity-dependent manner and play important roles in the higher brain function. A single miRNA drives hemimetabolan metamorphosis to a right end

Xavier Belles*, Jesus Lozano, Raul Montañez

Institute of Evolutionary Biology (CSIC-UPF), Barcelona, Spain. *E-mail: [email protected]

Previous studies carried out in our laboratory had shown that RNAi depletion of dicer-1, a key enzyme involved in miRNA maturation, inhibited metamorphosis in the cockroach Blattella germanica. Dicer-1 suppression in the last (6th) instar nymph triggered the depletion of miRNA expression, and the nymphs molted to a supernumerary 7th nymphal instar instead to molt to adults. In order to identify which miRNAs are involved in regulating metamorphosis, we focused our attention on Krüppel-h1 (Kr-h1), a JH-dependent transcription factor that represses metamorphosis. Indeed, a decrease of Kr-h1 expression in the pre-metamorphic instar is crucial for metamorphosis progression. The decrease is abrupt, and this abruptness is hardly explained only by mechanisms controlling Kr-h1 transcription, and might be driven by miRNAs. Using again B. germanica as model, we observed that RNAi of Kr-h1 carried out in dicer-1-depleted specimens rescued normal metamorphosis. This pointed to miRNAs affecting Kr-h1 as responsible of the metamorphosis inhibition triggered by dicer-1 depletion. We also observed that the 3’UTR of Kr-h1 mRNA contains predicted sites for miR-2 family miRNAs, and that the predicted sites are functional, according to the results of luciferase reporter assays. Moreover, depletion of miR-2a impaired the decrease of Kr-h1 expression leading to metamorphosis inhibition, and administration of miR-2a mimic rescued the normal metamorphosis phenotype in dicer-1-depleted insects. The whole data suggests that miR-2a plays the key role of radically removing Kr-h1 mRNA in the premetamorphic stage, as a prerequisite for the activation of the transcription factors repressed by Kr-h1 that are the ultimate inducers of metamorphosis.

Hormonal regulation of Krüppel homolog 1 and its mechanisms repressing metamorphosis in the silkworm Bombyx mori

Tetsuro Shinoda, Hideki Sezutsu and Takumi Kayukawa

National Institute of Agrobiological Sciences, Tsukuba 305-8634, Japan

Juvenile hormone (JH) and ecdysone coordinately regulate molting and metamorphosis during post-embryonic development in insects. Krüppel homolog 1 gene (Kr-h1) was known to be a stage-specific modulator of the prepupal ecdysone response, which is essential for Drosophila metamorphosis. Recently, Kr-h1 has been revealed to be an early JH-inducible gene that works as a repressor of metamorphosis in many insect species; however, the hormonal regulation of Kr-h1 in the development and the molecular mechanism of its anti-metamorphic action are little understood. We have analyzed the hormonal regulation of Kr-h1 and its molecular function in the epidermis of the silkworm, Bombyx mori. Allatectomy (removal of corpora allata (CA)) showed that Kr-h1 was induced principally by JH derived from CA in the penultimate larvae and adults. In contrast, allatectomy did not affect Kr-h1 expression during prepupal stage. Ecdysteroid alone was unable to induce Kr-h1, but synergized the induction by JH in the cultured epidermis, suggesting that Kr-h1 is induced cooperatively by ecdysone and JH derived from tissue(s) other than CA in the prepupal stage. To elucidate the molecular function of Kr-h1, we generated silkworms overexpressing Kr-h1 (Kr-h1O/E silkworms). The Kr-h1O/E silkworms grew normally until last instar larvae but arrested their development at the prepupal stage. Removal of CA in the penultimate instar larvae of Kr-h1O/E resulted in a precocious prepupal arrest rather than precocious pupation. Thus the ectopic expression of Kr-h1 indeed prevents metamorphosis but is not sufficient to achieve normal larval-larval molt. Moreover, the gene expression profiles of early ecdysone-inducible genes (E74, E75, and Broad), which could be modulated by the application of ectopic JH during the early stage of last instar larvae, were identical between wild type and Kr-h1O/E silkworms. These results suggest the presence of a Kr-h1 independent JH signaling pathway, which is necessary for the modulation of early ecdysone-inducible genes by JH and maintenance of larval status.

Key words: Krüppel homolog 1; juvenile hormone; ecdysteroid; molt; metamorphosis; ecdysone-inducible gene; Bombyx mori

The characteristics of juvenile hormone epoxide hydrolase genes in insects

Takahiro Shiotsuki and Takuya Tsubota (National Institute of Agrobiological Sciences, Tsukuba 305-8634, Japan) E-mail : [email protected]

In the regulation of a number of physiological processes such as molting, metamorphosis, reproductive maturation and pheromone biosynthesis, Juvenile hormones (JHs) play important roles. JH titer is mainly regulated by the control of its synthesis and of its degradation, and two enzyme groups are mainly known to degrade JHs: JH-specific epoxide hydrolase (JHEH) and

JH-specific esterase (JHE). In this study we attempted to evaluate related genes of JHEHs in several insects with complete genome database. The search of the genomic sequence revealed that both species have five or six jheh-related genes. Each gene was cloned and the enzymatic activity of those gene products in two model insects, that is, Bombyx mori and

Tribolium castaneum was investigated. We found that multiple jheh-related gene products in both species could degrade JH efficiently, suggesting that one insect species have multiple

JHEHs. Since it was found that each jheh-related gene was expressed in different tissues and stage-specificity, it is supposed that the JH titer by JHEH would be regulated separately in each tissue. It is different from JHE, only JHE from single jhe gene has JH-degrading activity secreted to hemolymph at the last larval stadium. The JHEHs hydrolyze a JH to JH diol, an irreversibly hydrolyzed product in contrast to JHEs hydrolyze methyl carboxylate to give JH acid, which is able to regenerate JH reversibly by JH acid methyl transferase. Our results indicate that the regulation of JH titer by JHEH and JHE is performed in a distinct manner.

Keyword: juvenile hormone epoxide hydrolase, genomic analysis, Bombyx mori, Tribolium castaneum Roles of the juvenile hormone receptors Methoprene-tolerant and Germ cell-expressed in Drosophila larval development and metamorphosis.

Lynn M. Riddiford, Aaron A. Baumann, Raechel S. Warner, Michael Texada, and Loren Looger Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA

In Drosophila melanogaster the ancestral juvenile hormone (JH) receptor Germ cell- expressed (Gce) has duplicated to form Methoprene-tolerant (Met). Initial studies suggested that the two receptors acted redundantly to direct larval development and metamorphosis such that metamorphosis was blocked at head eversion to the pupa only in the double Met-gce null mutant [Abdou et al., Insect Mol. Biol. Biochem. 41, 938-45 (2011)] similarly to blockage after removal of the corpora allata (CAX) [Liu et al., Development 136, 205-15 (2009); Riddiford et al., Development 137, 1117-26 (2010)]. In the latter study however we found that adult optic lobe development in the Met27 null mutant was accelerated in response to the pupariation peak of ecdysteroid similarly to that of CAX prepupae. In contrast, the gce null mutant showed no anomalous development of the optic lobe [Riddiford, Gen. Comp. Endocrinol. 179, 477–484 (2012)]. To study the tissue distribution of these two receptors, we have taken two approaches: 1) Preparation of an affinity-purified chicken polyclonal antibody to a recombinant peptide designed to be Met-specific. 2) Prepared transgenic D. melanogaster lines carrying BACs comprised of either the Met or gce ORF interrupted by a GAL4 or LexA driver in the first exon, flanked on either side by approximately 40 kb of genomic context. These driver lines have been used to drive fluorescent reporters either singly or in combination. Western immunoblotting analysis of whole Canton S 2nd and 3rd instar larvae with the polyclonal antiserum showed only one band of about 100 kD which was absent in the Met27-gce double mutant. Analysis of the single mutants showed that the same band was present in the Met27 null mutant, but not in the gce2.5K null mutant which instead showed one 106 kD band, indicating possible post-translational modification. Immunocytochemistry with this antiserum showed that late 2nd and early 3rd instar salivary glands had nuclear staining that decreased as they filled with glue. Fat body, imaginal discs, gut, and the central nervous system also showed nuclear staining that varied with developmental age. These data indicate that the antiserum was specific for the two receptors but did not distinguish between them. The BAC probes allowed us to distinguish tissue specificity of the two receptors as well as developmental specificity. Most striking was the change in the expression of the two in the ring gland during the 2nd and 3rd larval instars. In the late 1st and early 2nd instar larvae, only gce is expressed in the prothoracic gland (PTG) portion, but in the early 3rd instar Met appears in the corpora allata (CA) and the corpora cardiaca (CC) portions. Later in the 3rd instar Met is also found in a few PTG cells, whereas Gce continues to be present in the PTG throughout the feeding period, then disappears during wandering. These findings suggest JH feedback effects on the glands which are currently being tested. Changing patterns of the two receptors are also seen in the CNS, gut, salivary glands, fat body during larval life and the onset of metamorphosis and will be discussed. Interestingly, with these reporters the imaginal discs are devoid of staining except for a few cells in the eye disc. This finding is at odds with the immunocytochemical evidence and will be discussed. If true, the lack of JH receptors in the discs would explain why disc-derived adult structures are aloof to exogenous JH at metamorphosis. Supported by the Howard Hughes Medical Institute. Ecdysone meets juvenile hormone: nuclear receptors seven-up and FTZ-F1 control juvenile hormone biosynthesis during hemimetabolan metamorphosis

Ferran Borras, Claudia Nieva, Oscar Maestro, Jose Luis Maestro, Xavier Belles and David Martín

Institute of Evolutionary Biology (IBE, CSIC-Universitat Pompeu Fabra), Barcelona, Spain.

Ecdysone and juvenile hormone (JH) are critical coordinators of normal developmental transitions in insects. In the hemimetabolous insect Blattella germanica, as in all insects, the presence of JH during juvenile development ensures that the molt will produce another juvenile instar, whereas the natural fall of this hormone in the final instar leads to the imaginal molt. Interestingly, a characteristic endocrine feature of the final nymphal instar of B. germanica is the reappearance of JH at the molt to the adult stage, as this hormone has also a major role in the regulation of reproduction. Thus, the corpora allata, whose biosynthetic activity has been impaired during the final nymphal stage, is reactivated to synthesize JH during the adult stage. Unfortunately, the mechanism that accounts for the re-induction of the corpora allata biosynthetic activity at the adult molt has not been identified. Here, we show that two nuclear hormone receptors expressed in the corpora allata, BgFTZ-F1 (whose expression in the gland is tightly controlled by the ecdysone-dependent transcriptional cascade of nuclear hormone receptors) and seven-up (BgSvp), control the stage- specific activation of the JH-biosynthetic activity of the gland during the nymphal to adult transition. Animals with reduced levels of BgSvp or BgFTZ-F1 presented a dramatic reduction in JH biosynthesis due to the lack of activation of several key JH-biosynthesis enzymes, such as HMG-CoA Synthase and HMG-CoA reductase. Finally, we show that this complex cross-regulatory interplay between BgSvp and BgFTZ-F1 also controls JH biosynthesis during the successive gonadotrophic cycles of B. germanica.

Transcriptional regulation of two Drosophila early puff genes in the larval salivary gland by 20- hydroxyecdysone and juvenile hormone: the role of MET and GCE

Jenna Callender*†, Sarah Keppler*, Nathan Howell*, and Vincent C. Henrich*† *Dept. of Biology, University of North Carolina at Greensboro, Greensboro, NC 27402 †Center for Biotechnology, Genomics, and Health Research, UNCG

The interactive effects of 20-hydroxyecdysone (20E) and juvenile hormone (JH) on transcriptional regulation has been described in numerous studies, often by assessing the effects of mutations and hormonal treatments upon developmental processes. These studies have focused directly on the short- term effects of exogenous hormonal application in timed late larval salivary glands from Drosophila melanogaster on the transcript levels of two “early puff” genes: E74A/B and broad (br). E74 transcription involves two promoters, a “low dose” promoter (100 nM 20E) which regulates E74B transctription, and a “high dose” promoter (1 µM) that regulates E74A. The br gene is regulated by two promoters (distal and proximal) with subsequent splicing of four isoforms from its mRNA. Salivary glands (SGs) were dissected from morphologically staged early wandering third instar larvae. Staging was verified using previously established SG transcript profiles (Huet et al, 1993). One gland from each larva was incubated in Schneider’s medium for 2 h, while the other gland was incubated in medium supplemented with a hormonal supplement of: 100 nM 20E, 1 µM 20E, 5 µM JHIII, 100 nM 20E + 5 µM JHIII, or 1 µM 20E + 5 µM JHIII. Based on direct comparisons of transcript levels in paired SGs, induction of Broad Z1 (Br-Z1) and E74A transcript levels depended on 20E levels. At the lower dose (100 nM 20E), the additional presence of JHIII was associated with transcript levels of Z1 that approximated maximal induction (i.e. JH potentiation of 20E induction), whereas JH repression of maximal induction was observed at 1 µM 20E. As expected, E74B induction was maximal at 100 nM 20E, and repressed at 1 µM, but this was unaffected by the simultaneous presence of JHIII. Several Met and Gce mutant SGs displayed vastly altered responses when tested with the same paradigm. The 20E induction of Br-Z1 and E74A was reduced in Met27 (null) mutant SGs at low and high doses. Gce-RNAi knockdowns evoked a similar effect. E74B transcript levels, however, were dramatically higher than normal at both 20E dose levels, suggesting that Met normally is involved in the repression of E74B transcription. One point mutation of Met (Metw3) which phenotypically behaves as a dominant negative, did not affect 20E-induction, but eliminated the modulatory effects of JHIII on E74A and Br-Z1, indicating that Met has separable JH-independent and JH-dependent transcriptional functions. To test the possibility further that MET and GCE are required for 20E induction, and based on developmental profiles showing that transcript levels of Met and gce peak sharply at about the time that 20E titers reach a late larval peak, the effects of Met overexpression were examined. Met overexpression modestly affected basal transcript levels, and caused 20E-superinduction of E74A and Br-Z1 that was quantitatively correlated with the level of Met overexpression. In the case of Br-Z1, all of the hormonal regulation described here is attributable to the distal Broad promoter. Collectively, these results indicate that Met and Gce play an essential role for 20E-regulated transcription of E74 and broad, include both JH-independent and JH-dependent functions, and modulate transcript levels via inductive, potentiative, repressive, and derepressive mechanisms that depend upon MET/GCE function and hormonal, MET, and GCE titers. Subfunctionalization of duplicate juvenile hormone receptors in higher Diptera

Aaron A. Baumann1, Josh Benoit2, Michael Texada1, Lynn M. Riddiford1

1 Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20174 2 Department of Epidemiology of Microbial Diseases, School of Public Health, Yale University, New Haven, CT 06520

Despite juvenile hormone (JH) involvement in numerous aspects of insect physiology, including developmental progression and reproduction, the JH receptor was only recently conclusively identified as the bHLH PAS protein Methoprene-tolerant (Met) in Tribolium. While mosquitoes likewise carry a single Met ortholog, higher flies carry two paralogous JH receptor proteins; Met in these insects is the nascent paralog of another bHLH PAS protein called germ cell expressed (gce). Both Met and gce bind JH with nanomolar affinity and D. melanogaster mutants lacking both genes die as prepupae. In Drosophila, gce can substitute for Met to rescue some larval but not adult Met mutant phenotypes, supporting the notion of Met/gce subfunctionalization. We examined the functional divergence of these duplicate JH receptors via genetic and physiological studies in two brachyceran flies, D. melanogaster and the tsetse fly Glossina morsitans. In tsetse, a combination of RNAi knockdown and hormone treatments showed that JH acts through Met but not gce to mediate lipid homeostasis between bouts of lactation. In this case, JH-Met action is synergistic with insulin signaling. While RNAi suppression of Met impaired fecundity, suppression of gce expression had no effect on fecundity or milk homeostasis, suggesting that as in Drosophila, Met is the key player during reproduction. To study more rigorously distinct roles for Met and gce throughout development, we employed several transgenic D. melanogaster lines carrying BACs comprised of a Met or gce ORF interrupted by a GAL4 or LexA driver in the first exon, flanked on either side by approximately 40 kb of genomic context. Using this system to drive the expression of a series of fluorescent reporters, we have systematically surveyed the spatio-temporal expression patterns of Met and gce in both larval and adult D. melanogaster. We show that these paralogous JH receptors exhibit largely non-overlapping expression in both larval (Lynn Riddiford, this symposium) and adult tissues, favoring a model which includes tissue- and stage-specific Met or gce homodimer formation. In the adult CNS, individual or subsets of cell types show striking patterns of spatially partitioned Met or Gce expression. For instance, in the optic lobe, Met but not gce expresses abundantly in a single layer each of the lobula and medulla, corresponding to regions affected by a morphological defect characteristic of some Met alleles. Our work suggests that the origin of Met from its paralog gce in higher flies corresponds with subfunctionalization and partitioning of expression across cell types, clearly evident in the D. melanogaster adult central nervous system.

Supported by the National Institutes of Health (AI081774, F32AI093023, RF01228833) and the Howard Hughes Medical Institute.

The Contribution of Endocrine Genes to Latitudinal Population Differentiation in Drosophila melanogaster

Thomas Flatt1

1Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

E-mail: [email protected]

Natural populations of D. melanogaster exhibit major latitudinal clines for life history traits such as developmental time, body size, fecundity, stress resistance, lifespan and the ability to undergo reproductive diapause, for example along the North American and Australian east coastal clines. This differentiation is thought to be driven by differences in selection pressure at the opposite ends of the clines, yet the genomic basis underlying these patterns remains poorly understood. Since many traits showing pervasive clinality are physiologically tightly regulated by hormonal signaling, including insulin/insulin-like growth factor (IIS), ecdysone (20E) and juvenile hormone (JH) signaling, it is tempting to speculate that polymorphisms in these endocrine pathways might be important contributors to clinal patterns (cf. De Jong & Bochdanovits, 2003, J. Genet.). Here we have applied genome-wide next generation sequencing of DNA pools ("pool-seq") to three populations collected along the North American east coast (Southern Florida, Pennsylvania, Maine) to investigate genic patterns of latitudinal differentiation. Remarkably, we find indeed that many of the most strongly differentiated candidate genes are involved in hormone signaling and production, including numerous candidates in the IIS/TOR and 20E signaling pathways. A comparison to previous data from the Australian cline (Kolaczkowski et al., 2011, Genetics) shows that many of these endocrine candidates also show major differentiation along the parallel but independent Australian cline. Our results therefore strongly suggest that at least part of the clinal patterns in Drosophila are shaped by selection on pathways involved in life history physiology, including endocrine signaling pathways.

Juvenile hormone and circadian genes regulate reproductive diapause in Pyrrhocoris apterus.

Authors:

Adam Bajgar, Vlastimil Smykal, Marek Jindra, David Dolezel

Organisms living in temperate regions must synchronize their physiology and development with environmental conditions. Shortening day length informs insects to prepare for winter in advance, in many species by inducing diapause. The actual photoperiod-measuring mechanisms are elusive, mostly due to absence of reliable phenotypes in typical genetic models such as Drosophila or Tribolium. The adult diapause of the linden bug, Pyrrhocoris apterus, involves a robust reproductive arrest, accompanied by energy storage, reduction of metabolic needs, and preparation to withstand low temperatures. To switch from diapause to reproduction, diapausing females require a photoperiod-dependent juvenile hormone (JH) signal. Here, we show that diapause regulation of the insect gut relies on an interaction between JH signaling and circadian clock genes. The circadian factors Clock and Cycle as well as the JH receptor, Methoprene-tolerant (Met), are all required in the gut for activation of the Par domain protein 1, isoform 1 gene

(Pdp1iso1) during reproduction, and for simultaneous suppression of a mammalian- type cryptochrome2 gene (cry2) that promotes diapause. A non-periodic, organ- autonomous feedback between Pdp1iso1 and Cry2 orchestrates expression of downstream genes that mark diapause versus reproductive state of the gut. These results suggest a model in which circadian factors mediate a diapause response through a novel mechanism that is independent of daily oscillations and operates downstream of JH signaling (Bajgar et al. 2013). Next, we focused on the role of JH signaling in reproduction itself. Importantly, knockdown of either Met or Taiman suppressed ovarian development independently of Clock, Cycle, Pdp1iso1 and Cry2. Accordingly, stimulation of vitellogenesis by treating diapause females with a JH mimic methoprene required both Met and Taiman. In vitro culture experiments confirmed that the fat body autonomously required both components of the JH receptor for expression of yolk, vitellogenin (Vg) mRNA. The absence of JH (in diapause females) or RNAi-mediated deficiency in JH reception resulted in loss of Vg expression and a simultaneous up-regulation of hexamerin, a storage protein typical of diapause. Our results indicate that JH signals through distinct pathways, where Met interacts with different basic helix-loop-helix transcription factors, possibly in a tissue-autonomous manner.

Bajgar, A., Jindra, M., and Dolezel, D. (2013). Autonomous regulation of the insect gut by circadian genes acting downstream of juvenile hormone signaling. Proc. Natl. Acad. Sci. USA 110:4416-4421.

Ovary ecdysteroidogenic hormone: function and signaling in mosquitoes

Mark R. Brown, Monika Gulia-Nuss, Animesh Dhara, and Michael R Strand Department of Entomology, University of Georgia, Athens, GA, 30602, USA

Reproduction in most mosquitoes encompasses a highly regulated sequence of behavioral, metabolic, and synthetic processes that result in the production of eggs. This is because the females must steal a blood meal from a much larger vertebrate host to acquire amino acids for yolk production, but some do not and instead mobilize teneral reserves for this purpose. We now know that ovary ecdysteroidogenic hormone (OEH) is the central neuroendocrine signal that activates egg maturation across the Culicidae and in non- and blood fed females. Its name signifies its primary action, and it also plays a role in the mobilization of amino acids from blood or teneral reserves. Amino acids, as well, enhance the primary activity of OEH through ‘target of rapamycin’ (TOR) signaling, which is also linked to insulin signaling. Insulin-like peptides (ILPs) can also activate ovary ecdysteroid production. These shared characteristics suggested that OEH was in some way dependent on ILP interactions or signaling. Our recent studies show that OEH does not require ILPs or the insulin receptor for activity and provide leads to the identification of the OEH receptor and signaling pathway. (Research support by NIH AI33108 to MRB and MRS).

Hormonal Regulation of Reproduction in the Red Flour Beetle, Tribolium castaneum

Xu J, Sheng Z, Parthasarathy R and Palli SR Department of Entomology, University of Kentucky, Lexington, KY 40546

20 hydroxyecdysone (20E), Juvenile hormones (JH) and insulin-like peptides (ILPs) regulate reproduction in the red flour beetle, Tribolium castaneum. In the female, 20E regulates oocyte maturation, JH regulates vitellogenin (Vg) synthesis in the fat body, and the nutritional signals mediated by Insulin/IGF-1 (IIS) and TOR pathways play key roles in regulation of both Vg synthesis and oocyte maturation. Both nutrition and JH are necessary for synthesis of Vg, and both these signals work through ILPs and the downstream FOXO transcription factor. Eleven nuclear receptors (E75, E78, FTZ-F1, HR38, HR4, Knirps-like, HNF4, Tailless, HR51, Dsf and HR39) play key roles in regulation of male reproduction. E78 and HR39 are required for sperm production and their transfer to the females. E75 and HR38 are required for the maturation of male accessory glands (MAG) and the production of accessory gland proteins (Acps). Nutritional signals working through IIS play important roles in regulation of MAG maturation and Acp production. Molecular mechanisms involved in regulation of reproduction in Tribolium by 20E, JH and ILPs will be discussed. References Xu J, Sheng Z, Palli SR (2013) Juvenile Hormone and Insulin Regulate Trehalose Homeostasis in the Red Flour Beetle, Tribolium castaneum. PLoS Genet. e1003535. Xu J, Baulding J, Palli SR. (2013) Proteomics of Tribolium castaneum seminal fluid proteins: identification of an angiotensin-converting enzyme as a key player in regulation of reproduction. J Proteomics. 78:83-93. Xu J, Raman C, Zhu F, Tan A, Palli SR (2012). Identification of nuclear receptors involved in regulation of male reproduction in the red flour beetle, Tribolium castaneum. J Insect Physiol. 58:710-7. Sheng Z, Xu J, Bai H, Zhu F, Palli SR (2011). Juvenile hormone regulates vitellogenin gene expression through insulin-like peptide signaling pathway in the red flour beetle, Tribolium castaneum. J Biol Chem.286:41924-36. Parthasarathy R, Palli SR (2011). Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol. 41:294-305. Xu J, Tan A, Palli SR. (2010) The function of nuclear receptors in regulation of female reproduction and embryogenesis in the red flour beetle, Tribolium castaneum. J Insect Physiol. 56:1471-80. Parthasarathy R, Sheng Z, Sun Z, Palli SR. (2010) Ecdysteriod regulation of ovarian growth and oocyte maturation in the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol.40:429-39. Parthasarathy R, Sun Z, Bai H, Palli SR (2010) Juvenile hormone regulation of vitellogenin synthesis in the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol. 40:405-14. Keynote Address

Karlson Lecture

Alex Raikhel

The Role of JH and 20-E in Mosquito Reproduction

Reception 5-6:00 PM Indoor Club TCF Bank Stadium 2009 University Ave SE Minneapolis Lecture 6:00-6:50 PM The role of juvenile hormone and 20-hydroxyecdysone in mosquito reproduction Alexander S. Raikhel University of California Riverside Hematophagous , such as mosquitoes, require vertebrate blood for their egg development with each gonadotrophic cycle being tightly coupled to a separate blood meal. As a consequence, mosquitoes are vectors for numerous disease pathogens of human and domestic animals, most importantly Malaria, Dengue fever and West Nile virus. Deciphering regulatory pathways controlling mosquito egg development is of paramount importance for devising novel approaches for mosquito control. The mosquito Aedes aegypti has been used for these studies due to its exceptional features as a vector model organism. Female mosquitoes undergo post- eclosion development (PE) that is controlled by JH III. PE is critical for a female mosquito to attain competence for blood feeding and egg development. Microarray analysis of developmental gene dynamics in the female fat body has demonstrated that 6,146 genes are differentially expressed during JH-dependent PE. These genes show striking temporal and functional separation. The RNAi microarray screen revealed a differential action of Met in down- and up- regulation of genes expressed during early and late PE, respectively. Sequence motif analysis has revealed the consensus 9-mer Met-binding motif that was found in 5’regions of Met up-regulated Late PE genes. EMSA using a combination of mutational and anti-Met antibody super-shift analyses has confirmed the presence of Met-binding motifs in these genes. Thus, Met action in up-regulation of some of the JH-dependent genes appears to be direct. I will discuss our recent efforts in deciphering the mechanism of Met action in down-regulation of gene expression. After blood feeding, 20E orchestrates vitellogenic and post-vitellogenic events in a female mosquito. The nuclear receptor βFTZ-F1 is essential for attaining competence for 20E responsiveness by acting as a facilitator for the recruitment of SRC FISC (Taiman) to EcR/USP. Isoforms of early genes E74 and E75 play different roles in vitellogenesis. Isoforms of Broad, which has been considered the “metamorphic” factor, are involved in vitellogenesis, with Br2 being an activator and Br4 a repressor of the vitellogenic genes. Our recent microarray analysis of gene expression in the fat bodies has revealed that 7729 genes are differentially expressed over nine consecutive time points during 72 hr post blood meal (PBM). Significantly, 2,343 genes are up-regulated, but 3,227 genes are down-regulated at 24 hr PBM following the 20E peak. We are investigating whether the 20E hierarchy is involved in gene down-regulation in the presence of a high titer of 20E. Successive egg maturation cycles are activated by separate blood meals, providing a basis for pathogen acquisition and transmission. Thus, it is important to understand the mechanism governing a succession of gonadotrophic cycles. Our results have shown an important role of the orphan nuclear receptor HR3 in regulation of developmental switches during reproductive cycles of A. aegypti females. Overall, research of hormonal regulation of mosquito reproduction has revealed many general aspects of insect endocrinology.

Support by NIH grants R37 AI244716 and RO1 AI59492

Ecdysoneless – Why is it ecdysone-less?

Marek Jindra1, Ann-Katrin Claudius2, Patrizia Romani3, Tobias Lamkemeyer2 and Mirka Uhlirova2

1Biology Center ASCR, Ceske Budejovice, Czech Republic 2Institute for Genetics and CECAD, University of Cologne, Cologne, Germany 3Dep. of Evolutionary Biology, University of Bologna, Bologna, Italy

The temperature-sensitive ecdysoneless1 (ecd1) strain has traditionally provided Drosophila researchers with a steroid-deficient animal model. Since its discovery (Garen et al., 1977), the mutant has served to assess the impact of ecdysone on gene regulation, morphogenesis, reproduction, behavior, immunity, and homeostasis. The ecd1 mutants suffer from insufficient ecdysteroid production for unknown reasons. Our identification of the ecd gene and its causal lesions (Gaziova et al., 2004) showed conservation of the Ecd protein from fission yeast to humans without revealing its molecular function. Mammalian Ecdysoneless has since been implicated as a positive regulator of cell cycle and cancer progression, although the precise mode of its action remains unknown. Here, we show that expression of genes encoding critical ecdysone-biosynthetic enzymes, primarily of CYP307A2/Spookier (Spok), is compromised in the larval prothoracic gland lacking ecd function. Although this explains the hormone deficiency of ecd1 mutants, Ecd does not primarily regulate ecdysone biosynthesis. In agreement with a recent proteomic study (Guruharsha et al., 2011), we show that Ecd associates with several core protein components of the U5 snRNP spliceosomal complex in Drosophila cells. Loss of Ecd disrupts splicing of spok pre- mRNA, thus eliminating its protein product. Hypothetical reasons why spok pre-mRNA is particularly sensitive to loss of Ecd are that the Drosophila spok gene resides in the heterochromatin, harbors a vast intron, and is highly and exclusively expressed by the prothoracic gland cells. In contrast, expression of Ecd is broader and its loss affects tissues in a manner unrelated to ecdysone. Consistently with a role in pre-mRNA splicing, Ecd is cell-autonomously required for survival of proliferating epithelial cells within the imaginal discs. Transgenic expression of the human Ecd ortholog averts all defects caused by the lack of Drosophila Ecd in the imaginal discs and in the prothoracic gland, where it restores spok pre-mRNA splicing and protein expression. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

Garen, A., Kauvar, L. and Lepesant, J. A. (1977). Roles of ecdysone in Drosophila development. Proc. Natl. Acad. Sci. USA, 74:5099-5103. Gaziova, I., Bonnette, P. C., Henrich, V. C. and Jindra, M. (2004). Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis. Development 131:2715-2725. Guruharsha, K. G., Rual, J.-F., Zhai, B., Mintseris, J., Vaidya, P., Vaidya, N., et al. (2011). A protein complex network of Drosophila melanogaster. Cell 147:690-703.

A novel Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis in the prothoracic gland

Sora Enya1, Yuko Shimada-Niwa1, Fumihiko Igarashi2, Masatoshi Iga2, Hiroshi Kataoka2, Yoshinori Fujimoto3, Tetsuro Shinoda4, and Ryusuke Niwa1, 5

1) Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan 2) Graduate School of Frontier Sciences, University of Tokyo, Japan 3) Department of Chemistry and Materials Science, Tokyo Institute of Technology, Japan 4) Division of Insect Science, National Institute of Agrobiological Sciences, Japan 5) PRESTO, JST, Japan

In insects, the precise timing of molting and metamorphosis is strictly guided by ecdysteroids that are synthesized from dietary cholesterol in the prothoracic gland (PG). In the last decade, several ecdysteroidogenic enzymes, some of which are encoded in the Halloween genes, have been identified and characterized. Here we report a novel Halloween gene noppera-bo (nobo) that belongs to the glutathione S-transferase family. nobo was identified as a gene predominantly expressed in the PG of the fruit fly Drosophila melanogaster. We generated a nobo knockout mutant, which displayed embryonic lethality and a naked cuticle structure. These phenotypes are typical for Halloween mutants showing embryonic ecdysteroid deficiency. In addition, the PG-specific nobo RNAi larvae displayed an arrested phenotype and reduced 20-hydroxyecdysone (20E) titers. Importantly, both embryonic and larval phenotypes were rescued by 20E administration. These results suggest nobo is essential for ecdysteroid biosynthesis. The larval lethality of nobo RNAi animals was rescued by feeding not only 20E but also cholesterol. Furthermore, when noboKO heterozygous females were fed cholesterol, the embryonic lethality of noboKO homozygote offspring was rescued and noboKO offspring hatched to larvae. Considering that cholesterol is the most upstream material for ecdysteroid biosynthesis in the PG, we currently hypothesize that nobo plays a crucial role in cholesterol transport and metabolism in the PG. Further analyses investigating the molecular functions of nobo in ecdysteroid biosynthesis will be presented. Characterization of 3-oxo steroids as intermediates in the Black Box of the ecdysone biosynthetic pathway

Hajime Ono , Ryota Kimura, Yuya Kaieda, Sayo Morita, Ichiyoh Asakura, Ritsuo Nishida Graduate School of Agriculture, Kyoto University, Japan

Ecdysteroids, steroid hormones in insects, coordinate major developmental transitions. During postembryonic development, ecdysone is biosynthesized from dietary cholesterol in the prothoracic gland (PG). Despite extensive studies, the initial conversion process, the socalled “Black Box”, has not been characterized. The steps from 7dehydrocholesterol (7dC) to the ketodiol, which is corresponding to the Black Box, help to build the ecdysteroid skeleton. The structure of the ecdysteroid skeleton is characterized by a cis junction of rings A and B, a 7ene6one chromophore, and a trans junction of rings C and D. In contrast, ecdysteroid biosynthetic enzymes including of Neverland (Nvd) and Spookier (Spok) have been characterized in the last decade. RNAimediated knockdown of expression of a gene which codes for an ecdysteroid biosynthetic enzyme results in arrest of molting, the phenotype of which can be rescued by feeding ecdysone or appropriate intermediate(s) to the larva. Therefore, we examined potential activities of candidate intermediates in the RNAitreated larvae. We found that several 3oxo steroids including of the 4diketol and diketol triggered molting of the spok RNAi larvae. We also detected an enhancement of the amounts of ecdysteroids in the spok RNAi larvae by feeding the 4diketol or diketol, indicating that the dietary 3oxo steroids were incorporated and converted into ecdysteroids in vivo . Furthermore, 20hydroxyecdysone inducible genes were induced in the spok RNAi larvae by feeding the 4diketol or diketol. Of them, expression of E75A was induced in both 4diketolfed and diketolfed spok RNAi larvae at a level similar to that in control GFP larvae. These results indicate that 4diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok. Forward Genetic Analysis of Ecdysone-Triggered Responses During Metamorphosis Arash Bashirullah University of Wisconsin-Madison, Madison WI 53705

Pulses of steroid hormones regulate a diverse array of biological responses during development, but how these simple global signals are refined into specific local responses remains poorly understood. We have taken a forward genetic approach to identify mutations that disrupt a subset of ecdysone-triggered responses during metamorphosis in Drosophila melanogaster. This approach has allowed us to identify novel and unexpected regulators of ecdysone signaling, like the DEAD box RNA helicase belle, the chromatin remodeler Ino80 and the mediator subunit med24.

Characterization of these genes have provided new insights into different aspects of ecdysone signaling including mechanisms that regulate tissue-specificity, auto- regulation, transcriptional memory and developmental timing. This work has highlighted a critical role, not only for transcriptional, but also for translational control mechanisms within the ecdysone-triggered signaling cascade. Oxygen affects the developmental physiology of growth and metamorphosis initiation in Drosophila

Viviane Callier, Colin Brent, Jinkyu Kim, Shampa M. Ghosh, Alexander Shingleton, Jon Harrison

Rearing oxygen level is known to affect final body size in a variety of insects, but the physiological mechanisms by which oxygen affects size are incompletely understood. In Manduca and Drosophila, the larval size at which metamorphosis is initiated largely determines adult size, and metamorphosis is initiated when larvae attain a critical weight. We hypothesized that oxygen effects on final size might be mediated by oxygen effects on the critical weight and the ecdysone titres, which regulate growth rate and the timing of developmental transitions.

Our results show that oxygen affects critical weight, the basal ecdysone titres, and the timing of the ecdysone peak, providing clear evidence that oxygen affects growth rate and development rate. Hypoxic third instar larvae (10% oxygen) exhibit a reduced critical weight, slower growth rate, delayed pupariation, elevated baseline ecdysone levels and a delayed ecdysone peak that occurred at a lower larval weight. Hyperoxic larvae exhibit accelerated pupariation and increased basal ecdysone levels, but no change in critical weight compared with normoxic larvae. Previous studies have shown that nutrition is critical for regulating growth rate and the timing of developmental transitions. Here we show that oxygen level is one of multiple cues that together regulate adult size and the timing and dynamics of growth, development rate and ecdysone signaling.

A role for the IP3 receptor in neuropeptide-producing cells of Drosophila during larval development

Megha, Siddharth Jayakumar, Manivannan S and Gaiti Hasan National Centre for Biological Sciences, Bangalore, India

The Inositol 1,4,5-trisphosphate (IP3) receptor (itpr) is a ligand-gated Calcium channel of the ER that releases Calcium from ER stores, downstream of G- protein coupled receptor stimulation. This signaling pathway is one of the primary means by which cytosolic calcium levels can be transiently raised in order to potentiate various cellular processes that eventually lead to physiological events such as hormone release, neurotransmission, fertilization etc. We observe that an itpr mutant is unable to transition from larvae to pupae, when transferred in early third instar stage to sucrose-only media. Control larvae transferred at a similar point are able to transition normally, from larvae to pupae to adults, albeit emerging as smaller sized flies. As responses to metabolic stress, and a subsequent integration of nutritional information to development rely on neuropeptides, we sought to understand the role of a functional IP3 receptor in neuropeptide-secreting cells, marked by the dimm transcription factor. We find that knockdown of itpr in such cells phenocopies the mutant’s behaviour on sucrose-only media, and over- expression of the wild type itpr in these cells is sufficient to rescue lethality of the mutant on sucrose-only media. These observations suggest that IP3 mediated calcium release is important for an appropriate response to metabolic stress. Experiments are underway to determine if itpr function in specific neuropeptide-producing cells, that are known to be involved in metabolism, is essential. Our experimental setup also allows us to interrogate how itpr functions to couple nutritional information to development, both at the organ and molecular level. Preliminary studies on this line of investigation will be presented. Together, these studies we hope will elaborate a hitherto unknown role for IP3 receptor-mediated calcium signaling in neuropeptide- release, and consequently, metabolism in the context of development. Mapping the Drosophila peptide-hormone system

Michael J. Texada1 and Jim Truman1

1Janelia Farm Research Campus, Howard Hughes Medical Institute; Ashburn, Virginia, USA.

Like other animals, insects use an array of hormones to adapt their physiology and behavior to changing developmental or environmental circumstances. The largest class of these comprises the peptide hormones, which are encoded by at least 39 genes in Drosophila, some of which give rise to multiple independent peptides. These hormones act through about 60 different receptors to evoke stereotyped responses from tissues ranging from the nervous system to the gut and fat body.

Peptide hormones are diverse in sequence and are generally present in great abundance in their cells of origin, allowing these “upstream” cells to be identified easily using immunohistochemistry. Their receptor molecules, on the other hand, are similar in sequence to one another and may be expressed at low levels, making the production and use of specific antibodies difficult. In addition, since hormonal signaling may occur over great anatomical distances, physical proximity to producing cells is not an indicator or requirement for responsiveness. As a result, although tissue-level analyses have been conducted based on expression microarray data (e.g., FlyAtlas.org; V. R. Chintapalli et al., Nature Genetics, 2007), the cellular targets for many peptide hormones are poorly defined, inhibiting the investigation of events “downstream” of hormone release.

To fill this investigational gap, I have created a collection of genetic tools to allow the direct identification and manipulation of hormone-responsive cells. Using a BAC-based “recombineering” method, I have replaced a portion of the coding sequence of 55 known or predicted hormone-receptor genes with that of the strong trans-activator GAL4::p65, while leaving intact the introns, the 5’ UTR, and 10-40k base-pairs of sequence on each flank. Consequently, it is likely that the regulatory elements have been retained, to give transgene expression that faithfully reproduces that of the endogenous gene. To date, 32 of these constructs have been successfully integrated into the fly genome and their expression patterns determined. When complete, this collection of lines should be a valuable resource for the Drosophila hormonal-signaling community. Abstracts of Poster Presentations

in numeric order Identification and molecular cloning of three Halloween genes in the varroa mite, Varroa destructor (Anderson & Trueman) (Acari: Varroidae)

Ana R. Cabrera1, Paul D. Shirk2, Jay D. Evans3 and Peter E. A. Teal2

1University of Florida, Entomology and Nematology Department, Gainesville, FL 32611 2USDA-ARS CMAVE, Gainesville, FL 32608 3USDA-ARS BRL, BARC-E, Beltsville MD 20705

Biosynthesis of 20-hydroxyecdysone (20E) in insects involves the action of five cytochrome P450s collectively known as Halloween genes. The complete transcripts of 3 Halloween genes [spook (Vdspo), disembodied (Vddib) and shade (Vdshd)] from the varroa mite were identified, sequenced and mapped to their genomic sequences. As compared with spook from insects and crustacean which have a maximum of 2 introns, Vdspo contained 5 introns ranging from 166 to 1479 bp. Both, Vddib and Vdshd contained 8 introns. A phylogenetic analysis revealed these 3 Halloween genes derived from common ancestoral genes. Phantom and shadow orthologs have not been identified in the varroa genome. Similarly, phantom orthologs have not been identified in the spider mite Tetranychus urticae or the deer tick Ixodes scapularis genomes, but both acarine genomes contained shadow orthologs. Predicted amino acid sequences from Vdspo, Vddib and Vdshd coding regions shared 33.3, 32.1 and 29.6% identity with the Drosophila melanogaster orthologs, respectively. Vddib transcript was present in ovary/lyrate organ samples while Vdshd transcript was present in ovary/lyrate organ, Malpighian tubules and gut samples. Vdspo transcript was detected only in gut samples and remained at constant levels in phoretic and early reproductive mites (from pre-capping brood cells). The Vdspo transcript levels in phoretic mites were 7.8 and 7 times higher than those of Vddib and Vdshd, respectively. In contrast to Vdspo, Vddib and Vdshd transcript levels were significantly up-regulated by 1.87 and 2.05 fold in early reproductive mites when compared to phoretic mites. A brood cell invasion assay showed that transcript levels from Vdspo, Vddib and Vdshd were not significantly different between mites that entered a brood cell within 4 hr compared to mites that remained on adult bees. LC-MS analysis of the hemolymph from various honey bee stages and whole body phoretic mites revealed a high abundance of putative diketol (intermediate in the 20E biosynthesis) in all samples, but none had detectable levels of 20E. These results suggest that the expression of Vddib and Vdshd is not related with the initiation of the varroa mite brood cell invasion behavior, but is associated to the physiological shift from phoretic to reproductive mite.

Hormone titers change rapidly in response to environmental stimuli. Feedback effects among hormone concentration, behavior, and environment have been extensively studied in vertebrate systems. However, less is known about causal feedback effects in invertebrate systems.

In some insects, Juvenile Hormone (JH) regulates aggression and dominance. Some previous work suggests that JH titers change rapidly in response to conflict. However, whether these changes subsequently affect behavior, and on what time scale, is unclear. We used a social wasp (Polistes dominulus) with a behavioral continuum ranging from highly cooperative to extremely aggressive to quantify how social and aggressive behavior influence JH titers.

The transcription factor Vvl regulates steroidogenesis in the Drosophila prothoracic gland

E. Thomas Danielsen #1#, Morten E. Møller #1#, Rachel Harder#2#, Michael B. O’Connor#2# and Kim F. Rewitz #1#

#1# Department of Biology, University of Copenhagen, Denmark. #2# Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, USA.

In the Drosophila larva, molting and metamorphosis depend on the steroid hormone ecdysone, produced in the prothoracic gland (PG). Transcription of the genes involved in the biosynthesis of ecdysone is regulated in response to both developmental and environmental cues in order to time the production of the ecdysone pulses that coordinate molting and metamorphosis. Expression of these genes is spatially restricted to the PG, however, the underlying transcriptional mechanisms which dictate the cell specific program still remains to be elucidated. We have identified the transcription factor Ventral veins lacking (Vvl) to be predominantly expressed in the PG from the embryonic stage and throughout larval development and, have investigated the consequence of reducing its expression specific in the PG. Interestingly, the larva arrests in the first instar and fails to undergo metamorphosis. The PG is intact in these larvae suggesting that Vvl regulates aspects of steroid synthesis and not cell fate. We observed a dramatically reduced expression of the Halloween gene, phantom, and a reduced level of an ecdysone-responsive target gene suggesting that Vvl is implicated in transcriptional regulation of the steroidogenic pathway. We further demonstrate binding-sites for Vvl in the regulatory promoter-region of phantom in vitro and in vivo. In conclusion, our data suggest that Vvl is a key factor implicated in Drosophila steroid synthesis by regulating the expression of a PG-specific gene encoding an enzyme in the biosynthetic pathway of molting hormone, ecdysone.

Methyl Farnesoate Action, and Morphogenetic Signaling Through the Ligand Binding Pocket of the Ortholog of the Retinoid X Receptor, in Higher Diptera

Grace Jones,1 Jose Bocanegra,1 John Smolka, 1 Davy Jones,2 Peter Teal,3 Vince Henrich,4 Anna 5 5 6 6 6 Niewiadomska-Cimicka, Marian Kochman, Anna Krzywonos, Agnes Sapa, Mietek Wozniak 1Dept. of Biology, Univ. of KY, Lexington, KY 40506; 2Grad. Ctr for Toxicology, Univ. of KY, Lexington, KY 40536; 3USDA/ARS, Gainesville, FL, 32608; 4Ctr for Biotech, Genomics, Health Res., Univ. of NC at Greensboro, NC 27402; 5Dept. Biochem, Wrocław Univ. Tech., Wrocław, Poland; 6Dept. Clinical Chem., Wrocław Med. Univ.

Most attention on metamorphic signaling by small terpenoids has focused action by juvenile hormone (JH) through bHLH-PAS proteins (e.g., MET and GCE), especially as that signaling axis intersects with ecdysteroid action through the receptor EcR. However, a long-standing series of endocrine and pharmacological studies on pupariation in Diptera have remained persistently refractory to explanation with the above two-axis model.

Larval Corpora Allata Secretion Necessary for Puparium Formation. Endocrine transplantation studies of Vogt [1] and Possompes [2] demonstrated that secretion from the corpora allata of the ring gland during the mid-late 3rd instar feeding stage is necessary for morphogenesis of the shape, sclerotization, and color of the puparium. Implantation of ecdysone-secreting ring glands at the early 3rd instar resulted in apolysis of the epidermis but the 3rd instar cuticle manifested a highly larviform puparium [1, 2].

Normal and Altered Titers of Circulating Methyl Farnesoids. Using a highly sensitive GC-MS on methyl farnesoid extracts of D.melanogaster blood, we determined that during the mid-late 3rd instar feeding stage, a large increase to near half micromolar levels* occurs for methyl farnesoate (MF) and bisepoxy-JH III, but not JH III (Jones et al. [3]). We then genetically reduced methyl farnesoate to <1% of normal during late 3rd instar feeding; bisepoxy JH III was 4-26% of normal, and JH III 26-125% of normal. These 3rd instar larvae exhibited the typical behaviors of ceasing feeding and wandering, but then only feebly attempted puparium formation, instead remaining essentially larviform. These above studies on endogenous secretions suggest that at the mid- late 3rd instar, larval methyl farnesoids interact with a small peak in 20E to enable subsequent puparium formation. However, the above data do not chemically identify the necessary corpora allata secretion(s).

JH III Reception Not Required for Puparium Formation. Recently Abdou et al. [4] showed that D. melanogaster null for its two acknowledged JH III receptors (MET and GCE) formed a puparium essentially normal in shape, sclerotization and color. Dietary inclusion of JH III or methoprene during the 3rd instar feeding period (when JH is lowest vs. MF and bisJHIII) failed to prevent essentially normal puparium formation (Riddiford and Ashburner [5]).

Ligand Pocket of RXR Ortholog Ultraspiracle: Competent to Bind and Respond to Sesquiterpene Ligand. Clayton et al. [6] estimated even under crystal formation conditions, ca. 25% of the Drosophila USP molecules were physically in apo conformation, available to receive ligand. In functional performance, Drosophila USP prepared under less severe conditions is bound by the known RXR synthetic ligand tributyltin (TBT), and by various sesquiterpenes at the respective saturating concentration of each [6]. In fact, TBT displaces sesquiterpene binding to USP in an equilibrium manner [6]. Henrich et al. [8] showed that in cultured mammalian cells Drosophila USP is competent to transduce binding of sesquiterpene via a potentiation of 20E signaling to the USP heterodimer partner, EcR. Fang et al. [8] obtained similar results in insect Sf9 cells. In a plant cell system ligand transduced USP homodimerization and transcriptional activation [9]. Of the three methyl farnesoids confirmed to be in larval circulation, MF binds USP by far the most strongly (Kd ca. 50 nM [6], similar to 9-cis RA for vertebrate RXR) and is the only sesquiterpene circulating at a titer corresponding favorably to its Kd for USP [5]. In vivo, Chironomus TBT was recently shown to modulate 20E signaling in larvae of the dipteran (Morales et al. [7]). Ligand Pocket of Ultraspiracle: Required for Puparium Formation. When USP point-mutated for reduced affinity for MF was challenged to function in larvae genetically null for usp, the larvae developed through the ecdysone-driven first two larval molts. Although the 3rd instar larvae ceased feeding, wandered, and apolysed to the pupal body, the 3rd instar cuticle remained larviform, unsclerotized and untanned (Jones et al. [5]). Genome expression analysis (by microarray, confirmed by qPCR) showed that genes for enzymes (e.g. DDC) and structural proteins associated with formation of the late 3rd instar integument were being misexpressed.

The above data evoke a model for puparium formation in which MF and USP necessarily intersect with 20E/EcR signaling during late 3rd instar feeding, the JH/MET-GCE axis is not required, and the role of bisJHIII is unresolved.

*MF also circulates >200 nM in Orthoptera and Hemiptera (1) Biol. Zblt. 63 (1943) 395-446. (2) Arch. Zool. Exp. Gen. 89 (1953) 203-264. [3] Gen. Comp. Endocrinol. 182 (2013) 73-82.[4] Insect Biochem. Mol. Biol. 41 (2011) 938-45. [5] Gen. Comp. Endocrinol. 82 (1991)172-83. [5] Proc. Natl. Acad. Sci. USA 98 (2001) 1549-54. [6] FEBS J. 273 (2006) 4983-96. [7] Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2013 May 14. [9] Int Patent Application Number PCT/EP96/ 04224. [10] FEBS. J. 272 (2005) 1577–1589. Pupal commitment of a single celled Verson’s gland occurs gradually

Kiyoshi Hiruma1, Yu Kaneko1, and Lynn M. Riddiford2

1Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki 036-8561, JAPAN 2Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA

Commitment of a cell generally occurs on an all-or-none basis (Stamatoyannopoulos, 2005). It has also been reported that the same mechanisms could be applied in Pyrrhocoris apterus, as the structures of the cuticle of nymphal-adult intermediates was never observed in the “regionally-replaced cuticle” produced by the application of juvenile hormone (Sláma and Weyda, 1997). Yet Wigglesworth (1934) has shown in Rhodnius prolixus that a bristle-forming cell can produce an nymphal-adult intermediate, and later Lawrence (1966) and Willis et al. (1982) found similar results in Oncopeltus and lepidopteran cuticle respectively. The segmentally arranged Verson’s glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The secretory cell in the last instar stadium in Bombyx mori grows bigger than an adult Drosophila. In Manduca sexta the secretory cells produce stage-specific proteins (Lane et al., 1986; Horwath and Riddiford, 1988). Using Bombyx Verson’s glands, we applied DNA microarray analyses and found a number of larval- and pupal-specific genes, which we were able to use as molecular markers for pupal commitment of a single cell. Pupal commitment was assayed by implantation of Verson’s glands from varying stages of 5th (last) instar larvae into day 1 4th instar larvae, then assessed 12 hr before the host had molted to the 5th instar. The transcripts of larval-specific genes in the Verson’s glands began to decrease on day 1 and disappeared completely by day 3 of the 5th instar, whereas those of pupal-specific genes began increasing on day 2 and peaked over the next 24 hr. These results showed that the pupal commitment of Verson’s glands occurred 1-3 days after the last larval ecdysis. When we used a day 2 5th Verson’s gland, which expressed some larval- but no pupal-specific genes, for the commitment assay, the transcripts of both type of genes were co-expressed in a single secretory cell. Therefore, pupal commitment of a cell may not occur on an all-or-none basis, but rather gradually. Supported by the JSPS. PTTH promotes cellular cholesterol uptake of the prothoracic glands Bombyx mori.

Fumihiko Igarashi, Juri Hikiba, Takayoshi Nakaoka, Hiroshi Kataoka Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Japan

Ecdysone is an insect steroid hormone essential for development and metamorphosis. For the past decades, the regulation of the ecdysone synthesis in the prothoracic glands (PG) has been studied, and it was elucidated that the signaling pathway and the metabolic machineries of ecdysteroidogenesis. As ecdysone biosynthesis is started from cholesterol, the cholesterol transport system in the PG could also mediate an important role on the synthesis regulation. However, the mechanisms of the cholesterol transport in the PG, such as cellular uptake and intracellular traffic of cholesterol, have not been well understood. In order to understand the cholesterol transport in the PG, we first analyzed concentration changes of steroid metabolites around the stages of ecdysoteroidogenesis, by developing and using the LC-MS/MS for the quantification of whole steroid metabolites in the PG of the silkworms. As a result, we found the amounts of cholesterol and 7-dehydratecholesterol increased along with the capacity for ecdysone synthesis. As most of the cholesterol in the PG was derived from plasma lipoprotein (Lp), we next assumed that the cholesterol uptake from the Lp would increase with ecdysone synthesis. Then, we tested whether the PG cells cultured with prothoracicotropic hormone (PTTH) showed cholesterol uptake activity, and observed that the PG incubated with the PTTH contained higher amounts of cholesterol. Also, the transcript levels of the Lp receptor (LpR) increased corresponding to the cholesterol uptake activity. These results indicated that PTTH stimulation in vitro promoted the cellular cholesterol uptake of the PG. Moreover, we found that the PG incubated with the Lp produced higher amounts of ecdysone. This result implied that the cellular cholesterol uptake could regulate the rate of ecdysone synthesis, intriguing us further analysis of its function in ecdysteroidogenesis.

This study was partly supported by the grant from Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry and Japan Society for the Promotion of Science.

Poster Presentation

An endoparasitoid, Cotesia plutellae , alters insulin, juvenile hormone, and ecdysone signalings of the diamondback moth, Plutella xylostella

Yonggyun Kim 1, Ramjan Ali 1, Rahul Hepat 1, Xiaojun Gu 2

1Department of Bio-Sciences, Andong National University, Andong 760-749, Korea 2College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian Province, People's Republic of China

Developmental alteration due to parasitism is well explained in a host-parasitoid interaction of Plutella xylostella -Cotesia plutellae . Parasitized larvae undergo an extended larval period and fail to metamorphosis to pupal stage. Juvenile hormone esterase (JHE) activity was significantly suppressed and corpora allata was well developed in the last instar larvae of the parasitized. The prothoracic gland was hypotrophied and expression of ecdysone receptor (EcR) was suppressed in the parasitized larvae. The ecdysteroid-inducible E74B was suppressed in its expression compared to nonparasitized larvae. Expression of insulin-like peptide (ILP) was markedly reduced in the parasitized larvae. However, insulin receptor and FOXO were highly expressed in the end of larval period. Two main parasitic factors of polydnavirus and teratocyte induced the alterations of the endocrine signalings. Some viral protein tyrosine phosphatases were associated with the elevation of InR expression and suppression of ILP. Teratocytes suppressed JHE activity as well as EcR expression. Insulin-like peptides in the cockroach Blattella germanica. Cloning and expression studies.

José L. Maestro, Songül Süren-Castillo

Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Barcelona, Spain.

The insulin/insulin-like growth factor signaling (IIS) pathway is involved in essential processes such as growth, cell proliferation, longevity, metabolism or reproduction. In vertebrates insulin and insulin-like growth factors (IGFs) interact with insulin receptor and IGF-1 receptor. However, most insects possess a high number of insulin-like peptides (ILPs) (8 in Drosophila melanogaster, 8 in Aedes aegypti or up to 39 in Bombyx mori) but only one receptor. W hich is the reason for this high number of ILPs in insects? How do these peptides regulate the different processes in which they are involved? Preliminary steps in order to answer these questions have been performed for the German cockroach, Blattella germanica. The sequences of five different cDNAs coding for ILPs have been obtained during the analysis of transcriptomes representing different organs and stages of development of B. germanica. Expression studies reveal that they are differently expressed in different tissues. ILPs expression has also been studied in fed and starved adult females. Results show differences in mRNA levels for some ILPs when compared animals under different nutritional conditions.

Evolution of SUMO protein functions in insects

Enric Ureña1, Coralia Pérez2, Manuel S. Rodríguez2, Valérie Lang2, Lucia Pirone2, James D. Sutherland2, Rosa Barrio2 and David Martín1

1Institute of Evolutionary Biology (IBE, CSIC-Universitat pompeu Fabra), Barcelona, Spain. 2CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.

SUMOylation is a highly conserved post-translational modification that modulates target protein activity in a great number of cellular processes during development. In the holometabolous insect Drosophila melanogaster, SUMOylation has been shown to be necessary during the metamorphic transition. Reduction of the single D. melanogaster SUMO homologue (smt3) expression in the prothoracic gland prevents the larval to pupal transition due to reduced lipid content in the gland and low ecdysone titer1. Unfortunately, our knowledge on the role of SUMOylation in insect development exclusively derives from studies in D. melanogaster, which shows a highly derived type of insect development that may not be representative in the insect class context. Our research, therefore, is devoted to establish the role of SUMOylation in the hemimetabolous insect Blattella germanica. In contrast to D. melanogaster, we have identified two B. germanica SUMO homologs, BgSumo1 and BgSumo3 that are ubiquitously expressed throughout development. By using RNAi in vivo experiments we have shown that, whereas BgSumo3 is dispensable for the correct development of B. germanica, reduction of BgSumo1 levels resulted in severe defects during the metamorphic transition, including a marked developmental delay due to impaired activation of the ecdysone-triggered signaling cascade. Furthermore, we have shown that all the proteins belonging to the ecdysone-dependent transcriptional cascade of nuclear hormone receptors (BgEcR, BgRXR, BgE75, BgHR3 and BgFTZ-F1) are SUMOylated in vitro. Finally, to test whether the functions of SUMO proteins are evolutionary conserved between hemimetabolous and holometabolous insects, we performed functional analysis in vivo of the two B. germanica Sumo homologues using D. melanogaster as model system. These experiments showed that BgSumo3 contains all the elements necessary to substitute functionally for D. melanogaster smt3, while BgSumo1 does not.

1 Talamillo, A. et al. (2008) Development 135: 1659-1668.

Preliminary Profile of Nuclear di-phospho-Erk Expression in the Drosophila Prothoracic Gland During the 3rd Larval Instar

Zofeyah McBrayer, Mary Jane O’Connor, Michael B. O’Connor Department of Genetics, Cell Biology, and Development University of Minnesota, Minneapolis, MN 55455

Erk (Drosophila rolled) is a downstream kinase of the MAPK signaling cascade. It is also downstream of Torso signaling in the Drosophila Prothoracic gland (PG), a component of the Ring gland that produces the hormone Ecdysone, which controls larval transitions (instars), puparium formation, and metamorphosis in Drosophila. Prothoracicotropic Hormone (PTTH) is a neuropeptide produced in neurosecretory cells of the brain that innervate the PG. Activation of Torso by PTTH activates the MAPK signaling cascade leading to the phosphorylation and activation of Erk, which then enters the nucleus to interact with factors that regulate Ecdysteroid production. Loss of Torso or Erk in the PG or destruction of the PTTH-producing neurosecretory cells causes severe delays in developmental timing. Affected larvae show slight delays in larval transitions and a severe delay in puparium formation, due to a prolonged final (3rd) instar. The prolonged 3rd instar causes prolonged feeding, resulting in enlarged larvae, which then produce enlarged pupae and adults.

Hormone regulation is frequently entrained to light-dark cycles (circadian rhythms) in nature. In Drosophila the pdf-producing neurons (pigment dispersing factor) are part of the circadian clock. Axons of the pdf neurons are known to contact the dendrites of the PTTH-producing neurons. Furthermore, PTTH mRNA is known to have cyclic expression. In this experiment we are investigating the expression profile of the active form of Erk (diphospho-Erk) in the Drosophila Ring Gland in 3rd instar larvae raised on a 12 hour light-dark cycle as well the expression of an HA tagged PTTH protein.

In PTTH-HA transgenic larvae, our results show that active Erk appears to accumulate in the nucleus at “mid-day” during the first (6hr) and second (30hr) day of the 3rd instar, as well as at “sunrise” (24hr) of the second day. Additionally, nuclear active Erk is found at 20hr during the dark phase. All of these times correlate approximately with known peaks of Ecdysteroid release during the 3rd instar. By the wandering phase, larvae become highly unsynchronized and show variable localization of di-phospho- Erk. It appears that there is one more nuclear phase, but the timing of this event is unclear. Co-staining for PTTH-HA did not reveal any obvious rhythm or fluctuation of this protein and requires further analysis. Future experiments will reveal whether this pattern is also present in wildtype larvae. Dynamic feedback circuits generate the maturation-inducing steroid pulse in Drosophila . Morten E. Møller 1, E. Thomas Danielsen 1, Rachel Harder 2, Michael B. OConner 2, Kim F. Rewitz 1. 1) Department of Biology, University of Copenhagen, Copenhagen, Denmark; 2) Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota.

Pulses of steroid hormones act as temporal signals that drive the juvenile-adult transition, which transforms the developing organism to a reproductively mature adult. This transition, known as metamorphosis in Drosophila, is triggered by pulses of the steroid hormone ecdysone produced and released from the prothoracic gland (PG). Ecdysone is synthesized through a series of enzymatic reactions mediated by P450 enzymes in response to the neuropeptide prothoracicotropic hormone (PTTH) released from the brain. The shape and duration of the ecdysone pulses are defined by a genetic program which ensures a rapid ramp of hormone synthesis followed by mechanisms that shut down the production, but the underling mechanisms are not fully understood. We show an ecdysone-dependent feedback switch in the PG which is required for the rapid increase and following decline of the ecdysone titer. This switch consists of a feedforward and a feedback loop. Blocking the feedforward loop in the PG results in reduced levels of ecdysone and delayed puparation. The negative feedback is responsible for the following decline of the titer and together these processes are required for generating a pulse that drives developmental progression. The feedback is mediated through the ecdysone receptor (EcR) that induces the expression of a transcription factor called Broad, which regulates the expression of the ecdysone biosynthetic enzymes by binding onto their promoters. Different Broad isoforms are responsible for the transcriptional activation and repression that changes the capacity of the PG to produce ecdysone.

In conclusion: These findings demonstrate a feedback mechanism in the PG involving EcR and Broad, which is required to establishes the temporal boundaries of the ecdysone pulse and developmental transition to adulthood.

Keywords: Ecdysone feedback loop

Control of Body Size by TGF-β Signaling Lindsay Moss-Taylor1, Michael O’Connor2 1Molecular, Cellular Developmental Biology and Genetics Program, University of Minnesota; 2Department of Genetics, Cell Biology and Development, University of Minnesota

Body size is tightly regulated during development to maximize adult fitness. In Drosophila, final body size is mainly determined by growth rate and duration of growth during juvenile stages; once maturation occurs, body size is set. The rate of growth is determined by insulin-like peptide (dilp) signaling, while the duration of growth is regulated by the neuropeptide prothoraciotropic hormone (PTTH). Manipulation of these pathways alters final body size by either accelerating or delaying ecdysone production and the onset of metamorphosis. Under normal growth conditions, termination of Drosophila juvenile development is triggered when third instar larvae achieve “critical weight,” a size after which starvation no longer delays the time to metamorphosis. How larvae sense critical weight and the relationship of critical weight to dilp and PTTH signaling remains largely unknown. We are investigating the newly identified role that the TGF-β ligand Activinβ plays in regulating Drosophila body size and timing of metamorphosis. We have found that mutations in dActivinβ (dActβ) cause accelerated larval development and smaller final body size. Our preliminary results show that dActβ is expressed in the Insulin Producing Cells (IPCs) and motorneurons. Overexpression of Activinβ in either motorneurons or in endocrine tissues increases body size. Since increased insulin signaling advances metamorphosis, we will test the hypothesis that this TGF-β ligand is affecting body size and developmental timing by regulating insulin production and/or release.

Development of competence to undergo metamorphosis

Vlastimil Smykal1, Takaaki Daimon2, Takumi Kayukawa2, Keiko Takaki1, Tetsuro Shinoda2 and Marek Jindra1

1Biology Center ASCR, and University of South Bohemia, Ceske Budejovice, Czech Republic 2National Institute of Agrobiological Sciences, Tsukuba, Japan

In insects, juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. At that time, drop in JH secretion permits a metamorphic molt. In both holometabolous and hemimetabolous insects, JH acts through its receptor, Methoprene-tolerant (Met) to suppress precocious metamorphosis via activation of the Krüppel-homolog 1 (Kr-h1) gene. However, various methods of depleting JH fail to induce precocious metamorphosis during earliest larval instars, suggesting that the juvenile character of these youngest larvae may not depend upon JH signaling. To investigate how and when insect larvae gain competence to metamorphose, we employed two species with distant modes of development, the hemimetabolous true bug, Pyrrhocoris apterus, and the holometabolous silkmoth, Bombyx mori. Pyrrhocoris invariantly undergoes five larval instars. Systemic RNAi knockdown of either Met or Kr-h1 in the penultimate instar (L4) causes a metamorphic molt with precocious development of adult color pattern, wings, and genitalia (Konopova et al., 2011). However, Met RNAi administered during the L3 and L2 instars required two molts for the precocious metamorphosis phenotype to be observed at L5 and L4, respectively, and the earlier (L2) dsRNA injection only yielded an incomplete phenotype. Met RNAi during L1 had no heterochronic effect. Kr-h1 silencing at the L3 and L2 instars induced precocious metamorphic phenotypes already after a single molt, but again these were limited to minor changes of pigmentation and external genitalia. Kr-h1 RNAi administered maternally or during L1 did not accelerate development at all. Therefore, the sensitivity to disrupted JH signaling progressively increased from L1 (insensitive) towards the penultimate (L4) larval instar (most sensitive). To examine the anti-metamorphic signaling in a JH-free background, we utilized the silkmoth mod mutants unable to synthesize JH (Daimon et al., 2012). Control silkmoths developed through five larval instars, whereas 98% of mod larvae underwent four (and the 2% only three) larval instars before they pupated. We found that Kr-h1 mRNA expression in pre-final mod instars was roughly 10-fold lower than in control larvae, yet during the mod ultimate (L4) instar, it followed the natural decrease observable in control L5 larvae. Treatment of mod larvae with the JH mimic methoprene restored the normal, 5-instar development and increased Kr-h1 expression, showing that the normal response to JH in these mutants was retained. Our data also suggest that a minor component of Kr-h1 expression during pre-final mod larval instars appears not to depend on JH. Whether this JH-independent Kr-h1 expression is sufficient to prevent metamorphosis during the L1-L3 instars of insects cannot be currently resolved. However, we conclude that (i) insect larvae gain their competence to metamorphose gradually during the successive instars, and (ii) factors in addition to JH may safeguard insects against precocious metamorphosis during their earliest juvenile stages.

Daimon, T., Kozaki, T., Niwa, R., Kobayashi, I., Furuta, K., Namiki, T., Uchino, K., Banno, Y., Katsuma, S., Tamura, T., Mita, K., Sezutsu, H., Nakayama, M., Itoyama, K., Shimada, T. and Shinoda, T. (2012). Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori. PLoS Genetics 8, e1002486. Konopova, B., Smykal, V. and Jindra, M. (2011). Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects. PLoS ONE 6, e28728.

Genome-wide Ecdysone Receptor binding changes after exposure to the juvenile hormone mimic methoprene

Rebecca F. Spokony1, Robert Arthur1, Christopher D. Brown2, Nicholas Bild1, Jennifer Zieba1, Matthew Slattery1, Jesse Cohen1, Kevin P. White1 1Institute for Genomics and Systems Biology, University of Chicago, Chicago IL 60637 2Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104

Drosophila melanogaster development is controlled by two main hormones, ecdysone and juvenile hormone (JH). Ecdysone controls developmental transitions and acts primarily through a heterodimer of two nuclear receptors, Ecdysone Receptor (EcR) and Ultraspiracle (USP). Juvenile hormone controls the type of transitions and acts through a bHLH protein, Methoprene-tolerant (MET). In the presence of JH, MET has been shown to bind Taiman (TAI) and FTZ-F1, two nuclear receptors also involved in the ecdysone pathway. Ectopic juvenile hormone can disrupt normal ecdysone mediated processes and is used as an insecticide. Known ecdysone mediated process that are disrupted include polytene puffing and morphogenesis of the central nervous system and salivary glands. Drosophila melanogaster is most sensitive to ectopic juvenile hormone at the larval to prepupal transition (pupariation). We exposed white prepupae (WPP) to the juvenile hormone mimic, methoprene at WPP for 4-5 hours. Using ChIP-seq, we characterized EcR binding sites in the absence and presence of methoprene. Using 100 bp windows, there are 186 locations with 4-fold more EcR binding after exposure to methoprene and 825 locations with 4-fold less EcR binding after exposure to methoprene. Differential binding was found at many JH targets’ loci, such as Eip75B, ftz-f1, Kr-h1, and tai, as well differentially expressed genes under ectopic JH or methoprene exposure. EcR binding increases are enriched for TAI binding sites (characterized by late wandering third larval binding pattern, when JH is normally present). We predict that the lost EcR binding sites required TAI or FTZ-F1 that has been removed due to binding with MET. In order to test if these binding sites are responsible for the differential expression, we are constructing reporters using 1 kb intact-EcR binding regions and mutated-EcR binding regions and testing their responsiveness to methoprene.

Nuclear receptor DHR51, heme and ecdysone biosynthesis in Drosophila Brian Phelps, Qiuxiang Ou, Kirst King-Jones Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

An RNAi screen for regulators of ecdysone production in Drosophila melanogaster prothoracic glands resulted in the identification of spaztle5 (spz5). Loss of spz5 function did not only abolish ecdysone biosynthesis, but also led to a dramatic accumulation of heme precursors, indicating an unexpected link between the regulation of heme biosynthesis and steroid hormone production. The accumulation of heme precursors is a result of the upregulation of ALAS, which encodes the rate-limiting enzyme in the heme biosynthetic pathway. We show here that upregulation of ALAS is dependent on the nuclear receptor DHR51 (Drosophila hormone receptor 51), providing genetic evidence that this nuclear receptor may act as a novel heme sensor. Previous biochemical evidence established that DHR51 can bind heme in vitro, but no data has been brought forward supporting the idea that DHR51 acts as a heme sensor in vivo. Loss of DHR51 function via RNAi in the prothoracic gland results in a development delay or arrest depending on the strength of the knockdown, suggesting that DHR51 has a critical function in the prothoracic gland. However, DHR51 overexpression results in arrests from the first instar to pupae in a dose-dependent manner. We hypothesise that DHR51 activity is dependent on cellular heme concentrations and that low heme levels allow DHR51 to directly upregulate ALAS expression. Our results support this idea since a chimeric DHR51 ligand trap protein is active when cellular heme concentrations are lowered by genetic means. Taken together, our data identifies heme biosynthesis as a key regulatory process for steroid hormone production, a process that has remained largely unexplored.

Ecdysone conference 2013

Poster Abstract :

CHARACTERIZATION OF THE ECDYSONE RECEPTOR (EcR) FROM THE SALMON LOUSE, LEPEOPHTHEIRUS SALMONIS.

Liv Sandlund (1), Lars Hamre (1), Heidi Kongshaug (1), Rune Male (1), Frank Nilsen (1) and Sussie Dalvin (1, 2),

1. Sea Lice Research Centre, University of Bergen, Department of Biology PO box: 7803, NO-5020 Bergen [email protected]

2. Institute of Marine Research P.O. Box 1870 Nordnes, N-5201 Bergen, Norway

The salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae) is an important parasite in the salmon farming industry in the Northern Hemisphere causing annual losses of hundreds of million US dollars world-wide. In order to facilitate development of a vaccine or other novel control measures to gain control of the parasite, knowledge about molecular biological functions of L. salmonis is vital. In arthropods, developmental processes such as reproduction and oogenesis are mediated by binding of steroid hormones to a heterodimer of the ecdysone receptor (EcR) and a homolog of the retinoid X receptor, ultraspiracle (USP). In this study, full-length cDNA of the L.salmonis EcR (LsEcR) was obtained by 5`RACE PCR and characterized. LsEcR amino acid sequence demonstrated high sequence similarities level to other EcRs including Tribolium castaneum and Locusta migratoria. Moreover, in-situ analysis of adult female louse revealed LsEcR transcript to be localized in a wide variety of tissues such as ovaries, intestine and oocytes. Furthermore, the functional role of LsEcR was investigated using RNA interference.

Deciphering the patterns of gene expression during the gonadotrophic cycles of the yellow fever mosquito, Aedes aegypti Sourav Roy, Tusar Saha, Lisa Johnson, Jisu Ha and Alexander Raikhel Department of Entomology and Institute of Integrative Genome Biology University of California, Riverside. Abstract:

Female mosquitoes act as vectors for numerous devastating human diseases. Aedes aegypti, the yellow fever mosquito, is the primary vector for dengue and yellow fever. It is also considered to be the principal vector for chikungunya in Asia, and can transmit pathogens for diseases like filariasis, encephalitis etc. Mosquitoes are anautogenous, a vertebrate blood meal is necessary for the maturation of each batch of their eggs. It is during blood feeding that the mosquitoes ingest and transmit the pathogens from and to the human body. Their reproductive cycle can distinctly be separated into pre-vitellogenic (pre blood meal) and vitellogenic (post blood meal) periods. The pre-vitellogenic period, or the maturation phase, during which the mosquito becomes competent for host-seeking behavior, blood digestion, and egg development, is controlled by juvenile hormone III (JH). Whereas, the vitellogenic period, during which the fat body, a functional analogue of the vertebrate liver, produces massive amounts of yolk protein precursors for the oocytes, is under the influence of 20-hydroxyecdysone (20E). Microarray analysis of gene expression during nine different times points, post blood meal (PBM), ranging from 3h to 72h, show that, 7729 genes are differentially expressed by ≥ 1.75 folds when compared to their expression at 72h post eclosion (PE). Hierarchical clustering of these genes placed them in 12 different clusters, which can be consolidated into three broad ones, based on their expression profiles, viz. early-PBM (EPBM), mid-PBM (MPBM) and late-PBM (LPBM). The EPBM genes show high expression during 3h to 12h PBM, whereas, the MPBM and LPBM genes are expressed highly during 18h to 36h and 48h to 72h PBM, respectively. KEGG analyses for gene ontology have shown clear functional separation between the clusters. A striking correlation between the clusters and the different titers of 20E (which peaks at 18h and then subsides by 30h PBM), during this period (3h – 72h PBM) can also be noticed. While, the MPBM genes are up-regulated at the high titer of 20E, the EPBM and the LPBM genes show an inverse trend. Our goal is to determine the effects of 20E on the EPBM, MPBM and the LPBM genes. We are also using 2kb upstream regions of 200 highly expressed and co-regulated genes in each of the different clusters for the identification of putative transcription factor binding sites, by de-novo motif discovery. This study should provide a better understanding of the complex gene regulatory mechanism during the different time points in a gonadotrophic cycle of the female Aedes aegypti. Title: Comparative genomics of peptide hormone receptors from five dipteran genomes.

Abstract: Peptide hormones play essential roles in coordinating development, reproduction and maintaining homeostasis in insects. These hormones rely on membrane-bound receptors to transmit signals to target tissues and potentiate a response. These receptors are predominated by three protein families: G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and receptor guanylyl cyclases (GCCs). Comprehensive analysis of the distribution and evolutionary relationships of these receptors has been limited to a small number of related proteins across a broad spectrum of animals or to a large group of receptors within a single species. A phylogenetic analysis of a large group of peptide hormone receptors across deep evolutionary divides could help to inform understanding of these important proteins. We implemented phylogenetic methods to examine the relationship of GPCRs, RTKs, and GCCs across five dipteran genomes – Drosophila melanogaster, D. mojavensis, Aedes aegypti, Anopheles gambiae and Culex quinquefaciatus, which encompassing over half a billion years of evolution. Our analyses reveal relative stasis among RTKs and GCCs, while demonstrating frequent lineage-specific gains and losses in the GPCRs. Moreover, we demonstrate that major divisions within receptor types correspond to conservation of ligand type. These data should aid in attempts to further de-orphanize receptors by narrowing the potential targets for experimental investigation. Additionally, the patterns of receptor gain and loss may provide clues to important lineage specific hormonal processes. Finally, the data generated will help in improving genome annotation by clarifying the relationships of orthologous and paralogous receptors in dipteran genomes. The juvenile hormone receptor Methoprene-Tolerant (Met) controls gene expression dynamics in the yellow fever mosquito, Aedes aegypti

Tusar T. Saha, Zhen Zou, Sourav Roy, Sang Woon Shin, Tyler W. H. Backman, Thomas Girke, Kevin P. White and Alexander S. Raikhel.

Mosquitoes are vectors of devastating diseases worldwide. They require vertebrate blood for their egg maturation and therefore, an in depth understanding of mosquito reproductive biology can lead to developing novel vector control strategies. One of key aspects of adult female development is the post eclosion (PE) period that is controlled by juvenile hormone III (JH). This JH-controlled PE period is required for post blood feeding reproductive events controlled by 20- hydroxyecdysone. Our recent research efforts have been focused on deciphering the underlying molecular mechanism in JH action in A. aegypti. We performed a detailed temporal gene expression analysis of PE fat-body (FB) using microarray technology. Hierarchical clustering of the differentially expressed genes revealed three distinct expression patterns: early-PE (EPE) genes maximally expressed at 6 h PE, mid-PE (MPE) genes at 24 h PE, and late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for Met showed that 27% of EPE and 40% of MPE genes were up-regulated whereas 36% of LPE genes were down-regulated in the absence of this receptor. Met repression of EPE and MPE and activation of LPE genes were validated by an in vitro fat-body culture experiment using Met RNAi. The clusters were also found to be functionally distinct, with carbohydrate, lipid, and xenobiotics metabolism belonging to the EPE and MPE clusters and transcription and translation to the LPE cluster. Sequence motif analysis revealed the consensus 9-mer Met-binding motif, CACGC/TGA/GT/AG. Met-binding motif variants were overrepresented within the first 300 bases of the promoters of Met RNAi–down- regulated (LPE) genes but not in Met RNAi–up-regulated (EPE) genes. EMSAs using a combination of mutational and anti-Met antibody super-shift analyses confirmed the binding properties of the Met consensus motif variants. This study represents a significant advancement in the understanding of the regulation of gene expression by JH and its receptor Met during female mosquito reproduction. Binding of Methoprene‐tolerant Protein to Juvenile Hormone Response Element

Jinsong Zhu and Meng Li

Department of Biochemistry, Virginia Tech

Accumulating evidence supports the notion that the Methoprene‐tolerant (Met) gene product is a juvenile hormone (JH) receptor. Our previous study of Aedes aegypti has shown that activation of JH target genes require Met and its protein partner, FISC/TAI/SRA. The Met‐FISC complex is associated with the JH responsive promoters in vivo when JH titers increase after eclosion. While JH response elements are identified from a handful of genes, the DNA binding properties of Met have not been fully characterized.

Using purified recombinant proteins, our current work indicates that Met alone can’t bind the JH response element identified from the mosquito early trypsin gene. DNA binding of Met requires FISC and the presence of JH. As shown in other bHLH‐PAS transcription factors, the conserved basic amino acid residues in the first α‐helix of the bHLH domains of Met and FISC are essential for the sequence‐specific DNA binding. This result suggests that FISC acts as a DNA binding partner of Met, not as a coactivator.

The consensus binding site for the Met‐FISC complex is determined from an unbiased set of degenerate oligonucleotides using CASTing (cyclic amplification and selection of targets). The core sequence is GCACGTGY, well conserved in the JH response elements identified from the early trypsin gene and the kruppel homologue 1 gene. This study illustrates how the JH receptor functions as a transcriptional activator. A Single Amino Acid Substitution in Methoprene-Tolerant (Met) Alters Juvenile Hormone Use by Insects and Crustaceans

Hitoshi Miyakawa1, Kenji Toyota1, Ikumi Hirakawa1, Yukiko Ogino1, Shinichi Miyagawa1, Shigeto Oda1, Norihisa Tatarazako2, Toru Miura3, John K. Colbourne4 and Taisen Iguchi1

1Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences

2Environmental Quality Measurement Section, Research Center for Environmental Risk, National Institute for Environmental Studies

3Laboratory of Ecological Genetics, Graduate School of Environmental Science, Hokkaido University

4School of Biosciences, University of Birmingham

Juvenile hormone (JH) is an essential regulator of major developmental and life history events in arthropods. Most of insects use JH III as the innate JH ligand. By contrast, crustaceans use methyl farnesoate (MF). Despite this difference that is tied to their deep evolutionary divergence, the process of this ligand transition is unknown. Here we show that a single amino acid substitution plays an important role during evolution of the arthropod JH pathway. We found that microcrustacea Daphnia pulex and D. magna share a JH signal transduction pathway with insects, involving Methoprene-tolerant (Met) and Steroid Receptor Co-activator (SRC) proteins that form a heterodimer in response to various juvenoids. JH-binding pockets of the orthologous genes differ by only 2 amino acids, yet a single amino acid substitution within Daphnia Met enhances the receptor’s responsiveness to the juvenoid used by insects. These results indicate that this single mutation within an ancestral insect lineage contributed to the evolution of a JH III receptor system.

20-hydroxyecdysone regulated cellular trafficking in the larval salivary gland of Drosophila melanogaster Kathryn M. Lantz1, Benjamin F. B. Costantino1, John R. Merriam2, and Andrew J. Andres1 1 School of Life Science, University of Nevada, Las Vegas, Las Vegas, Nevada 2 Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California

The salivary gland of Drosophila melanogaster synthesizes and secretes several glue polypeptides that function to adhere a pupa to a surface during metamorphosis. The steroid hormone, 20-hydroxyecdysone (20E), regulates the synthesis and secretion of glue polypeptides. After glue synthesis, the polypeptides are sorted and packaged into secretory vesicles. The movement of vesicles and eventually the secretion of the glue cargo is a complex interplay of many proteins involved in cellular trafficking, yet how 20E impacts this process is largely unknown. We have observed that the overexpression of Guanosine diphosphate Dissociation Inhibitor (GDI) causes a secretion defect by completely blocking the exocytosis of glue. This is a complex phenotype resulting in the formation of irregular shaped secretion vesicles that fail to acidify, and an expansion of the endoplasmic reticulum is seen within these cells. Because there is only one GDI in the Drosophila genome, we have focused on downstream secretory molecules; specifically the Rab GTPases thought to be regulated by GDI. We have thus far identified the localization of 31 Rabs in the salivary gland during three different developmental time points. In addition, next generation RNA sequencing data that we have collected suggests that only 20 of the 31 Rabs are endogenously expressed in the salivary gland, with perhaps 13 being induced by 20E and 3 repressed. Using UAS-transgenes from the Drosophila community, we have tested the function of all 31 Rabs for the affects on secretion using dominant-negative, constitutive-active, and RNAi-hairpin transgenes. The analysis of these results indicates an important role by Rab 1, Rab11, Rab30 and Rab35 in this tissue specific, 20E-regulated secretion pathway. The role of hormone nuclear receptor DHR96 in ageing in Drosophila melanogaster

Sonita Afschar1, Dan Magner1, Matthew Piper2, Adam Antebi1, Linda Partridge1,2

1Max Planck Institute for Biology of Ageing, Cologne, Germany 2Institute of Healthy Ageing, University College London, England

The insulin/insulin-like growth factor like signaling (IIS) pathway is involved in lifespan regulation of different organisms. In C. elegans, D. melanogaster and M. musculus, a reduction in IIS signalling leads to lifespan extension. This effect, however, is blocked in the absence of the transcription factor dFOXO in Drosophila. Furthermore, only xenobiotic metabolism is affected, but not body size, fecundity or oxidative stress resistance, which are altered in IIS- compromised animals, independently of dFOXO. Therefore it is suspected that the detoxification process plays a key role in longevity. In addition, many detoxification genes which are upregulated in long-lived IIS mutants are enriched for the putative binding site of the hormone nuclear receptor DHR96. The findings that DHR96 is a direct target of dFOXO, and that the DHR96 orthologue DAF-12 modulates longevity in C. elegans, further suggests an important role of this nuclear receptor in ageing.

DHR96 protein is detected predominantly in tissues that metabolize xenobiotic compounds: the malphigian tubules, fat body, salivary glands and gastric caeca of the midgut. In addition to the xenobiotic response, DHR96 functions in Triacylglycerol homeostasis (TAG) and cholesterol metabolism. DHR96 null mutants are short-lived, and characterized by decreased xenobiotic resistance. We have shown that ubiquitous overexpression of wild type as well as ligand- insensitive DHR96 increases resistance to the xenobiotic DDT, which is a key phenotype of long-lived IIS mutants. Moreover, these animals are long lived compared to controls. Thus DHR96 is a key regulator of longevity, downstream of FOXO and IIS. Further experimental approaches are targeted to discover how this transcription factor functions, and is regulated.

SONITA AFSCHAR MARK BROWN ROBERT-KOCH-STRASSE 21 DEPT ENTOMOLOGY, BIOSCI BLDG COLOGNE, GERMANY 120 CEDAR ST [email protected] ATHENS, GA 30602 [email protected]

ANDREW ANDRES ANA CABRERA UNLV SCHOOL OF LIFE SCIENCES 1700 SW 23RD DRIVE 4505 MARYLAND PARKWAY GAINVESVILLE, FL 32608 LAS VEGAS, NV 89154-4004 [email protected] [email protected] VIVIANE CALLIER MASAKO ASAHINA 1811 E APACHE BLVD 600 16TH ST. APT 3099 UCSF MISSION BAY GH-S574 TEMPE, AZ 85281 SAN FRANCISCO, CA 94158 [email protected] [email protected] KATHERINE CROCKER HUA BAI 830 NORTH UNIVERSITY AVE 34 OLIVE ST. BMC502 ANN ARBOR, MI 48109-1048 PROVIDENCE, RI 02912 [email protected] [email protected] TAKAAKI DAIMON ROSA BARRIO OWASHI 1-2 BIZKAIA TECHNOLOGY PARK TSUKUBA BUILDING 801A TSUKUBA, JAPAN DERIO, SPAIN [email protected] [email protected] SUSSIE DALVIN ARASH BASHIRULLAH NORDNESGATEN 50 777 HIGHLAND DRIVE BERGEN, NORWAY MADISON, WI 53705 [email protected] [email protected] E. THOMAS DANIELSEN AARON BAUMANN UNIVERSITETSPARKEN 15 19700 HELIX DR. BYGNING 3 ASHBURN, VA 20147 COPENHAGEN, DENMARK [email protected] [email protected]

XAVIER BELLES WU-MIN DENG INSTITUT DE BIOLOGIA EVOLUTIVA FLORIDA STATE UNIVERSITY PASSEIG MARíTIM 37, 08003 BARC DEPARTMENT OF BIOLOGICAL BARCELONA, SPAIN SCIEN [email protected] TALLAHASSEE, FL 32306-4295 [email protected]

DAVID DOLEZEL SAMANTHA HINDLE ENTU, BIOLOGY CENTER, ASCR,V.V 600 16TH STREET CZ60077344 GENENTECH HALL, S302A 37005 CESKE BUDEJOVI, CZECH SAN FRANCISCO, CA 94158 REPUBLIC [email protected] [email protected] KIYOSHI HIRUMA SORA ENYA FACULTY OF AGRICULTURE AND RYUSUKE NIWA'S LAB, TENNOUDAI LIF TSUKUBA, JAPAN HIROSAKI UNIVERSITY [email protected] HIROSAKI, JAPAN [email protected] THOMAS FLATT UNIVERSITY OF LAUSANNE MASATOSHI IGA DEPARTMENT OF ECOLOGY AND 5-1-5 KASHIWANOHA, KASHIWA EVOL CHIBA 277-8562 LAUSANNE, SWITZERLAND KASHIWA, JAPAN [email protected] [email protected]

ARPAN GHOSH FUMIHIKO IGARASHI 321 CHURCH ST. SE KASHIWANOHA 5-1-5 6-160 JACKSON HALL KASHIWA, JAPAN MINNEAPOLIS, MN 55455 [email protected] [email protected] HIROSHI ISHIMOTO REWATEE GOKHALE E324 GRADUATE SCHOOL OF 240 NATURAL SCIENCES, SCIENC MICHIGAN STATE UNIVERSITY FURO-CHO, CHIKUSA-KU EAST LANSING, MI 48824 NAGOYA, JAPAN [email protected] [email protected]

SHI-HONG GU ERIN JIMENEZ DEPARTMENT OF ZOOLOGY, 3400 NORTH CHARLES STREET NATIONA 302 MUDD HALL TAICHUNG, TAIWAN BALTIMORE, MD 21218 [email protected] [email protected]

VINCENT HENRICH MAREK JINDRA 3704 MHRA BUILDING BIOLOGY CENTER ASCR GREENSBORO, NC 27402 CESKE BUDEJOVICE, CZECH [email protected] REPUBLIC [email protected] RONALD HILL CAFHS PO BOX 52 DAVY JONES NORTH RYDE, AUSTRALIA 304 MORGAN BIOLOGY BLDG [email protected] LEXINGTON, KY 40506-0225 [email protected] GRACE JONES JOLIENE LINDHOLM 304 MORGAN BIOLOGY BLDG 1630 LINDEN DR LEXINGTON, KY 40506-0225 ROOM 740 [email protected] MADISON, WI 53706 [email protected] HIROSHI KATAOKA KASHIWANOHA5-1-5 JOSE MAESTRO KASHIWA, JAPAN PG MARITIM BARCELONETA 37-49 [email protected] BARCELONA, SPAIN [email protected] YONGGYUN KIM MAJOR IN PLANT MEDICINE, DAVID MARTíN SCHOO PASSEIG MARíTIM DE LA BARCELON 388 SONGCHON-DONG BARCELONA, SPAIN ANDONG, KOREA, REPUBLIC OF [email protected] [email protected] ZOFEYAH MCBRAYER KIRST KING-JONES 6-160 JACKSON HALL G504 BIOLOGICAL SCIENCES BLDG 321 CHURCH ST. SE EDMONTON, AB T6H 5V3 MINNEAPOLIS, MN 55455 [email protected] [email protected]

TAKETOSHI KIYA MEGHA MEGHA KAKUMA-MACHI, KANAZAWA, LAB 14, NCBS-TIFR ISHIKA GKVK CAMPUS KANAZAWA, JAPAN BANGALORE, INDIA [email protected] [email protected]

TAKASHI KOYAMA HITOSHI MIYAKAWA RUA DA QUINTA GRANDE, 6 5-1, HIGASHIYAMA, MYODAIJI OEIRAS, PORTUGAL OKAZAKI, JAPAN [email protected] [email protected]

KATHRYN LANTZ LINDSAY MOSS-TAYLOR 4505 S. MARYLAND PKWY 414 7TH AVE SE C207 MAIL STOP 4004 MINNEAPOLIS, MN 55414 LAS VEGAS, NV 89154-4004 [email protected] [email protected] MORTEN E. MØLLER SHENG LI UNIVERSITETSPARKEN 15 CENSORED BY SANITIZE: BYGNING 3 STANDARD COPENHAGEN, DENMARK SHANGHAI, CHINA [email protected] [email protected]

RYUSUKE NIWA SUBBA PALLI TENNOUDAI 1-1-1 S225 AG. SCIENCE N TSUKUBA, JAPAN ENTOMOLOGY [email protected] LEXINGTON, KY 40504 [email protected]

MARYJANE O'CONNOR XUEYANG PAN 321 CHURCH ST SE 6-160 JACKSON HALL 6-160 JACKSON HALL 321 CHURCH ST. SE MINNEAPOLIS, MN 55455 MINNEAPOLIS, MN 55455 [email protected] [email protected]

MICHAEL O'CONNOR BRIAN PHELPS 564 OWASSO HILLS DR. 5 ESCALLIER PL. ROSEVILLE, MN 55113 ST. ALBERT, AB T8N 5T1 [email protected] [email protected]

TAKAHIRO OHDE VICTORIA POCIUS 334 EAST 25TH STREET #402 2411 LOUISIANA ST. NEW YORK, NY 10010 L143 [email protected] LAWRENCE, KS 66046 [email protected] YUYA OHHARA SHIZUOKA 422-8526 ALEXANDER RAIKHEL SHIZUOKA, JAPAN 115 SWEETWOOD COURT [email protected] RIVERSIDE, CA 92506 [email protected] NAOKI OKAMOTO 2-2-3 MINATOJIMA-MINAMIMACHI, KIM REWITZ KOBE, JAPAN UNIVERSITETSPARKEN 15 [email protected] COPENHAGEN, DENMARK [email protected] HAJIME ONO KITASHIRAKAWAOIWAKE-CHO LYNN RIDDIFORD SAKYO-KU 19700 HELIX DRIVE KYOTO, JAPAN ASHBURN, VA 20175 [email protected] [email protected]

QIUXIANG OU EREZ ROMI G502 BIOLOGICAL SCIENCES BLDG. MANDELBLAT 1 UNIVERSITY OF ALBERTA HERZLIYA, ISRAEL EDMONTON, AB T6G2E9 [email protected] [email protected]

SOURAV ROY REBECCA SPOKONY 337 ENTOMOLOGY BUILDING 900 E 57TH STREET UNIVERSITRY OF CALIFORNIA, RIV CHICAGO, IL 60637 RIVERSIDE, CA 92346 [email protected] [email protected] YUICHIRO SUZUKI DEPARTMENT OF BIOLOGICAL SCIEN TUSAR SAHA WELLESLEY COLLEGE 1122 WEST LINDEN STREET WELLESLEY, MA 02481 APT # 205 [email protected] KOLKATA, CA 92507 [email protected] JASON TENNESSEN 15 N 2030 EAST LIV SANDLUND SALT LAKE CITY, UT 84112 CENSORED BY SANITIZE: [email protected] STANDARD BERGEN, NORWAY MICHAEL TEXADA [email protected] 19700 HELIX DRIVE ROOM 3E.235 HIROKO SANO ASHBURN, VA 20147 1-1 HYAKUNEN-KOEN [email protected] KURUME, FUKUOKA, JAPAN [email protected] JAMES TRUMAN 19700 HELIX DRIVE TETSURO SHINODA JANELIA FARM RESEARCH CAMPUS 1-2, OOWASHI ASHBURN, VA 20147 NATIONAL INSTITUTE OF [email protected] AGROBIOL TSUKUBA, JAPAN HITOSHI UEDA [email protected] TSUSHIMA-NAKA 3-1-1 OKAYAMA, JAPAN TAKAHIRO SHIOTSUKI [email protected] 1-2 OWASHI TSUKUB, JAPAN AMBUJ UPADHYAY [email protected] 315 5TH ST APT 104 GUY SMAGGHE MINNEAPOLIS, MN 55414 COUPURE LINKS 653 [email protected] GHENT, BELGIUM [email protected] KEVIN VOGEL 413 BIOLOGICAL SCIENCES VLASTIMIL SMYKAL ATHENS, GA 30602 BRANISOVSKA 31 [email protected] CESKE BUDEJOVICE, CZECH REPUBLIC [email protected] NAOKI YAMANAKA 6-160 JACKSON HALL 321 CHURCH ST. SE MINNEAPOLIS, MN 55455 [email protected]

XIAO-FAN ZHAO NO 27, SHANDA SOUTH ROAD SCHOOL OF LIFE SCIENCES JINAN, CHINA [email protected]

JINSONG ZHU 313 ENGEL HALL BLACKSBURG, VA 24061 [email protected]