Molecular Biology of bHLH PAS Genes Involved in Dipteran Juvenile Hormone Signaling Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Aaron A. Baumann. B.S. Graduate Program in Entomology The Ohio State University 2010 Dissertation Committee: Thomas G. Wilson, Advisor David Denlinger H. Lisle Gibbs Amanda Simcox Copyright by Aaron A. Baumann 2010 Abstract Methoprene tolerant (Met), a member of the bHLH-PAS family of transcriptional regulators, has been implicated in juvenile hormone (JH) signaling in Drosophila melanogaster. Met mutants are resistant to the toxic and morphogenetic defects of exogenous JH application. A paralogous gene in D. melanogaster, germ cell expressed (gce), forms JH-sensitive heterodimers with MET, but a function for gce has not been reported. DmMet orthologs from three mosquito species are characterized and, based on sequence analysis and intron position, are shown to have higher sequence identity to Dmgce than to DmMet. An evolutionary scheme for the origin of Met from a gce-like ancestor gene in lower Diptera is proposed. RNAi-driven underexpression of Met in the Yellow Fever mosquito, Aedes aegypti, results in the concomitant reduction of putative JH-inducible genes, suggesting involvement in JH signaling. The viability of D. melanogaster Met mutants is thought to result from functional redundancy conferred by gce. Therefore, genetic manipulation of gce expression was used to probe the function of this gene. Overexpression of gce was shown to alleviate preadult, but not adult Met phenotypes. RNAi-driven underexpression of gce resulted in ii preadult lethality in both Met+ and Met mutant backgrounds. Therefore, unlike Met, gce is a vital gene. Evolutionary analysis of 12 Met and gce orthologs showed that these genes are conserved across the genus Drosophila. Additionally, distinct signatures of selective pressure were identified in Met and gce via dN/dS analysis. The paucity of introns in Met relative to gce supports the notion of a retrotransposition mechanism of duplication, through which Met arose from a gce-like ancestor following the divergence of higher and lower Diptera. Furthermore, RT-PCR analysis revealed discrete embryonic expression profiles for Met and gce. Together, these results show a degree of post-duplication subfunctionalization. iii Dedication To my parents and grandfather for their love and encouragement. To my wife, Rachelle, for her patience during my pursuit of this endeavor. iv Acknowledgements I am indebted to my major advisor, Dr. Thomas Wilson, for his guidance and support throughout my graduate studies. When my confidence was shaken during bouts of frustrating results, a brief consultation with Tom allowed me to regain my poise, to try again, and ultimately to succeed. His mentorship afforded me valuable opportunities to explore and grow within the laboratory, and encouraged development of a self-motivated attitude that will no doubt serve me in the future. I thank the members of my dissertation committee, Drs. Dave Denlinger, Amanda Simcox, and Lisle Gibbs, for their comments and suggestions during committee meetings. Additionally, I extend thanks to Dr. Woodbridge Foster for guiding me through the process of rearing mosquitoes and for allowing me to maintain A. aegypti colonies in his laboratory space. I am extremely grateful for Dr. Shaoli Wang’s instruction in various molecular techniques that were instrumental to my ability to design and perform much of the work presented herein. Finally, I would like to thank Dr. Joshua Benoit for his assistance and encouragement, and my good friend Chris Herman for helping me with various manual tasks. v Vita March 30, 1982……………………………………. Born-West Allis, Wisconsin 2000………………………………………………... Centerville High School 2003-2004………………………………….. ……... Student Research Assistant The Ohio State University 2004……………………………………………….. B.S., The Ohio State University 2005-2010…………………………………………. Graduate Teaching / Research Associate The Ohio State University Publications 1. Baumann, A., Wilson, T.G., Barry, J., Wang, S. Juvenile hormone action requires paralogous genes in Drosophila melanogaster. Genetics. 185:1327-1336. 2. Baumann, A., Fujiwara, Y., Wilson, T.G. 2010. Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila. Journal of Insect Physiology. 56:1445-1455. 3. Wang, S., Baumann, A., Wilson, T.G. Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: members of PAS transcriptional factor family. Journal of Insect Physiology. 2007; 53:246–253. Field of Study Major field: Entomology vi Table of Contents Abstract…………………………………………………………………….. ……... ii Dedication………………………………………………………………….. ……... iv Acknowledgements………………………………………………………………… v Vita…………………………………………………………………………………. vi List of Tables………………………………………………………………………. vii List of Figures……………………………………………………………………… ix Chapters 1. Introduction………………………………………………………… ……... 1 Juvenile hormone in insect growth and development……………… 1 Juvenile hormone in insect reproduction…………………………... 3 Juvenile hormone structure………………………………………… 4 Synthesis and metabolism………………………………………….. 6 JH agonists: insecticidal use……………………………………….. 7 Molecular mechanisms of JH signal transduction…………………. 9 Molecular crosstalk between 20E and JH signaling pathways…….. 13 Directions………………………………………………………….. 16 Research goals……………………………………………………... 16 References………………………………………………………….. 18 Illustrations………………………………………………………… 24 2. Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: Members of PAS transcriptional factor family………………………………………………………………. ……....26 Abstract…………………………………………………………….. 26 Introduction………………………………………………………… 27 Materials and Methods…………………………………………….. 30 Results……………………………………………………………… 35 Discussion………………………………………………………….. 39 References………………………………………………………….. 43 vii Illustrations………………………………………………………… 47 3. Paralogous Genes Involved in Juvenile Hormone Action in Drosophila melanogaster……………………………………………………………….. 57 Abstract…………………………………………………………….. 57 Introduction………………………………………………………… 58 Materials and Methods……………………………………………... 60 Results……………………………………………………………… 69 Discussion………………………………………………………….. 77 References………………………………………………………….. 83 Illustrations………………………………………………………….87 4. Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila………………. 98 Abstract…………………………………………………………….. 98 Introduction………………………………………………………… 99 Materials and Methods……………………………………………... 102 Results……………………………………………………………… 109 Discussion………………………………………………………….. 117 References………………………………………………………….. 122 Illustrations………………………………………………………….128 Conclusions………………………………………………………………………… 140 Appendix A: Revision of the genomic sequence of D. melanogaster gce………….147 Bibliography……………………………………………………………………….. 154 viii List of Tables Table Page 1.1 Representative table of some JH inducible genes in Drosophila melanogaster………………………………………………………………. 24 2.1 Gene-specific primers used for 5’ and 3’ RACE PCR amplification of mosquito Met homologs……………………………………………………………… 47 2.2 Amino acid identities among mosquito Met and Drosophila Met and gce genes…………………………………………………………… ……... 48 2.3 Primers used for RT-PCR analysis of dsMet and dsß-gal-injected Aedes aegypti…………………………………………….. 49 3.1 Number of defective eye facets in gce overexpressing and underexpressing strains of D. melanogaster………………………………………………………. 87 3.2 Larval and pupal survival rates of gce-dsRNA strains of D. melanogaster………………………………………………….……... 88 3.3 Pupal survival (%) of gce-dsRNA strains of D. melanogaster…………….. 89 4.1 Flybase (http://www.FlyBase.org) annotation symbols for Met and gce orthologs of 12 species of Drosophila……………………………………... 128 4.2 Primer sequences used for RT-PCR of selected gce orthologs……………. 129 4.3 Sequence identity matrices for gce and CG15032 orthologs in 12 species of Drosophila………………………………………………………………….130 4.4 Sequence identity matrices calculated from the amino acid sequences of functional domain and open reading frames of Met and gce orthologs from D. melanogaster, three mosquito species, and Tribolium castaneum….…….. 131 ix List of Figures Figure Page 1.1 Representative juvenile hormones and juvenile hormone analogs………… 25 2.1. Multiple alignment of mosquito Met and D. melanogaster Met and gce amino acid sequences………………………………………………………. 50 2.2 RT-PCR products amplified from D. melanogaster and Aedes aegypti using degenerate or D. melanogaster Met-specific primers……………….. 53 2.3 Phylogenetic tree produced using Met and gce amino acid sequences from members of Culicidae and Drosophilidae………………………………….. 54 2.4 Relative expression of putative JH-inducible genes in dsMet and dsß-gal-injected A. aegypti………………………………………………… 55 2.5 Developmental expression profile of AaMet………………………………. 56 3.1 Northern blot analysis of the developmental expression of gce during D. melanogaster development………………………………………………… 90 3.2 RT-PCR analysis of Met and gce in selected tissues of D. melanogaster………………………………………………….…………91 3.3 Expression of gce in UAS-gce-overexpressing flies………………………. 92 3.4 Enhanced methoprene pupal toxicity of gce-overexpressing flies………… 93 3.5 Rescue of resistance to methoprene-induced male genitalia malrotation during pupal development in gce-overexpressing flies …………………… 94 3.6 Rescue of eye phenotype in Metw3 flies overexpressing