MOLECULAR CHARACTERIZATION OF ADULT DIAPAUSE IN THE NORTHERN HOUSE MOSQUITO, CULEX PIPIENS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Rebecca M. Robich, M.S. ***** The Ohio State University 2005 Dissertation Committee: Professor David L. Denlinger, Advisor Approved by Professor Donald H. Dean ________________________ Professor Glen R. Needham Advisor Graduate Program in Entomology Professor Brian H. Smith ABSTRACT In the northern United States, Culex pipiens (L.), a major avian vector of several arthropod-borne viruses, spends a good portion of the year in a state of developmental arrest (diapause). Although the physiological and hormonal aspects of Cx. pipiens diapause have been well-documented, there is little known on the molecular aspects of this important stage. Using suppressive subtractive hybridization (SSH), 40 genes differentially expressed in diapause were identified and their expression profiles were probed by northern blot hybridization. These genes have been classified into 8 distinct groupings: regulatory function, food utilization, stress response, metabolic function, cytoskeletal, ribosomal, transposable elements, and genes with unknown functions. Among 32 genes confirmed by northern blot hybridization, 6 are upregulated specifically in early diapause, 17 are upregulated in late diapause, and 2 are upregulated throughout diapause. In addition, 2 genes are diapause downregulated and 5 remained unchanged during diapause. Two regulatory genes upregulated in late diapause, ribosomal protein (rp) S3A and rpS6, are of particular interest for their potential involvement in developmental arrest. In other mosquito species, these genes are upregulated prior to oogenesis, and their suppression leads to a disruption in ovarian development. Since arrested ovarian development is a key characteristic of Cx. pipiens diapause, the lack of expression in early diapause may be key to this developmental ii arrest. Of equal interest is the upregulation of two stress-response genes in late diapause. The molecular chaperone, heat shock protein 23 (hsp23) is slightly upregulated in late diapause and may be involved in protecting females from environmental stresses. In addition, a gene encoding an enzyme involved in oxidizing certain insecticides in insecticide-resistant strains of Cx. quinquefasciatus, aldehyde oxidase, is strongly upregulated in Cx. pipiens late diapause. SSH and northern blot analysis also demonstrated the strong upregulation of a muscle-specific actin gene in Cx. pipiens early diapause. Although the upregulation of this cytoskeletal gene in early diapause may seem counterintuitive, females actively fly during diapause preparation in search of sugar meals and a protective site for overwintering. In addition, two ribosomal genes (large ribosomal subunit L18 and large ribosomal subunit) are upregulated in early diapause suggesting that their function is restricted to diapause preparation. Curiously, two genes encoding transposable elements, transposon T1-2 and Mimo-Cp2, are upregulated during late diapause in Cx. pipiens, but the function of these genes during diapause is unknown. Nine genes were also isolated by SSH with unknown identities, and all but one are upregulated in late diapause, as identified by northern blot hybridization In addition to the aforementioned genes, two genes encoding the blood digestive enzymes, trypsin and chymotrypsin-like, are downregulated in early diapause, and concurrently a gene associated with the accumulation of lipids, fatty acid synthase, is highly upregulated. As females enter diapause, fatty-acid synthase is only sporadically expressed, while the expression of trypsin and chymotrypsin-like remain undetectable iii until late diapause, when females prepare to take a blood meal upon diapause break. This is the first molecular evidence demonstrating that diapause in Cx. pipiens evokes a molecular switch from blood-feeding in nondiapausing individuals to sugar feeding in diapause-destined females. The increase in sugar feeding in diapause-destined Cx. pipiens and subsequent accumulation of lipid reserves likely requires a high amount of energy. This may be reflected in the upregulation of genes encoding two respiratory enzymes, cytochrome c oxidase subunit I (COI) and cytochrome c oxidase subunit III (COIII), during diapause preparation. Although transcript levels decline in mid-diapause when females enter an inactive state, levels again rise in late diapause, just prior to diapause break. There are no differences in mtDNA levels between nondiapausing and diapausing Cx. pipiens, thus suggesting that mitochondrial numbers are not reduced during diapause. Regulation of COI and COIII is thus likely to be under transcriptional control. In addition to genes obtained through SSH, heat shock protein 70 was isolated from Cx. pipiens by 5- and 3- rapid amplification of cDNA ends. This gene is upregulated upon heat shock and recovery from cold shock in diapausing and nondiapausing females, but is not upregulated as a component of the diapause program. However, diapausing females reared at 18°C survive cold exposure (-5°C) nearly twice as long as its nondiapausing counterparts reared at 18°C and 10 times as long as nondiapausing mosquitoes reared at 25°C. Diapausing females are also more desiccation resistant (1.6 to 2 fold) than nondiapausing females, regardless of rearing temperature. iv These results may be useful when comparing the molecular aspects of diapause across different taxa and developmental stages. In addition, this work may be helpful in understanding the transseasonal maintenance of viruses that utilize overwintering insects. v For Michael vi ACKNOWLEDGMENTS I am grateful to my advisor, David L. Denlinger, Ph.D., for his intellectual guidance and continued support for this project, and for his assistance in editing each chapter. I would like to thank my committee members, Drs. Donald H. Dean, Glen R. Needham, and Brian H. Smith, for their ideas and comments in bettering this thesis. I am also grateful for the assistance of Joseph P. Rinehart, Ph.D., and Linda Kitchen in helping to make this dissertation come together. I would like to thank those involved in the surveillance of West Nile virus in overwintering mosquitoes, including Richard Gary and Robert A. Restifo of the Ohio Department of Health's Vector-Borne Disease Program, and Joseph Lynch of the Cuyahoga County Board of Health. I appreciate the efforts of Woodbridge Foster, Ph.D., in helping to establish the Culex pipiens (Buckeye strain) colony, who collected the original larvae from his backyard in September 2000. vii VITA September 20, 1974 .…....... Born – Painesville, Ohio 1992 ..…………………..… Bachelor of Science, Environmental Biology Ohio University 1997-1998 .………………. Graduate Teaching Associate Ohio University 1998 .……………………... Master of Science, Environmental Studies Ohio University 1999-2000 .………………. Pre-Doctoral Fellow Virology Division U.S. Army Medical Research Institute of Infectious Diseases 2000-2004 .………………. Graduate Teaching and Research Associate The Ohio State University 2001 .……………………... Medical Entomology Summer Intern Ohio Department of Health PUBLICATIONS Moll, R.M., Romoser, W.S., Modrzakowski, M.C, Moncayo, A.C., and Lerdthusnee, K. 2001. Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. Journal of Medical Entomology 38:29-32. Romoser, W.S., Moll, R.M., Moncayo, A.C., and Lerdthusnee, K. 2000. The occurrence and fate of the meconium and meconial peritrophic membranes in pupal and adult mosquitoes (Diptera: Culicidae). Journal of Medical Entomology 37:893-6. FIELDS OF STUDY Major Field: Entomology viii TABLE OF CONTENTS Abstract ………………………………………………………………………………….. ii Dedication ………………………………………………………………………………. vi Acknowledgements …………………………………………………………………...... vii Vita ……………………………………………………………………………………. viii List of Tables …………………………………………………………………………… xi List of Figures ………………………………………………………………………….. xii Chapters: 1. Introduction ……………………………………………………………………… 1 References ……………………………………………………………… 12 2. Diapause-specific gene expression in adults of the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization. Abstract ………………………………………………………………… 16 Introduction …………………………………………………………….. 17 Materials and Methods …………………………………………………. 19 Results ………………………………………………………………….. 23 Discussion ……………………………………………………………… 27 References ……………………………………………………………… 38 3. Diapause in the mosquito Culex pipiens evokes a metabolic switch that shuts down genes encoding blood digestive enzymes and upregulates a gene associated with sugar utilization and lipid storage. Abstract ………………………………………………………………… 49 Introduction …………………………………………………………….. 50 Materials and Methods …………………………………………….…… 52 Results ……………………………………………………………….…. 57 Discussion ……………………………………………………………… 63 References ……………………………………………………………… 69 ix 4. Downregulation of mitochondrial mRNA expression, but not mitochondrial number, during adult diapause in the northern house mosquito, Culex pipiens. Abstract ………………………………………………………………… 82 Introduction ………………………………………………………….…. 83 Materials and Methods ……………………………………………….… 85 Results ………………………………………………………………….. 90 Discussion ……………………………………………………………… 94 References ……………………………………………………………… 99 5. Enhanced cold and desiccation tolerance in diapausing adults of Culex pipiens, and a role for hsp70 in response