Genetic analysis of cold tolerance in Drosophila albomicans. Kotoha Isobe Department of Biological Sciences Tokyo Metropolitan University 2014 CONTENTS Page GENERAL INTRODUCTION ...... 1 LITERATURE CITED............ 4 Part I: Cold tolerance and metabolic rate increased by cold acclimation in Drosophila albomicans from natural populations 7 SUMMARY.......................................................... 8 INTRODUCTION 9 MATERIALS AND METHODS 13 RESULTS................................... 19 DISCUSSION 25 LITERATURE CITED 34 TABLES...................... 42 FIGURES 47 MENTARY 53 Part II: ansgenic analysis for identification of Drosophila albomicans genes response to cold acclimation 63 SUMMARY....................... 64 INTRODUCTION 65 MATERIALS AND METHODS 68 RESULTS................................... 74 DISCUSSION 79 LITERATURE CITED 85 TABLES...................... 92 FIGURES 95 SUPPLEMENTARY 105 GENERAL DISCUSSION 109 LITERATURE CITED.... 113 ACKNOWLEDGEMENTS 115 GENERAL INTRODUCTION Organisms inhabit various environments on the earth and have acquired characters that are needed to adapt to their living environment. Natural selection plays a central role in the acquisition process of such adaptive characters (Darwin, 1859), which is often referred to as adaptive evolution. Adaptive evolution is an important aspect of the evolution of organisms influenced by various environmental factors such as temperature, intensity of ultraviolet light, microbiota, etc. (e.g., Tanaka and Nei, 1989; Jablonskia and Chaplinb, 2000; Bonduriansky, 2001 for review). An objective of evolutionary genetics is to provide a genetic basis of adaptive evolution. In contrast to endothermic organisms, which have thermoregulatory systems to adjust their physiological conditions and functions, such as energy utilization, growth, reproduction, and locomotion, in response to changes in ambient temperature (Bullock, 1955; Johnston et al., 1996; Angilletta et al., 2002), in ectothermic organisms, physiological conditions are strongly influenced by the environmental temperature (Guschina and Harwood, 2006 for review). A correlation between distribution boundary in latitude and low temperature tolerance is frequently reported in many species (Stevens, 1992; Addo-Bediako et al., 2000; Hoffmann et al., 2003 for review), whereas there no such association is seen between latitude and high temperature tolerance in insect species (Addo-Bediako et al., 2000). Therefore, it is suggested that in ectothermic organisms, low temperature adaptation plays an important role in determining their habitat. However, in most of these reports on thermal adaptation, the authors examined adaptive traits already fixed in the populations. Therefore, it is still unclear how gene 1 and/or gene expression changes by natural selection have contributed to the adaptive evolution. It is important to study on-going adaptive evolution, which will bring a new prospect for better understanding the mechanisms of environmental adaptation by organisms at the molecular level. A fruit fly species, Drosophila albomicans belonging to the D. nasuta subgroup, had a wide geographic distribution in Southeast Asia until late 1980's (Kitagawa et al., 1982) but has expanded the distribution to Japanese archipelago in the temperate regions recently (Mikasa, 1991; Chen et al., 1994; Fujino et al., 2006; Hoshina et al., 2007), whereas its closely related species of the D. nasuta subgroup have stayed in the tropics or subtropics. This distribution expansion suggests the possibility that D. albomicans is in the process of adaptive evolution. According to Dobzhansky (1950), adaptation to lower temperatures especially in winter, is required for distribution expansion of Drosophila species from a subtropical to temperate zone. Therefore, it is likely that D. albomicans has undertaken an increase in cold tolerance to adapt to the lower temperature environment during the course of the in distribution expansion. In addition, it has been reported that cold acclimation that enhances the cold tolerance is one of the important factors in insects (Salt, 1961). Therefore, the molecular mechanism of cold acclimation response is a key to understand the cold adaptation of D. albomicans from tropical or subtropical habitats to temperate climate zone. In this thesis, for better understanding the molecular mechanisms of evolutionary adaptation of organisms, I investigated whether the distribution expansion of D. albomicans is attributable to adaptive evolution of cold tolerance and cold acclimation response, and their molecular mechanisms. In Part I, I examined the phenotypic variation in cold tolerance and cold acclimation response between geographical 2 populations of D. albomicans as well as between D. albomicans and its closely related species, and found that there were differences in the cold tolerance between the geographical populations of D. albomicans. I also found that the cold acclimation had a significantly strong effect on the cold tolerance. In addition, I detected a strong positive correlation between the cold tolerance change and the metabolic rate change in response to the cold acclimation, suggesting their strong physiological association by shared genetic factors. In Part II, using a transcriptome analysis and transgenic technique in D. melanogaster, I searched for the genes involved in the cold acclimation response to investigate the molecular mechanism of the cold tolerance improved by the cold acclimation. As the result, I found that Sdr and CG14153 genes, whose expression levels were significantly altered in response to the cold acclimation in D. albomicans, functioned in the transgenic D. melanogaster flies to improve the cold tolerance. 3 LITERATURE CITED Addo-Bediako, A., Chown, S. L., and Gaston, K. J. (2000) Thermal tolerance, climatic variability and latitude. Proc. Biol. Sci. 267, 739-745. Angilletta, M. J., Niewiarowski, P. H., and Navas, C. A. (2002) The evolution of thermal physiology in ectotherms. J. Therm. Biol. 27, 249-268. Bonduriansky, R. (2001) The evolution of male mate choice in insects: A synthesis of ideas and evidence. Biol. Rev. Camb. Philos. Soc. 76, 305-339. Bullock, T. H. (1955) Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30, 311-342. Chen, W., Zhang, J., Geng, Z., and Zhu, D. (1994) Invasion of Drosophila albomicans into Shanghai and areas nearby and a study on its mitochondrial DNA polymorphism. Yi Chuan Xue Bao 21, 179-187. Darwin, C. (1859) On the origin of species by means of natural selection. John Murray, London. Dobzhansky, T. (1950) Evolution in the Tropics. Am. Sci. 38, 209-221 Fujino, Y., Beppu, K., and Nakamura, H. (2006) Stratification of the Drosophilid assemblage in the research forest of AFC, Shinshu University. Bulletin Shinshu University Alpine Field Center 4, 47-55. Guschina, I. A., and Harwood, J. L. (2006) Mechanisms of temperature adaptation in poikilotherms. FEBS Lett. 580, 5477-5483. 4 Hoffmann, A. A., Sorensen, J. G., and Loeschcke, V. (2003) Adaptation of Drosophila to temperature extremes: bringing together quantitative and molecular approaches. J. Therm. Biol. 28, 175-216. Hoshina, H., Yamada, C., Uomi, H., and Terashima, M. (2007) Actual distribution of Drosophila (Drosophila) albomicans Duda, 1924 (Diptera: Drosophilidae) in Fukui pref., Honshu, Japan. Bulletin of The Fukui City Museum of Natural History 54, 79-82. Jablonskia, N. G., and Chaplinb, G. (2000) The evolution of human skin coloration. J. Hum. Evol. 39, 57-106. Johnston, I. A., and Bennett, A. F. (1996) Animals and temperature: Phenotypic and evolutionary adaptation. Cambridge University Press, Cambridge. Kitagawa, 0., Wakahama, K. I., Fuyama, Y., Shimada, T., Takanasi, E., Hatsumi, M., Uwabo, M., and Mita, Y. (1982) Genetic studies of the Drosophila nasuta subgroup, with notes on distribution and morphology. Jan. J. Genet. 57, 113-141. Mikasa, K. (1991) A statistical analysis of seasonal fluctuations of population size in Drosophila populations near human habitation at Himeji city. J. Arts Sci. Meikai University 3, 8-18. Salt, R. W. (1961) Principles of insect cold-hardiness. Annu. Rev. Entomol. 6, 55-74. Stevens, G. C. (1992) The elevational gradient in altitudinal range: an extension of Rapoport's latitudinal rule to altitude. Am. Nat. 140, 893-911. Tanaka, T., and Nei, M. (1989) Positive darwinian selection observed at the 5 variable-region genes of immunoglob ulins. Mol. Biol. Evol. 6, 447-459. 6 Part I Cold tolerance and metabolic rate increased by cold acclimation in Drosophila albomicans from natural populations 7 SUMMARY Cold acclimation is one of the important factors in temperature adaptation for insects needing to make rapid adjustment to the seasonal temperature changes in their living environment. In a fruit fly species, Drosophila albomicans, which has a tropical origin and currently has a wide geographic distribution extended into Asian temperate regions, cold tolerance in terms of survival time at 1 °C of adult flies reared at 25 °C was substantially improved by a cold acclimation at 20 °C for several days. Examining 29 isofemale lines from widely distributed natural populations, I observed a substantial variation in their acclimation response. However, the acclimation response was not necessarily stronger in the strains from the recently colonized temperate regions. A significantly stronger acclimation response was detected in male flies of the temperate strains when compared to those of the tropical strains. D. albomicans also