The Influence of Developmental Temperature on Sperm Form and Function in Callosobruchus Maculatus
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THE INFLUENCE OF DEVELOPMENTAL TEMPERATURE ON SPERM FORM AND FUNCTION IN CALLOSOBRUCHUS MACULATUS Ramakrishnan Vasudeva A thesis submitted in partial fulfilment of the requirements of the University of Lincoln for the degree of Doctor of Philosophy MARCH 2014 ii I dedicate this work to my Mother, Maya, Paul & Charles iii Acknowledgements First and foremost, I sincerely thank both my supervisors Dr. Paul Eady and Dr. Charles Deeming for their support and encouragement during the past three years, without which I could never have been able to write this. In 2006, I came to Lincoln to embark on a research project to study sexual conflict coevolution in the Bruchid beetle Callosobruchus maculatus. Thus, with the completion of my first research project the scene was set for a PhD in January 2010. I am forever in admiration of my insect populations as they provided me with a means for the past six years to learn about their Biology and apply it to study the processes of evolution. In that spirit I am truly indebted to my beetles (and to all those hundreds of beetles which I dissected). Paul and I have worked together for over six years now and throughout he has been an ace mentor and a great inspiration. He played a significant role in welding together the multifarious branches of knowledge. He also went to great troubles in making me understand the importance of maintaining consistency in all my work (lab assays, data management, statistical analysis and writing). Regular meetings turned out to be the lynchpin of my understandings and the primary source to clarify doubts and avail research ideas. He taught me to maintain a balance and focus on the facts: a way to replace my inconsistencies. I shall remain indebted to him forever for being a superb guide and I am fortunate to be able to call Paul a colleague and friend. Special thanks go to Dr. Charles Deeming for guiding me during the past three years. I shall remain forever grateful to him for providing support and insightful discussions on thermal switch experiments, ectothermic development and scientific writing. Similarly, I thank Dr. Daniel Pincheira- iv Donoso; his advice on various aspects of the PhD were extremely helpful. He made valuable suggestions on acquiring key references in thermal biology and evolutionary biology. Thanks are due to Prof. Geoff Parker for being patient with my requests for papers on several occasions. I also thank the departmental regulars at 'Cake Fridays' and lunchtime conversations; they brightened me up. I will remain grateful to Dr. William Hayes for being an excellent advisor on all aspects of scientific investigation. Throughout my time doctoral research, Bill has offered wonderful tutelage and sharing a work space with him was indeed great fun. I will miss him and our conversations over coffee and lunch breaks. In the same breath, I thank Mrs. Amanda Mylett for her help which made life easier. I thank my family and friends who have unconditionally supported me to pursue my dreams. In this regard, my sincere thanks are due to my mother, Mrs. Gomathy Ramaswamy, Kenny & Alison Bell, who I think were my parents in England. Ken and Ali always looked after me like one of their own. I am forever grateful to my friends Dr. Kandasamy Bharathi and Dr. Needhirajan for their support. Thanks also go to Miss. Jacqueline Mayer who has been extremely supportive and many-a-time the driving force. Thanks are due to my Uncle Mr. R. Perumalswamy, whose unhindered financial support helped me focus just on my PhD. I also thank Gopal and Mahua for being there and supporting me always. Thanks are due to my wife Maya Kumar, her parents (Mr. K. S. Kumar and Mrs. Radha Kumar) and sister (Miss. Nidhi Kumar) for their support (in a myriad ways) during the writing up stages, which let me complete what I had started. Maya has been an excellent support in every way. I admire her steadfastness and those uplifting moments v whenever I lost focus. Now, I can spend more time with her and we look forward to our future together. In conclusion, I gratefully acknowledge the funding I received towards my PhD from the University of Lincoln. I remain forever grateful to numerous researchers worldwide for their valuable contributions and my thanks are due to the Miss. Denise Bateman and the Library acquisitions team at the University of Lincoln for their support. vi Abstract This thesis examines the effects of the thermal rearing environment on larval development and its influence on the expression of primary reproductive traits in Callosobruchus maculatus. Chapter 1 provided basic outline to the thesis, starting with a general introduction to sexual selection and specifically its role in the evolution of genitalia and primary reproductive traits. These traits appear to exhibit a level of phenotypic plasticity and thus the opening chapter introduces this concept and its role in evolution and diversification. Phenotypic plasticity in the primary reproductive traits of insects and the evolutionary consequences of this plasticity are discussed. Chapter 2 examined the effect of larval rearing temperature on larval development and offspring size. Larvae go through 4 larval instars prior to pupation. Unsurprisingly larvae reared at 17°C took approximately four times longer to develop than those larvae reared at 33°C but those beetles that completed their development at the coolest temperature were the largest, in agreement with Bergmann’s rule. This chapter paved way for the experiments carried out in subsequent chapters. Chapter 3 investigated the effect of developmental temperature on the expression of primary reproductive traits (e.g. sperm length and egg size) and life-history traits (e.g. adult longevity and fecundity). Sperm length exhibited phenotypic plasticity, being shortest when larvae developed at the temperature vii extremes (17°C & 33°C). Beetles raised at 17°C inseminated the fewest sperm despite the fact that these males had the largest absolute testes size. Developmental temperature affected female fecundity; females that underwent larval development at 17°C laid the fewest eggs. These females along with those reared at 33°C also laid the largest eggs. In general egg size tended to increase with decreasing developmental temperature (in agreement with Bergmann’s rule) although sperm size gives a more mixed response being largest at an intermediate temperature. This is in agreement with the effect of developmental temperature on sperm size in dung flies, but in guppies and a species of snail sperm size increased with decreasing temperature. Chapter 4 used thermal switch experiments to identify when, during larval development, the expression of sperm length was sensitive to temperature. Thermal switching involves an abrupt shift in the thermal environment experienced by the developing larvae, such that development starts at one environment and is completed at either a higher or lower thermal rearing environment. There appeared to be two thermal sensitive periods (TSP) for the expression of sperm length: one during the very stages of ontogeny and one around the time of instar III to instar IV. This is the first demonstration of a TSP in relation to the phenotypic expression of the sperm. The findings of this chapter are discussed in relation to previous shift-once or shift-twice experiments on reptilian embryos and a handful of insects. Chapter 5 studied the consequences of thermal environment during larval development and as adults on copula duration. Both larval rearing temperature viii and post-eclosion temperature affected copula duration; males reared at the lowest temperature taking longer to complete copulation. This is the first demonstration that larval rearing temperature affects copulatory behaviour. It is argued that because thermal heterogeneity is likely to be common in nature, this under-examined component of male behaviour could account for much variation in copulatory behaviour in nature. Chapter 6 examined the effect of larval rearing temperature on the outcome of sperm competition. Males reared at 17°C were less successful at sperm defence and sperm offence, whilst males reared at 33°C were less successful at sperm offence in comparison to males reared at 27°C. This is the first study to report an effect of developmental temperature on the outcome of postcopulatory sexual selection. The results are discussed in light of the physiological consequences of developmental temperature on male ejaculatory characteristics. Chapter 7 discusses the possible mechanisms and consequences of phenotypic plasticity in reproductive traits as a result of experiencing heterogeneous environments during development, and how these processes might interact with postcopulatory sexual selection. Further work on understanding the role of phenotypic plasticity in the evolution of primary reproductive traits is required. ix Contents Acknowledgements iii Abstract vi CHAPTER 1. GENERAL INTRODUCTION 1.1 Sexual selection 1 1.2 Postcopulatory sexual selection 5 1.3 Thermal biology, phenotypic plasticity and the temperature-size rule 12 1.4 Adaptive plasticity or pathological effects? 18 1.5 Ecological consequences of phenotypic plasticity 20 1.6 Phenotypic plasticity in primary reproductive traits 26 1.7 Thesis aims & objectives 31 CHAPTER 2. THE EFFECT OF TEMPERATURE ON LARVAL GROWTH AND DEVELOPMENT 2.1 Introduction 32 2.2 Materials and Methods 37 2.2.1 Model system and experimental design 37 2.3 Results 39 2.4 Discussion 45 CHAPTER 3. THE EFFECT OF THERMAL ENVIRONMENT ON THE EXPRESSION OF SPERM LENGTH 3.1 Introduction 53 3.2 Materials and Methods 58 3.2.1 The effect of larval rearing temperature on the adult phenotype 58 3.2.2 The effect of post-eclosion temperature on primary reproductive traits 60 3.3 Results 61 3.4 Discussion 68 x CHAPTER 4. THERMAL SWITCH EXPERIMENTS REVEAL WHEN DURING DEVELOPMENT, SPERM LENGTH IS TEMPERATURE SENSITIVE 4.1 Introduction 76 4.2 Materials and Methods 83 4.2.1 Measurements 85 4.3 Results 85 4.4 Discussion 90 CHAPTER 5.