Population dynamics and sex-determining mechanisms in the marine amphipod, Echinogammarus marinus Yasmin Zara Guler Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy of the University of Portsmouth November 2012 Declaration Whilst registered as a candidate for the above degree, I have not been registered for any other research award. The results and conclusions embodied in this thesis are my own and have not been submitted for any other academic award. Word Count: 47,222 P a g e | 1 Abstract Despite their huge diversity, abundance and ecological importance, very little is still known about sex determining mechanisms within Crustacea. Sex determination in crustaceans is known to be influenced by environmental factors, via parasitic infection and genetically, however, it is possible that all three mechanisms can be involved in a single species. The gonochoristic marine amphipod Echinogammarus marinus (Leach, 1815) is currently being used for the development of biomarkers to measure the influence of environmental contamination on crustacean sex determination and differentiation pathways. To truly understand whether anthropogenic disruption of sex determination is currently an issue, it is critical that all the mechanisms governing the process in E. marinus are fully evaluated. Therefore, the aim of this project was to fill gaps in our knowledge of the general population dynamics of E. marinus, with a particular focus on elucidating the mechanisms of sex determination in this ubiquitous amphipod. Sex determination in E. marinus has been linked with feminising parasites, however, to date, no such studies have linked this species with environmental sex determination (ESD) or genetic sex determination (GSD). This project investigated two E. marinus populations that differed in population structure. The Langstone Harbour E. marinus population (Southern England, UK) revealed no presence of parasitic sex determination (PSD). However, this study has shown that the population has a seasonal breeding pattern, with population growth and decline closely related to environmental parameters (temperature) and parasites (trematodes) respectively. The population data also revealed seasonally altered sex ratios, ranging from 36% to 71% males. ESD was recorded for the first time in an E. marinus population by revealing that photoperiod was the cue for sex determination. This finding was validated by a laboratory study that showed a male bias in broods that developed in long day light regimes (16h light: 8h dark) and a female bias in broods that developed in a short day light regime (8h light: 16h dark). The laboratory data and the seasonally altered sex ratios found in the field showed significant correlation with each other supporting these findings. A new species of trematode P a g e | 2 parasite belonging to the Microphallidae family has been identified that encysts in the amphipod brain and demonstrates clear capacity for behavioural changes in its host. Individuals infected with the trematode parasite displayed distinct positive phototaxic and negative geotaxic behavioural alterations that could potentially increase susceptilbility to predation. These behavioural alterations have been linked to changes at the level of gene expression suggesting modulation of neuronal genes in the infected individuals. Putative serotonin receptor 1A, inebratied neurotransmitter, tryptophan hydroxylase and amino acid decarboxylase like genes displayed the most dramatic change in their gene expression. This represents the first study to record such changes in the neuronal pathways of parasite infected amphipods. Another E. marinus population investigated from Invertkeithing (Scotland, UK) displayed a high female bias and high levels of intersexuality. The project has strengthened the evidence that PSD is present in this population with 40.4 % of the population being infected by either Paramyxea or microsporidia parasites. From the infected individuals 75% of that infection were female bias and 88.5% of intersexes, also presented an infection. The investigation explored the transmission pathways and efficiency of the parasites involved. Vertical transmission of a Paramyxean sp. was shown for the first time in an amphipod host and also showed the highest transmission efficiency from the mother to the eggs (96.8%). This has lead to the question of whether the microsporidian D. duebenum is a feminiser and has highlighted another parasite candidate for E. marinus sex distortion. Despite the range of genomic techniques employed, the attempt to determine genomic sexual determination in E. marinus did not reveal any sex specific genomic regions. However, considering the preliminary nature of the work, this study has provided insight for future directions. Several key genes involved in sexual differentiation that presented sex exclusive expression were identified. In addition, crucial method development was performed that will allow future investigations of genetic variation in E. marinus. The transcriptome of the E. marinus has now been sequenced and along with population models enabling a greater understanding of the P a g e | 3 links between genome and population ecology. With such a large investment in E. marinus as an ecological model species, it is crucial that basic biological questions and gaps in the field are addressed. Consequently, the data presented within this thesis will aid in the study of E. marinus and other crustaceans from the level of genetics to population effects. P a g e | 4 Acknowledgements I must initially thank Alex Ford and Pete Kille for firstly obtaining the funding from NERC (NE/G004587/1) for this research and then giving me the opportunity to undertake the project. Especially to Alex who has been extremely supportive and given me the freedom to explore my own research ideas. I would also like to give a special thanks to Stephen Short for all the time, guidance, discussions and patience given throughout my PhD. His training has been invaluable not only for my PhD but instilled the skills and attributes necessary for my future career. I am very grateful to Darren Gowers for his time, help and discussion with the AFLP study. It has been great working at IMS and would like to thank all my co-workers and the technicians during my time here. I cannot thank my mum and awesome friends enough for their continued support, encouragement and belief during my time at university. I would particularly like to thank Amaia Etxabe who has helped me with the majority of my sampling (she is great in the field) and has been a constant support, freely giving advice or discuss ideas to overcome obstacles in my work. To have such a close friend going through a PhD together has been such a blessing and made the journey a lot more fun. I would also like to give a special mention to Ashley Jones who has been a great housemate and a best friend through this period of my life. P a g e | 5 Contents Declaration ............................................................................................................... 1 Abstract .................................................................................................................... 2 List of Figures .......................................................................................................... 9 List of Tables .......................................................................................................... 14 Abbreviations ......................................................................................................... 16 1. General introduction ............................................................................................ 17 1.1 Echinogammarus marinus ........................................................................... 18 1.2 Intersexuality .................................................................................................... 21 1.3 Sex determination in Crustacea ........................................................................ 26 1.3.1 Sex chromosomes ...................................................................................... 29 1.3.2 Sex hormones ............................................................................................. 35 1.3.3 Sex distorting parasites .................................................................................. 38 1.3.4 Environmental sex determination .................................................................. 41 1.4 Aims and objectives ......................................................................................... 44 2. The population dynamics of Echinogammarus marinus at Langstone harbour .................................................................................................................................... 45 2.1 Introduction .......................................................................................................... 45 2.2 Materials and Methods ......................................................................................... 47 2.2.1 Population Study ........................................................................................... 47 2.2.2 Parasite prevalence and screening ................................................................. 50 2.2.3 Statistical analysis ......................................................................................... 53 2.3 Results .............................................................................................................