Pheromones in the Round Goby, Neogobius Melanostomus
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University of Alberta Olfactory and Behavioural Responses to Putative Steroidal Pheromones in the Round Goby, Neogobius melanostomus Cheryl Anne Murphy O A thesis subrnined to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Physiology and Ce11 Biology Department of Biological Sciences Edmonton, Alberta Spnng, 1998 National Library Bibliothèque nationale I*I of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1AON4 Ottawa ON KIA ON4 Canada Canada Your hie Votre milifence Our fi& Notre relerence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/filrn, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyight in ths thesis. Neither the droit d'auteur qui protège cette thése. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be pruited or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT To identify potential hormonal pheromones of the round goby (Neogobius melanostomus), a species recently introduced to the Great Lakes, 1used elecuo- olfactogram (EOG)recording to examine olfactory responsiveness to approximately 150 steroids and prostaglandins. Neogobius detects free and conjugated 1 a-, 19- and 2 1- carbon steroids. but no prostaglandins. EOG cross-adaptation, used to determine if Neogobius cmdiscriminate the detected compounds at the sensory level, suggested that the detected steroids act via four classes of olfactory receptor rnechanisrns named according to the rnost potent ligand for each: estrone, 1713-estradiol-3-glucuronide, etiocholanolone, and dehydroepiandrosterone-3-sulphate. Aithough none of the detected steroids induced reproductive behaviours, exposure to steroids from three of the four receptor classes (estrone, estradiol-glucuronide, or etiocholanolone) increased ventilation rate in males, whereas only etiocholanolone increased ventilation in females. The estrone- and estradiol- glucuronide-induced ventilation increase in males appears to be androgen-dependent because, in females, androgen implants induced the ventilatory response to these steroids within two weeks. Using the ventilation increase as a behavioral bioassay of steroid detection, behavioral cross-adaptation studies in males demonstrated that steroids discriminated at the sensory level are also discriminated behaviourally. These findings, the first olfactory study of hormonal pheromones in a perciform species. suggest the round goby has evoived a complex steroidal pheromone system. ACKNOWLEDGEMENTS 1 wish to express heartfelt thanks to rny supervisor. Dr. Norm E. Stacey, for al1 his suppon. advice and guidance dunng the course of my Master's thesis. 1 have learned much from him. and hold a tremendous amount of respect and admiration for his work and himself. 1 would also like to thank him for having patience with my decision to finish writing while in another locale. and for his support, input, and encouragement during the final stages. Thank-you Norm. I am not sure 1 would have finished without a final 'push'. This research was funded by an NSERC operating grant to Dr. Norm Stacey. 1 would also like to thank the Department of Biological Sciences for suppon through a Teaching Assistantship. Dr. Lynda Corkum has contributed much to this project. Not only did Dr. Corkum and her students. Andrew MacImis and Bill Ray provide live gobies, but she invited me to visit her laborator). and home where I leamed much about round goby ecolom. habitat and behaviour. Dr. Corkum has also ken a valuable source of information on round goby biologp. Thd-you very much! 1 would like to ihank my cornmittee members. Dr. Bev Mitchell. Dr. Jan Murie, Dr. Cindy Paszkowski for guidance. and also like to thad each committee member along with rny esternal examiner. Dr. Dallas Treit. for input on the final drafi of my thesis. 1 would also like to thank the chairman of my examining committee. Dr. Warren Gallin. for a smooth defence. 1 would like to thank Dr. Jiin Cardwell for help with EOG recordings. and for advice dunng the earl y stages of my degree. Bruce Wakeford provided much assistance wi th fish maintenance. Dr. Ian Stirling provided a hydrophone. and Wendy Calvert and Aiana Phillips helprd with equipment setup and spectrograph interpretation. 1 also thank Shelley Humphries for her photographie skills. and Figure 5.2. Dr. Peter Sorensen has been a great source of information. and assisted with the final witinp stages of the thesis by providing input and resources. Mernbers of the Sorensen lab: Leah Hanson. Judy Olson. Chris Appelt and Lance Vrieze also provided valuable input and put up with my manic behaviour dunng the final stages of thesis preparation. Thank-you vrp much. I uould like to thank rny good friend and office mate. Kim Christopher for being rny contact in the department while 1 was away. We started the Master's degree together. and kept each other poing. The Iaughs, advice and suppon helped me get through, thanks Kim! Lynne James. Mike Norton. Pam Boppel. Kim Howland. Alison Little and Shelley Humphries also have been great fiecds and were very supportive. Finally. 1 would like to thank my parents, Janet and Peter, and my sisters. Kelly and Erin for al1 their love and suppon. 1 really could not have done this degree without you. TABLE OF CONTENTS Chapter Page -.3 FISH 3. OLFACTORY RESPONSE TO PUTATIVE STEROIDAL PHEROMONES 3.1 . Methods 3.1.1. Electro-olfactogram (EOG) procedure 3.1 -2. Tested odours 3.1 -3. EOG concentration response studies 3.1.3. EOG cross-adaptation 3.2. Results 3 2.1 . Response to test odours 3 2.2. EOG concentration response results . EOG cross-adaptation results 4. BEHAVIOURAL RESPONSE TO PL'TATIVE STEROIDAL PHEROMONES 3.1. Methods 4.1.1. Behavioural repertoire 1.1.1.1. Spontaneous behaviours observed in the laboratory 4.1.1 -1. Brhaviours of potential reproductive significance 4.1 .1 3. Feeding behaviours 4.1.1.4. Behavioural measures of activity 41.2. Behavioural responses to detected steroids 4.1 -2.1. Testing apparatus 4.1 -2.7. Testing- procedure 41.2.3. I est odours 4.1..4 Data analysis 4.3Behavioural concentration response studies 4.1.4. Behavioural cross-adaptation 3.2. Results 4.2.1. Behavioural responses to detected steroids 42.2. Behavioural concentration response studies 4.5.3. Behavioural cross-adaptation 5. BEHAVIOURAL MASCULNIZATION OF FEMALES WlTH METHYLTESTOSTERONE (MT) 5.1 . Methods 5.3. ResuIts TABLE OF CONTENTS (cont.) Chapter Page 6 Discussion 6.1. Olfactory responses to steroids and steroid metabolites 6.1.1. Cross-adaptation 6.1 2. Proposed classes of olfactory receptors 6.1 -2.1. Estrone (El ) receptor 6.1 2.2. Estradio l (EZ-3g) receptor 6.1 2.3. Dehydroepiandrosterone-sulphate (DHEA-s) receptor 6.1.2.4. Etiocholanolone (Etio) receptor 6.1.3. Olfactory insensitivity to prostaglandins 6.1 -4. Olfactory insensitivip to maturation inducing steroids 6.2. Behaviour 6.2.1. Additional cues 6.2.2. Cheniical mixtures 6.1.3. Pherornones with pnming efkcts 6.2.4. Inappropnate reproductive condition 6.2.5. Ventilation behaviour 62.6. Relationship between EOG and behavioural responses 6.2.7. Possible pheromonal function of detected steroids 6.2.7.1 . C 1 8 steroids (estropens and their metabolites) 6.2.7.2. C 1 9 and C2 1 steroids 6.3.7.3. DHEA-s 6.2.7.4. Steroidal ses pherornones of the round goby 6.3. Sumrnary APPENDIX A. List of Steroids and Prostaglandins trsted in EOG APPESDlX B. Spectrograph of Gaping behaviour APPENDIX C. Spectrograph of Coughing behaviour APPENDIX D. Structure of steroids detected by the four receptor mechanisrns LIST OF TABLES Table Page 3.1. Detected steroids. grouped according to chernical structure with EOG responses 30-3 1 3.2. Detected steroids. grouped according to receptor class 32-34 * * 3.3 Detected steroids that act via two receptor mechanisms 35 4.1. Duration of ventilation rate response of male and femaie gobies in response to steroids LIST OF FIGURES Figure Page EOG responses Cornparison of male and female EOG responses Concentration response profiles for selected steroids Typical EOG concentration response profile EOG response to 2-sec steroid pulses delivered 30 min apart Effect of adaptation to E 1 on EOG response to E2 and EI-3g EOG responses to steroids before and during adaptation to El. E2-3g. and DHEA-g EOCi responses to steroids before and during adaptation to Etio and DHEA-s Ventilation rate response of male and female gobies to EtOH and T-g Ventilation rate response of male and female gobies to E 1 and EZ-3g Ventilation rate responsc of male and female gobies to Etio. Etio-g and Andr-s Ventilation rate response of male and female gobies to DHEA-S. DHEA-g and male response to steroid mixture Ventilation rate response of male and female gobies to Lalanine and food odour Eating frequency response of gobies to food odour, L-alanine, and EtOH Ventilation rate response of male gobies to increasing concentrations of El. EZ-3g. and Etio LIST OF FIGURES (cont) Figurc Page Prestimulus ventilation rates in cornparison IO post-stimulus ventilation rates in male gobies Ventilation rate response of male gobies to 1 M Andr-s and E2 Ventilation rate response of male gobies to El and sequential 1 M steroids Ventilation rate responsc of male gobies to Etio and sequential 1 M steroids Ventilation rate response of male gobies to DHE.4-s and sequential 1 M Andr-s Hopping frequenct of male gobies in response to El and sequential IO-^ M steroids Ventilation rate response of implanted (MT and Blank) female gobies to sequential steroids Ancsthrtizrd implantcd (MT and E3lan.k) female gobies 1.