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THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR EGGS Lesley Lynne Keiko Hamamoto B.S., University of California, Davis, 2001 THESIS Submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE in BIOLOGICAL SCIENCES (Biological Conservation) at CALIFORNIA STATE UNIVERSITY, SACRAMENTO SPRING 2010 iii © 2010 Lesley Lynne Keiko Hamamoto ALL RIGHTS RESERVED ii THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR EGGS A Thesis by Lesley Lynne Keiko Hamamoto Approved by: __________________________________, Committee Chair Ronald M. Coleman, PhD __________________________________, Second Reader Jamie M. Kneitel, PhD __________________________________, Third Reader James W. Baxter, PhD Date:____________________ iii Student: Lesley Lynne Keiko Hamamoto I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis. _________________________, Graduate Coordinator _________________ James W. Baxter, PhD Date Department of Biological Sciences iv Abstract of THE INFLUENCE OF PARENTAL CARE BEHAVIOR BY CONVICT CICHLIDS ON THE INCIDENCE OF OOMYCETE INFECTION OF THEIR EGGS by Lesley Lynne Keiko Hamamoto Infection of fish eggs by oomycete watermolds has been documented among numerous fish species occupying diverse aquatic habitats. In fact, watermolds are considered to be ubiquitous in freshwater systems and it seems that all species of fish eggs are susceptible to infection. Oomycete infection can result in the loss of large numbers of viable eggs because it can quickly spread from one infected egg to many others. To date, the majority of studies have been conducted using salmonid eggs under artificial rearing conditions, and there has been virtually no research on reproductive ecology or parental care behavior in fish as it relates to watermold infection. Additionally, few studies have utilized microscopy to elucidate the causes or pathways of infection. My research project had two major objectives. First, I looked at two aspects of convict cichlid (Archocentrus nigrofasciatus) behavior, fanning and v egg cleaning, in an attempt to quantify the individual and collective effectiveness of each behavior in preventing the spread of infection within a clutch of eggs. Effectiveness was evaluated by comparing egg mortality under different care regimes. Second, I used microscopy and histology techniques to look at and pictorially document modes of egg infection and spatial patterns of egg mortality. My evaluations of parental care effects on watermold infection did not yield any statistically significant differences between treatments, possibly due to unforeseen design flaws and inadequately controlled variables. I discuss these flaws and offer suggestions for additional research that will provide a reference for future studies on this important topic. Additionally, egg samples were evaluated using a variety of histologic and microscopic techniques, including scanning electron microscopy, mortal staining, and paraffin sectioning in an attempt to elucidate the oomycete’s modes of infection and spread. I present the results of this study as a photographic atlas, which may lead to a better understanding of this phenomenon and suggest alternative methods for control. Although the results of my study do not provide definitive approaches toward controlling oomycete infection, they do contribute to the limited body of information on the incidence of watermold infection in fish eggs. vi __________________________________, Committee Chair Ronald M. Coleman, PhD ACKNOWLEDGEMENTS I would like to acknowledge and thank the Pacific Coast Cichlid Association Mark Tomasello Research Fund and the American Cichlid Association Guy Jordan Endowment Fund for their generous financial support for this project and the Albert Delisle Family Scholarship for their contribution toward my academic expenses. I would like to thank Jim Ster from the CSUS Engineering Department and Grete Adamson and Pat Kysar from the UC Davis School of Medicine Electron Microscopy Laboratory for their assistance with scanning electron microscopy, and Dr. Judy Jernstedt from the UC Davis Plant Sciences Department and Sue Nichol from the UC Davis Plant Biology Department for their assistance and contributions toward histology and sectioning. I would like to thank the members of my graduate committee, Dr. Jamie Kneitel and Dr. James Baxter, for their support and assistance and their thoughtful comments on various drafts of my thesis. Lastly, I would like to express my extreme gratitude to my committee chair, Dr. Ronald Coleman, whose help, guidance, and persistent encouragement have gotten me through this process. vii TABLE OF CONTENTS Page Acknowledgements .................................................................................................. vii Chapter 1 .................................................................................................................... 1 INTRODUCTION ..................................................................................................... 1 Definition of Parental Care ................................................................ 1 Egg Laying and Parental Care Behavior by Convict Cichlids ........... 2 Parental Care and Pathogenic Infection ............................................. 4 Pathogenic Oomycete Watermolds .................................................... 5 Oomycete Infection ............................................................................ 6 Hypotheses ......................................................................................... 8 MATERIALS AND METHODS ............................................................................. 10 Oomycete Culture ............................................................................ 10 Aquarium Set-Up ............................................................................. 10 Experimental Design ........................................................................ 13 Data Collection................................................................................. 16 Data Analyses................................................................................... 16 RESULTS ................................................................................................................ 20 Comparison of Egg Survival Among Treatments ............................ 20 Comparison of Egg Survival with Respect to Distance ................... 20 viii DISCUSSION .......................................................................................................... 27 Comparison of Egg Survival Among Treatments ............................ 27 Comparison of Egg Survival with Respect to Distance ................... 29 Suggestions for Future Research ...................................................... 32 Chapter 2 .................................................................................................................. 34 INTRODUCTION ................................................................................................... 34 MATERIALS AND METHODS ............................................................................. 35 Oomycete Culture and Aquarium Set-Up ........................................ 35 Histology and Microscopy ............................................................... 35 RESULTS ................................................................................................................ 38 DISCUSSION .......................................................................................................... 46 Scanning Electron Microscopy ........................................................ 46 Mortal Staining with Evan’s Blue .................................................... 47 Paraffin Sectioning ........................................................................... 47 Progression of Infection over Time ................................................. 48 Appendices ............................................................................................................... 50 Appendix A. Egg Count Data for Survival Analysis .............................................. 51 Appendix B. Egg Count Data for Proximity Analysis ............................................ 52 Literature Cited ........................................................................................................ 56 ix LIST OF TABLES Page Table 1. ANOVA (single-factor) summary for the comparison of cichlid egg survival among different parental care treatments…………..….......22 Table 2. ANCOVA summary for the comparison of cichlid egg survival among different parental care treatments ……...……………….……….24 Table 3. ANOVA summary for the comparison of percent egg mortality in inner circles (near inoculation point) versus outer circles (farther from inoculation point)....……………………………………………….26 x LIST OF FIGURES Page Figure 1. Spawning structures were constructed as a substrate for egg-laying ....... 12 Figure 2. Exclusionary barriers were used to restrict parental access to egg clutches.. .................................................................................................... 14 Figure 3. An example of an image used to identify and mark eggs for spatial analyses ...................................................................................................

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