EVALUATING THE EFFECTS OF ELEVATED WATER CONDUCTIVITY ON THE SOUTHERN REDBELLY DACE (CHROSOMUS ERYTHROGASTER) AT DIFFERENT LIFE HISTORY STAGES By Austin Hannah A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology Austin Peay State University 2020 Thesis Committee: Dr. Mollie Cashner, Committee Chair Dr. Rebecca Johansen Dr. Chris Gienger Statement of Permission to Use In presenting this (type of paper) in partial fulfillment of the requirements for the (type of degree) at Austin Peay State University, I agree that the library shall make it available to borrowers under the rules of the library. Brief quotations from this (type of paper) are allowable without special permission, provided that accurate acknowledgement of the source is made. Permissions for extensive quotation or reproduction of this (type of paper) may be granted by my major professor, or in his/her absence, by the Head of the Interlibrary Services when, in the opinion of either, the proposed use of the material is for scholarly purposes. Any copying or use of the material in this (type of paper) for financial gain shall not be allowed without my written permission Austin Hannah 07/31/2020 kkkkk iii ACKNOWLEDGEMENTS I would like to first thank my supervisor, Dr. Mollie Cashner, for her continual support and much-needed guidance throughout the course of my graduate school experience. Thank you for facilitating my transition back into school and helping me develop the tools necessary to be successful scientist; skills that I will carry with me for the rest of my career. I would like to thank Dr. Leslie Hiatt for her help answering my (many, many) questions involving analytical and instrumental chemistry. I would like to thank Erin Schwarzbach for all her help in the field and lab and for being an outlet for the many frustrations encountered throughout my project. I would like to thank Austin Peay State University Co-STEM as well as North American Native Fish Association for their financial assistance; without which my project would have not been possible. I want to thank my family for their support and for kindling within me a passion for all living things that helped sustain my focus throughout this project. Lastly, I’d like to thank my wife, Michelle, for being a solid foundation, for offering constant reassurance, and for having incomparable patience over the last two years. iv ABSTRACT The Blackside Dace (Chrosomus cumberlandensis; BSD) is a federally threatened minnow that has been extirpated from 31 streams in Tennessee and Kentucky since 1978. Although there is strong evidence linking elevated conductivity levels and low BSD occurrence, the mechanistic relationship between the two variables is poorly understood. The goal of this project is to further elucidate proximate explanations for the progressive extirpation and contemporary genetic disjunction of BSD populations using a closely related species, Chrosomus erythrogaster (Southern Redbelly Dace, SRBD). We hypothesize that elevated water conductivity affects reproduction of SRBD and causes sub-lethal effects which contribute to reduced fitness in adults. First, we reconstituted water chemically analogous to streams within the historic range of BSD reflecting varying levels of mining related impairment. Using reconstituted water, we subsequently performed five separate experiments on SRBD at different life stages testing both acute and chronic stress responses to elevated water conductivity. We found a significant negative relationship between water conductivity level and proportion of SRBD egg hatch (GLM; p <0.001, n = 600 eggs from 7 individuals). Our acute, sub-lethal test results support the hypothesis that elevated conductivity elicits an acute stress response from adult SRBD exhibited by detection of significantly elevated waterborne cortisol (ANOVA; F 2,9 = 7.34, p = 0.013, n = 12) as well as increased oxygen consumption (ANOVA; F 2,17 = 11.5, p <0.001, n = 27). Furthermore, evidence of chronic stress to adult SRBD was detected after prolonged exposure to high conductivity; individuals in impaired treatments experienced reduced growth over a five week period compared to controls (ANOVA; F 1,18 = 7.11 , p = 0.016, 95% CI: -0.27, -0.04) and, although not significant, we detected a trend indicating hypothalamic-pituitary-interrenal axis disruption reflected by reduced cortisol response in high conductivity groups (Control: 6.96 ng g- v 1 60 min-1, SD = 4.6; High conductivity: 4.03 ng g-1 60 min-1, SD = 2.28; T-test; p = 0.16, n = 12). Our results provide the first evidence demonstrating the potential negative fitness costs associated with elevated water conductivity related to surface mining on a representative fish species at multiple life-history stages. vi TABLE OF CONTENTS I: INTRODUCTION ........................................................................................................................1 II: METHODS .................................................................................................................................7 A: Egg Hatching Experimentation ...........................................................................................7 i: Fish collection and maintenance ......................................................................................7 ii: Induction of captive spawning ........................................................................................8 iii: Collection and distribution of eggs ...............................................................................9 iv: Preparation of reconstituted water .................................................................................9 B: Acute Stress: Cortisol Experiments ...................................................................................11 i: Acute Experiment 1: consecutive day sub-lethal testing ................................................11 ii: Acute Experiment 2: singe day testing ..........................................................................12 iii: Experimental protocol and cortisol collection .............................................................13 C: Chronic Stress: Growth and HPI Tests .............................................................................14 i: Growth experiment .........................................................................................................14 ii: HPI axis assessment ......................................................................................................15 III: STATISTICAL ANALYSIS ..................................................................................................15 IV: RESULTS ................................................................................................................................17 A: Egg Hatching Tests ........................................................................................................17 i: Reconstituted water parameters ....................................................................................17 ii: Egg-hatching results......................................................................................................18 B: Acute Sub-lethal Experiment Results ............................................................................18 i: Acute Experiment 1: consecutive day tests .....................................................................18 ii: Acute Experiment 2: single day testing .........................................................................20 C: Chronic 6-week Growth Study and HPI Test Results ....................................................20 i: Fish growth over 5 weeks ...............................................................................................20 ii: HPI axis testing .............................................................................................................21 V: DISCUSSION ...........................................................................................................................21 A: Acute Egg Toxicity ........................................................................................................21 vii B: Sub-lethal Stress .............................................................................................................24 VI: CONCLUSIONS AND FUTURE DIRECTIONS .................................................................27 VII: LIST OF TABLES: Table 1: Target Water Parameters for Egg Hatching Experiments ...................................31 Table 2: Water Quality Characteristics of SRBD Collection Sites ....................................31 Table 3: Water Quality of Reconstituted Treatments in Egg-Hatching Experiments .......32 Table 4: Egg-Hatching Test Results ..................................................................................32 Table 5: Water Quality of Reconstituted Treatments in Acute Experiment 1 & 2 ............39 VII: LIST OF FIGURES: Figure 1(a-d): Pictures of Test Chambers Used in Egg and Acute Experiments...............30 Figure 2: Box-and-Whisker Plot of Egg Hatching Test Results ........................................33 Figure 3(a-d): Egg Mortality Rates per Treatment in Egg Hatching Experiments ............34 Figure 4: Box-and-Whisker Plot of Pooled Cortisol Release in Acute Experiment 1 .......35 Figure 5: Box-and-Whisker Plot of Cortisol Rates by Day in Acute Experiment 1 ..........36 Figure 6: Box-and-Whisker Plot of Oxygen Consumption in Acute Experiment 1 ..........37 Figure 7: Box-and-Whisker Plot