Larval Oyster Settlement and Distribution in a Freshwater-Dominated Estuary - Haley Nicholson Gancel

LARVAL OYSTER (CRASSOSTREA VIRGINICA) SETTLEMENT AND DISTRIBUTION IN A FRESHWATER-DOMINATED AND HUMAN- INFLUENCED ESTUARY A Dissertation Submitted to the Graduate Faculty of the University of South Alabama in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Marine Science by Haley Nicholson Gancel B., University of Miami, 2013 May 2020 I dedicate this dissertation to my parents, Stephanie and Brendon Nicholson, and to my husband Jake. ii ACKNOWLEDGMENTS I would like to acknowledge my funding sources: The Food and Drug Administration-Dauphin Island Sea Lab Fellowship (award numbers: 5U19FD005923-04 and 5U19FD004277-04) and the Mississippi-Alabama Sea Grant Consortium (project number #R/SFA-03). I would like to thank committee members Kevin Calci, Bill Burkhardt, Kyeong Park, Jeffrey Krause, Marcus Drymon, and Kelly Dorgan, as well as, Scott Rikard, Jiabi Du, and John Lehrter for teaching me new techniques and providing helpful comments. This work could not have done without the many hours of assistance from Carmichael Lab personnel, interns, and volunteers: Elizabeth Hieb, Kayla DaCosta, Carl Cloyed, Heather Patterson, Elizabeth Darrow, Allen Aven, Noel Wingers, Casey Fulford, Ashley Frith, Audrey McQuagge, Lauren Willis, Neil Burglund, Jamie Thompson, Victoria Drumm, Max Han, Chris Williams, Josh Millwood, Pavel Dimens, Jake Hall, and Anika Knight.

I would also like to thank Marine Science graduate students and technicians for also donating much of their time to this project: Caitlin Wessel, Maddie Kennedy, Laura Stone, Sydney Acton, and Steve Dykstra. Thank you to Dauphin Island Sea Lab and FDA technical support staff including Renee Collini, Yantzee Hintz, Grant Lockridge, Laura Linn, and George Doup. I would also like to thank Scott Rikard and the Auburn University Shellfish Laboratory for help with experimental design and use of their hatchery. Thank you to Jacob Blandford and The iii Nature Conservancy for collecting oysters off their reefs for this dissertation work.

A special thanks is deserved for Jake Gancel, Elizabeth Hieb, Kayla DaCosta, Maddie Kennedy, Ashley Frith, and Casey Fulford for getting me through my time as a Ph. Finally, I would like to thank my advisor, Ruth Carmichael, for being a kind and supportive advisor. iv TABLE OF CONTENTS Page LIST OF TABLES. vii LIST OF FIGURES.

xii LIST OF SYMBOLS AND ABBREVIATIONS .1 CHAPTER 1: FIELD MARK-RECAPTURE OF CALCEIN-STAINED LARVAL OYSTERS (CRASSOSTREA VIRGINICA) IN A FRESHWATER-DOMINATED ESTUARY .34 CHAPTER 2: USE OF SETTLEMENT PATTERNS AND GEOCHEMICAL TAGGING TO TEST POPULATION CONNECTIVITY OF EASTERN OYSTERS (CRASSOSTREA VIRGINICA) IN A FRESHWATER-INFLUENCED ESTUARY .71 CHAPTER 3: STRAIGHT TO THE SOURCE: UNDERSTANDING WASTEWATER INPUTS IN A FRESHWATER-DOMINATED SYSTEM. Chapter 1 supplemental figures and tables. Chapter 2 supplemental figures and tables. Chapter 3 supplemental figures and tables .254 vi LIST OF TABLES Table Page 1.

Recapture success from mark-recapture studies with invertebrate and fish larvae free- released in the field: ALC = Alizarin complexone; TC = Tetracycline. Predictions of larval origins (May–June 2016) from the larval origin prediction linear discriminant function analysis using trace element (TE) ratios in recent adult shell (i., proxy for natal site TE ratios). Field studies done in open coast and estuarine environments that determined spatial and temporal variability of trace element (TE) ratios in bivalve shells for use in larval connectivity studies. Characteristics of wastewater treatment plant (WTP) and river sources sampled at high and low flow subsystems and at additional sites sampled for estuarine-scale analyses.

Results of three-way ANOVAs used to determine if wastewater indicator (nutrient and indicator microbe) concentrations and loads were different in the WTP versus the river within each high and low flow subsystem through time using sources (WTP, river), seasons (warm, cold), and years (2015, 2016) as factors. Results of three-way ANOVAs used to determine if wastewater indicator (nutrient and indicator microbe) concentrations and loads in WTPs and rivers were different between subsystems (high, low), seasons (warm, cold), or years (2015, 2016). Results of three-way ANOVAs used to determine if wastewater indicator (nutrient and indicator microbe) concentrations were different among downstream receiving sites (MB1, MB2, BLB1, BLB2), seasons (warm, cold), or years (2015, 2016). Candidate models for model selection to investigate nutrient and indicator microbial concentrations in high and low flow subsystems at the receiving sites MB1 and MB2 vii (high flow) and BLB1 (low flow), with outliers included and excluded from the models.

Model averaging output for candidate models associated with Table 9 for model selection of nutrient and indicator microbial concentrations in high and low flow subsystems at the receiving sites MB1 and MB2 (high flow) and BLB1 (low flow), with outliers included and excluded from the models. Nutrient loads (mol d-1) from studies that compared WTP and river loads to estuaries for different nutrient species. Percent N and P from WTP or sewage point sources and from direct riverine discharge or agricultural runoff into estuaries worldwide. Number of stained and unstained oysters found in Niskin samples at 1, 2, 3, and 5 days following the release of stained larvae on May 19 (lower salinity) and July 28 (higher salinity), 2014.

Sites for settlement plate (“S”) and native adult oyster (“A”). Intercept statistics for negative binomial general linear model (2014) and zero-altered negative binomial linear model (2016) lines in Fig. Intercept statistics for regression lines in Fig. 9a, bottom panel showing salinity variation among sites with time in 2016.

Slope and intercept statistics for regression lines in Fig. A3 showing salinity variation with time in 2014 and 2016. MANOVA (multivariate) and ANOVA (univariate) results for recent (~single year) and whole (~multiple years) shell used to determine if there were differences in multi-elemental (MANOVA) and individual (ANOVA) trace element ratios among sites. Standardized coefficients explaining the relative contribution of elements to discriminate among sites for recent (~single year) and whole (~multiple years) shell linear discriminant function analyses.

Validation results of recent (~single year) and whole (~multiple years) shell linear discriminant function analyses. Two-way MANOVA results of spat shells during three time periods to determine if multi-elemental trace element ratios between larval and settled shell of spat were different among sites and between shell types. ANOVA (univariate) results following two-way MANOVAs (Table A8) of spat shells during three time periods to determine which individual trace element ratios differed among sites and between the larval and settled shell of spat. MANOVA (multivariate) and ANOVA (univariate) results for larval and settled shell during three time periods used to determine if there were differences in multi- elemental (MANOVA) and individual (ANOVA) trace element ratios among sites.

Standardized coefficients explaining the relative contribution of elements to discriminate among sites for larval and settled shell linear discriminant function (LDA) analyses for time periods that had significant MANOVAs. Validation results of larval and settled shell linear discriminant function (LDA) analyses for time periods that had significant MANOVAs. MANOVA (multivariate) and ANOVA (univariate) results in recent adult shell using trace element (TE) ratios used in the larval origin prediction analyses (i., TE ratios present in both larval and recent shell). Validation results from the larval origin prediction linear discriminant function analysis using trace element ratios in recent adult shell.

Standardized coefficients explaining the relative contribution of elements to discriminate among sites for the larval origin prediction linear discriminant function analysis using trace element ratios in recent adult shell. GPS coordinates of sources (wastewater treatment plants [WTP] and rivers) and downstream receiving sites sampled in high and low flow subsystems and at additional sites sampled for estuarine-scale analyses. Explanatory variables considered in each set of wastewater indicator models (dependent variables) for model selection analyses. Nutrient concentrations (µM) measured at wastewater treatment plants (WTP) and rivers in high and low flow subsystems and at nearby receiving sites, separated by season and year sampled.

Indicator microbial concentrations (CFU or PFU 100 mL-1) measured at wastewater treatment plants (WTP) and rivers in high and low flow subsystems and at nearby receiving sites, separated by season and year sampled. Environmental attributes measured at downstream receiving sites (salinity, temperature, dissolved oxygen [DO], chlorophyll a) and from nearby weather stations (wind direction, tidal amplitude, rainfall) in high and low flow subsystems, separated by season and year sampled. Nutrient loads (mol d-1) calculated from wastewater treatment plants (WTP) and rivers in high and low flow subsystems, separated by season and year sampled. Indicator microbial loads (CFU or PFU d-1) calculated from wastewater treatment plants (WTP) and rivers in high and low flow subsystems, separated by season and year sampled.

Candidate models for model selection to investigate nutrient concentrations in the high flow subsystem at the receiving site MB1. Model averaging output for candidate models associated with Table A24 for model selection to investigate nutrient concentrations in the high flow subsystem at the receiving site MB1. Full candidate models for model selection to investigate nutrient and indicator microbial concentrations in high and low flow subsystems at the receiving sites MB1 and MB2 (high flow) and BLB1 (low flow), with outliers included and excluded from the models. Full model averaging output for candidate models associated with Table A26 for model selection of nutrient and indicator microbial concentrations in high and low flow subsystems at the receiving sites MB1 and MB2 (high flow) and BLB1 (low flow), with outliers included and excluded from the models.

Candidate models for model selection to investigate nutrient concentrations in the high flow subsystem at the receiving site MB2. Model averaging output for candidate models associated with Table A28 for model selection to investigate nutrient concentrations in the high flow subsystem at the receiving site MB2. Candidate models for model selection to investigate nutrient concentrations in the low flow subsystem at the receiving site BLB1. Model averaging output for candidate models associated with Table A30 for model selection to investigate nutrient concentrations in the low flow subsystem at the receiving site BLB1.

Candidate models for model selection to investigate nutrient concentrations in the low flow subsystem at the receiving site BLB2. Model averaging output for candidate models associated with Table A32 for model selection to investigate nutrient concentrations in the low flow subsystem at the receiving site BLB2. Candidate models for model selection to investigate nutrient concentrations in the low flow subsystem at the receiving site BLB3. Model averaging output for candidate models associated with Table A34 for model selection to investigate nutrient concentrations in the low flow subsystem at the receiving site BLB3.

Candidate models for model selection to investigate indicator bacterial concentrations in the high flow subsystem at the receiving site MB1. Model averaging output for candidate models associated with Table A36 for model selection to investigate indicator bacterial concentrations in the high flow subsystem at the receiving site MB1. Candidate models for model selection to investigate indicator bacterial concentrations in the high flow subsystem at the receiving site MB2. Model averaging output for candidate models associated with Table A38 for model selection to investigate indicator bacterial concentrations in the high flow subsystem at the receiving site MB2.

Wastewater treatment plant (WTP) and river nutrient loads (mol d-1) to the whole system and to the whole system with the exclusion of the high flow WTP and river. Wastewater treatment plant (WTP) and river indicator microbial loads (CFU or PFU d-1) to the whole system and to the whole system with the exclusion of the high flow WTP and river.251 xi LIST OF FIGURES Figure Page 1. Larval oyster sampling sites in the Mobile Bay-eastern Mississippi Sound system, AL for the field study in 2014. Mean changes in maximum linear length (± SE) of larval oysters compared to (a) water manipulations (salinity offset from hatchery control conditions) and (b) holding tank effects through time.

Observed versus expected number of larvae detected using an Olympus BH2-RFCA fluorescent microscope and a FlowCam®VS series .