Reproductive Development of Female Bonefish (Albula Spp.)

REPRODUCTIVE DEVELOPMENT OF FEMALE BONEFISH (ALBULA SPP.)

FROM THE BAHAMAS

Cameron Alexander Luck

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

In Partial Fulfilment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, Florida

December 2018

ACKNOWLEDGEMENTS

I sincerely appreciate the guidance of my advisor Dr. Matthew Ajemian for his continued support throughout this endeavor as well as the input from my committee members: Dr. Sahar Mejri, Dr. Aaron Adams, and Dr. Paul Wills. I am extremely grateful for the collaboration and support of Justin Lewis. Field collections would have been impossible without his local knowledge, willingness to be available, passion for bonefish, and incredible work ethic. I am also grateful for the words of wisdom and comedic relief often provided by fellow graduate students, lab members, and interns of the Fish Ecology and Conservation Lab: Grace Roskar, Breanna Degroot, Steve Lombardo, Rachel Shaw,

Mike McCallister, Alex Allen, and many others.

This project was financially supported by Bonefish & Tarpon Trust (BTT) and

National Fish and Wildlife Foundation (NFWF). We are grateful for the lodging and water access provided by East End Lodge, Andros South, and Hank’s Place. The experimental protocol for this study received approval from Florida Atlantic University’s

Institutional Animal Care and Use Committee (Animal Use Protocol #A16-34).

ABSTRACT

Author: Cameron Luck

Title: Reproductive Development of Female Bonefish (Albula spp.) from the Bahamas

Institution: Florida Atlantic University

Thesis Advisor: Dr. Matthew J. Ajemian

Degree: Master of Science

Year: 2018

Bonefish (Albula spp.) support an economically important sport fishery, yet little is known regarding the reproductive biology of this genus. Analysis of oocytes histology and sex hormone levels was conducted on wild female bonefish sampled during and outside the spawning season in Grand Bahama, Central Andros, and South Andros, The

Bahamas to assess reproductive state. Bonefish are commonly found along shallow water flats, or in pre-spawn aggregations (PSA) during spawning months. 17β-estradiol levels suggest vitellogenic consistency between habitats. However, fish are more reproductively developed at PSA based on the occurrence of larger, more prevalent vitellogenic oocytes and evidence of final maturation. Variability in hormone levels and spawning readiness existed between Grand Bahama and Andros PSAs, suggesting peak spawning may differ

by region. Findings from this study will contribute baseline data to the captive bonefish restoration project at Harbor Branch Oceanographic Institute and to the limited ecological data regarding bonefish reproduction.

REPRODUCTIVE DEVELOPMENT OF FEMALE BONEFISH (ALBULA SPP.)

FROM THE BAHAMAS

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

BACKGROUND ...... 1

Spawning strategies in the marine environment ...... 1

Historic Anthropogenic Impact on Aggregate Spawners ...... 3

Physiological Indicators of Spawning in Marine Fishes ...... 4

Project Objectives ...... 8

CHAPTER 1 ...... 9

INTRODUCTION ...... 9

METHODS ...... 12

Initial sample collection and preservation ...... 12

Histological preparation of oocytes...... 13

Sex-hormone plasma level determination ...... 14

Statistical analysis ...... 15

RESULTS ...... 16

Histology ...... 16

Sex-hormone concentration ...... 16

I. Seasonal differences...... 16 vii

II. Flats vs. PSA ...... 17

DISCUSSION

Seasonal differences...... 17

Flats vs. pre-spawn aggregation...... 18

CHAPTER 2 ...... 23

INTRODUCTION ...... 23

METHODS ...... 27

Sample collection ...... 27

Histological preparation of oocytes...... 28

Reproductive phase classification ...... 28

Sex-hormone plasma level determination ...... 29

Statistical analysis ...... 31

RESULTS ...... 32

Histology ...... 33

Sex hormone concentrations ...... 33

I. Seasonal differences...... 33

II. Spatial differences ...... 33

III. Reproductive phase classification ...... 34

DISCUSSION ...... 35

Seasonal development ...... 35

Spatial trends ...... 36

CONCLUSION AND FUTURE DIRECTIONS ...... 41

APPENDICES ...... 44

viii

APPENDIX A. FIGURES ...... 45

APPENDIX B. TABLES ...... 58

REFERENCES ...... 61

LIST OF FIGURES Figure 1. Map showing sampling locations for both flats and pre-spawn aggregation

(PSA) sites during spawning (February, March, and April 2017) and non-

spawning (September 2017) months of bonefish (Albula vulpes) in Grand

Bahama Island, Bahamas ...... 45

Figure 2. Micrographs of histologically sectioned oocytes sample collected from a

wild female bonefish (Albula vulpes) on a shallow water flat during full

moon (April 2017) ...... 46

Figure 3. Proportional occurrence for each of the three oocyte stages: late vitellogenic

(LV), cortical alveolar (CA), primary growth (PG) at the two sites (flats and

pre-spawn aggregation [PSA]) sampled during the spawning month of April

2017...... 47

Figure 4. Monthly plasma concentrations of 17β–estradiol and Testosterone (mean 

SEM) from bonefish (Albula vulpes) females sampled in the flats during

spawning (February and April 2017) and non-spawning (September 2017)

months (mean  SEM) ...... 48

Figure 5. Plasma concentrations of 17β–estradiol and testosterone (mean  SEM) at

flats and pre-spawn aggregation (PSA) sites in female bonefish (Albula

vulpes) sampled during three spawning months (February, March, and

April 2017) ...... 49

Figure 6: Map showing sampling locations across four Bahamian islands and the

respective flats and/or pre-spawn aggregation (PSA) sites sampled for each.

All islands were sampled during the full moon of a spawning month

(Grand Bahama: March and April 2018; Central Andros: January 2018;

South Andros: December 2017) as well as one sampling event outside

the spawning season (Grand Bahama: June 2018) of bonefish

(Albula vulpes). Exact PSA locations have been generalized for

conservation purposes (Adams et al. 2018) ...... 50

Figure 7: Chronological schematic of different oocyte stages present during the

reproductive development of bonefish (Albula vulpes.) including: primary

growth (PG), cortical alveolar (CA), primary vitellogenic (Vtg 1),

secondary vitellogenic (Vtg 2), tertiary vitellogenic (LV), germinal

vesicle breakdown (GVBD), and germinal vesical migration (GVM) ...... 51

Figure 8: Diameters of vitellogenic oocytes collected from female bonefish

(Albula spp.) sampled at two different habitats (Flats, PSA) across three

different islands (Grand Bahama, Central Andros, South Andros). Black

line indicates median for each site sampled. Letters indicate significant

differences in mean oocyte diameters between sites across island ...... 52

Figure 9: Proportional distribution of primary growth (PG), cortical alveolar (CA),

primary vitellogenic (Vtg 1), secondary vitellogenic (Vtg 2), tertiary

vitellogenic (LV), and atretic oocytes found in female bonefish

(Albula spp.) sampled from both the flats and PSAs of Grand Bahama,

Central Andros, and South Andros ...... 53

Figure 10: Seasonal variation in both 17β-estradiol and testosterone concentrations

(mean ± SEM) for Bonefish (Albula spp.) sampled during both spawning

(March/April 2018) and non-spawning months (June 2018, September

2017) along the flats of Grand Bahama. Black letters indicate significant

differences in 17β-estradiol. Grey letters indicate significant differences

in testosterone ...... 54

Figure 11: Figure 11: Spatial variation in both 17β-estradiol, testosterone, and

luteinizing hormone (LH) concentrations (mean ± SEM) for Bonefish

(Albula spp.) sampled across three islands and two sites (Flats, PSA)

within each Island. Black letters indicate significant differences ...... 55

Figure 12: 17β-estradiol and testosterone concentrations (mean ± SEM) for each

reproductive phase observed in flats sampled female bonefish (Albula

vulpes) across all islands (Grand Bahama, South Andros, Central Andros).

Letters indicate significant differences in mean concentration of

17β-estradiol across phases ...... 56

Figure 13: Linear regression (power) assessing correlation between vitellogenic

oocyte diameter and 17β-Estradiol values from all mature female bonefish

sampled from Grand Bahama (2017, 2018), Central Andros (2017), and

South Andros (2018) ...... 57

xii

LIST OF TABLES Table 1: Sample sizes for all fish blood plasma (hormone) and oocyte (histology)

collected at each island (Grand Bahama, March/April 2018; Central Andros,

December 2017; South Andros, January 2018) and respective habitat (Flats,

PSA) ...... 58

Table 2: Proportional distribution (mean ± SD) of each major oocyte stage (primary

growth [PG], Cortical Alveolar [CA], Vitellogenic [Vtg]) found across both

island (Grand Bahama, Central Andros, South Andros) and habitat (Flats vs.

PSA). Letters have been applied to indicate significant differences among

both island and habitat for each oocyte stage ...... 59

Table 3: Statistical results. Significance threshold for all analyses based on 95 %

confidence, p < 0.05. Significant tests results have been bolded ...... 60

xiii

BACKGROUND

Marine teleosts (hereafter “fishes”) represent about 58 percent of the approximate total 20,000 extant fish species that inhabit the world’s oceans, estuaries, and lagoons

(Moyle and Cech 1982). Environmental variability, nutrient access, and habitat availability within and across each of these marine ecosystems has spurred the evolution of species-specific life history characteristics. This adaptability, in part, is due to the plasticity and compensatory abilities of each species’ reproductive strategy (Garrod and

Horwood 1984). Fishes optimize reproductive success through the development of unique reproductive traits, including: gender system (gonochoristic or hermaphroditic), oogenesis patterns, shifts in habitat utilization (migratory or stationary), seasonality, periodicity of spawning, behavior, spawning site selection and preparation, spawning frequency, egg characteristics, and type of embryonic development (Helfman et al. 1997;

Lowerre-Barbieri 2009). In doing so, species become specialized within an environment, and maximize the likelihood of reproductive success to maintain population densities. For the introduction of this thesis, I provide a general overview of the known spawning strategies used by marine teleosts as a preface to our understanding of the reproductive biology and ecology of the bonefish (family: Albulidae; Albula spp.: Linnaeus 1758).

Spawning Strategies in the Marine Environment

Despite the specializations exhibited by different species of fish during their reproductive cycle, spawning strategies can generally be categorized based on the presence or absence of a migratory event (Johannes 1978). Individuals that remain within

the safety of their home-range, typically a trait of smaller species (e.g. Tetraodontidae,

Blennidae, and Gobiidae), are considered to be “resident” spawners. During a spawning event, female fish typically release gametes either onto substrate or within the water column (Randall and Randall 1963; Barlow 1975; Fishelson 1975). Eggs laid along substrate may continuously be guarded from predators by parents (Bohlke and Chaplin

1968) and tend to hatch later than those released pelagically (Breder 1962). This tactic of extended development is speculated to maximize development of larval motility for avoiding lethal bottom-dwelling organisms (Swerdloff 1970; Hobson and Chess 1978;

Johannes 1978). Eggs released over bottom structure, or well into the water column minimize larval mortality by distributing larvae away from benthic predators. Sexually active individuals achieve this by releasing gametes during a vertical rush into the water column (Randall 1961; Randall and Randall 1963; Ehrlich 1975). This strategy also places larvae within the dispersing flow of passing currents and has been documented in up to 50 species from 15 different families (Helfman et al. 1997; Claydon 2004; Sadovy de Mitcheson and Colin 2012).

Alternatively, many species have been classified as “transient” spawners, a behavior that requires the transport of eggs to specific habitats prior to spawning. The specific selection of spawning habitat is thought to be adaptive to maximize larval feeding (Slotte and Fiksen 2000; Agostini and Bakun 2002; Bakun 2006), reduce egg and larval predation (Bakun 2013), and ensure larval transport brings recruits to suitable nursery habitats (Symonds and Rogers 1995; Bailey et al. 2005; Karnaukas et al. 2011).

Species that migrate to spawning grounds often do so in large species-specific groups known as spawning aggregations. Domeier (2012) defines a spawning aggregation as “a

repeated concentration of conspecific marine animals, gathered for the purpose of spawning, that is predictable in time and space. The density/number of individuals participating in a spawning aggregation is at least four times that found outside the aggregation and results in a mass point source of offspring”. For many species, spawning aggregations occur in distinct locations outside of normal home ranges and are generally ephemeral (Sadovy De Mitcheson et al. 2008; Cianelli et al. 2015). The temporal and spatial consistency of these aggregation events, combined with a lack in proper management, can leave populations particularly susceptible to an assortment of negative anthropogenic impacts and often result in population decline or collapses (Sadovy De

Mitcheson et al. 2008).

Historic Anthropogenic Impacts on Aggregate Spawners

Arguably one of the most studied aggregate spawning fishes are anadromous salmonids, which migrate from offshore feeding waters to aggregate staging areas at the mouths of rivers before heading upstream to spawn. During this migration, stocks are heavily exposed to numerous anthropogenic related threats such as fisher over- exploitation and the manipulation or destruction of critical migratory pathways, spawning grounds, and juvenile habitats (Young 1999; Allan et al. 2005; Waples et al. 2009). Either alone or in concert, these impacts have played a major role in the elevated extinction risk associated with most remaining anadromous populations (Parrish et al. 1998; Rand et al.

2012). Atlantic salmon (Salmo salar), in particular, experienced substantial overfishing from the 1960s through the 1990s, which was thought to play a substantial role in initial population declines (Parrish et al. 1998). This overfishing, combined with habitat degradation through watershed development, pollution, and constructed barriers, impeded

both spawning capable adults and ocean bound juveniles from reaching critical habitat and has thus severely impacted recruitment in many salmonid populations (National

Research Council 1996; Knowler et al. 2003; Young et al. 2011). Similar declines have been observed in marine aggregating species as well. Many species that aggregate retain some level of commercial or recreational value (e.g., snapper-grouper species) and are at an inherently high risk of overfishing and/or stock collapses due to advances in fishing techniques and/or failed fisheries management (Sala et al. 2001; Claro and Linderman

2003; Sadovy and Domeier 2005). The most well-documented decline is that of the

Nassau grouper (Epinephelus striatus), which was the first reef fish to be listed as endangered due to fishermen targeting aggregations (Cornish and Eklund 2003). Similar scenarios have been documented in other grouper and snapper species, including 20 species which were classified by the International Union for the Conservation of Nature

(IUCN) Red List Report as at risk for extinction should current trends in fishing pressure continue (Albins et al. 2009; Sadovy de Mitcheson et al. 2013). Although salmon

(salmonids), grouper (seranids), and snapper (lutjanids) species have received tremendous attention due to their commercial harvest values, there are still other important aggregate spawning fishes at risk of fishing pressure and habitat degradation.

In the tropical marine environment, one of the most popular recreational sportfish is the bonefish (Albula spp.). The name “bonefish” is applied across twelve morphologically similar, yet genetically distinct, coastal species found within estuaries or along nearshore sandflats easily accessible to fishermen (Colburn et al. 2001; Wallace and Tringali 2010; Wallace et al. 2014). Despite such speciation and the vast geographic distribution of these various species (e.g. Palau, Hawaii, The Bahamas, Florida, etc.),

aggregation by this genus has been historically documented in many of these regions

(Johannes 1978; Johannes and Yeeting 2000; Danylchuk et al. 2011). As a result, these aggregations are frequently targeted recreationally as part of the catch-and-release (C&R) fishery (South Florida, The Bahamas, Belize), commercially (Cuba, Belize), or for subsistence (The Bahamas and other locations) (Larkin et al. 2011; Perez et al. 2018;

Rennert et al. 2018). These aggregation events, similar to those observed in the aforementioned species, conceivably play a critical role in the reproductive success of bonefish but have yet to be studied in comprehensively detail. Throughout The Bahamas,

Cuba, Mexico, Belize and South Florida, A. vulpes (hereafter “bonefish”) is the primary species of bonefish that supports fishing lodges, guiding services, and the ecotourism industry (Fedler 2010; Ault et al. 2008). In the Florida Everglades alone, the recreational fishery has an annual economic value of approximately $1 billion (Fedler 2013). In the

Florida Keys, the flats fishery alone has an economic impact of $465 million (Fedler

2013). However, recent assessments have indicated the Florida bonefish populations has been in decline for the last 20 years with the stock close to maximum sustainable yield from either high fisher-related mortality or declines in larval recruitment (Larkin 2011;

Santos et al. 2017). In 2013, Florida Fish and Wildlife Conservation Commission

(FFWCC) passed regulations to prohibit the retention of any bonefish by both commercial and recreational fisheries in an attempt to address fishing related mortality.

Currently, management lacks information regarding bonefish spawning biology, habitat preference and the impacts physical habitat loss due to shoreline development may be having on the habitat connectivity of this species.

Physiological Indicators of Spawning in Marine Fishes

In concert with spawning habitat characterization and identification, understanding the reproductive development of a species throughout the spawning season is equally informative for management; particularly in regard to fecundity, spawning frequency, and spawning duration (Lowerre-Barbieri et al. 2011). Within the spawning season, significant physiological changes take place in sexually mature fish as hormonal stimulation and secretion promote oocyte development (Nagahama 1994). In fish, gonadotropin (GTH) is released from the pituitary in two separate instances; first as a follicle-stimulating hormone (FSH) and secondly as a luteinizing hormone (LH). FSH stimulates the release of 17ß-estradiol, a sex steroid primarily responsible for vitellogenic growth of the follicles as vitellogenin is sequestered into yolk protein within the oocyte

(Wallace 1985; Babin et al. 2007; Schreck 1973). Testosterone is also secreted in response to FSH occurring around the same time as 17ß-estradiol. This similarity is thought be a result of a decreasing catalytic reaction called aromatization, where testosterone is converted to 17ß-estradiol (Kagawa 2013) as 17ß-estradiol sequestration slows. The final stage of oocyte maturation takes place with the release of LH and results in the production of an important maturation inducing steroid, which results in germinal vesicle breakdown and allows oocytes to reach complete maturation (Nagahama 1994;

Nagahama and Yamashita, 2008).

Fish reproductive condition as a product of these physiological processes can also be assessed using an assortment of tools including gonadal indices, oocyte size-frequency distributions, and sex steroid quantification (Lowerre-Barbieri et al. 2011; West 1990).

Gonadosomatic index (GSI = gonad mass / somatic mass × 100%) (DeVlaming et al.

1982) is a common metric of reproductive condition in fisheries management for

categorizing a fish’s reproductive status (Zeyl et al. 2013). However, this metric requires fish to be sacrificed so that full gonads can be extracted. Alternatively, histological staging of biopsied or cannulated gonad tissue via microscopy can also provide a static view of the developmental stages of gametes at the time of sampling (Rottman et al.

1991; Ferraz et al. 2004). Specifically, measurements of both vitellogenic oocyte diameter and the proportional occurrence of various oocyte stages can help quantify reproductive development status (Crabtree et al. 1997, Brown-Peterson et al. 2011).

Finally, concentrations of testosterone and 17-estradiol from individuals at the time of sampling can also be quantified by using either radioimmunoassay (RIA) or an enzyme- linked immunosorbent assay (ELISA) (Scott et al. 1980; Zupa et al. 2017). The benefit of examining hormone concentrations in concert with gonad histology is that individuals can be non-lethally sampled to obtain high-resolution information regarding both reproductive status and developmental potential (Ueda et al. 1983). This approach is particularly helpful for rare and/or sensitive species (e.g., protected species, gamefish) where the culling of high numbers of fish is not feasible.

Historical assessment of bonefish spawning seasonality in South Florida waters suggested year-round spawning based on the presence of ripe and near ripe males and females (Bruger 1974). However, more recent work done in Cape Eleuthera, Bahamas found decreases in whole-body lipid content and energy densities in the winter months that coincided with a greater gonadal development, suggesting seasonality in reproductive activity of bonefish (Murchie et al. 2010). In the Florida Keys, Crabtree et al. (1997) found a similar trend, with elevated monthly median GSIs and higher frequencies of vitellogenic oocytes from November to May. Trends in abundance, age,

and recruitment of larvae in both the Bahamas and Florida Keys also indicate similar evidence of winter and spring spawns (Mojica et al. 1995). Despite these trends in the temporal aspects of spawning, there is little known about the physiology that regulates these processes or how they vary in both time and space.

Project Objectives

The current dearth of knowledge regarding the reproductive requirements of bonefish significantly hinders our ability to adequately apply management strategies for the recovery of this species. Through the use of sex hormone quantification and histological analysis of wild captured female bonefish, this project aims to characterize the reproductive development of bonefish from a seasonal, spatial, and physiological perspective. Findings from this study will contribute baseline data to both a much larger captive bonefish restoration project underway at Harbor Branch Oceanographic Institute and to the currently limited ecological knowledge that exists regarding reproduction in bonefish.

CHAPTER 1 Sex Hormone Profile and Oocyte Development in Spawning Ready Female Bonefish: A

Case Study from Grand Bahama

INTRODUCTION

Bonefishes (Albula spp.) occur in shallow waters throughout the tropical regions of the world (Crabtree et al. 1996, Johannes and Yeeting 2000, Murchie et al. 2010).

Within the subtropical to tropical waters of the northwest Atlantic, including the Florida

Keys (Florida, USA), Bahamas, and Caribbean Sea (Hildebrand 1963), Albula vulpes is the most common species present and supports an economically important recreational fishery (Fedler 2010, Fedler 2013, Wallace and Tringali 2016). Within these regions, A. vulpes (hereafter “bonefish”) commonly inhabit interconnected, nearshore, shallow (<2 m) tidal flats, which include mangrove forests, seagrass and macroalgal beds, sand shoals, and occasionally patch reefs (Murchie et al. 2013). The variability in flats habitat provides bonefish diverse foraging opportunities for prey items such as crabs, fishes, bivalves, polychaetes, and small crustaceans (Colton and Alevizon 1983, Crabtree et al.

1998, Layman and Silliman 2002). From October to May, bonefish migrate from flats to aggregation sites in many of these regions (Danylchuk et al. 2011, Boucek et al. 2018,

Adams et al. 2018). Based on the presence of developed gonads and increases in lipid storage around the liver and gonads in individuals at both flats and PSAs, this aggregating behavior and habitat selection likely play a critical role in the reproductive strategy of bonefish (Murchie et al. 2010, Danylchuk et al. 2011).

Although pre-spawn aggregation (PSA) sites are still being identified for bonefish, they can generally be characterized as deep (>4 m), protected nearshore basins, in close proximity to both flats habitat and the continental shelf (Adams et al. 2018).

Movement to these sites occurs largely with lunar periodicity (full and new moon) as schools of fish (~1000 or more individuals) assemble at the PSA site, while non-spawners remain on the flats (Danylchuk et al. 2011). It is important to note that bonefish are not commonly observed at PSA sites outside of the spawning season (J. Lewis, pers. obs.).

Neither flats nor PSAs are thought to act as spawning grounds for bonefish. This is based on the general absence of fully mature and/or hydrated oocytes typically associated with imminent spawning at these sites (Crabtree et al 1997; Larkin et al.

2011), and no observations of gamete release in the PSAs (Danylchuk et al. 2011;

Danylchuk et al. 2018; Adams et al. 2018). Observational and acoustic tracking data also indicate fish migrate offshore to spawn, possibly off the continental shelf during the late- evening during the full moon of the reproductive months (Crabtree et al. 1997,

Danylchuk et al. 2011). Therefore, the role that flats and PSA habitats play in facilitating the reproductive development of bonefish prior to spawning remains unclear.

It is widely accepted that oocyte development in vertebrates during spawning is heavily regulated by a cascade of hormones along the hypothalamus-pituitary-gonadal

(HPG) axis (Sower et al. 2009). Along this axis, gonadotropins released from the pituitary stimulate the synthesis and secretion of critical reproductive hormones by the gonads. Cyclical changes in the occurrence and concentrations of reproductive hormones are widely known to occur in association with both reproductive behavior and gonadal development. In females, oocyte development (oogenesis) occurs as germ cells develop

into oogonia and are eventually released as ova during spawning (Lubzen et al. 2010).

The reproductive hormones primarily responsible for the growth of oocytes during early and advanced oogenesis are 17β-estradiol and testosterone (Nagahama and Yamashita

2008).

During this time, vitellogenin synthesis is stimulated in the liver, facilitating the uptake of yolk protein within the oocyte and promoting substantial oocyte growth prior to final maturation (Lubzen et al. 2010). However, this process can vary greatly by fish species and depends on reproductive strategy, synchrony of oocyte development, and spawning frequency (Rocha and Rocha 2006).

To date, no studies have assessed bonefish reproductive sex hormone and gonadal development. Therefore, the objectives of this study were to: (1) provide the first reproductive profile in wild bonefish via simultaneous analysis of ovary development and plasma sex hormone levels (17β-estradiol and testosterone), and (2) describe differences in the reproductive status of fish at flats versus PSA habitat during the spawning season.

This work was conducted in the relatively pristine central and eastern end of Grand

Bahama Island, The Bahamas, where substantial flats habitat exists and a known PSA has been identified. Based on our current understanding of the spawning strategy of migratory fishes, elevated levels of both testosterone and 17β-estradiol were expected for sexually mature females sampled during the spawning season (Ueda et al. 1984,

Barannikova et al. 2004). Also, since PSAs are thought to act as staging areas for spawning capable fish, more progressed oocyte development were expected from these individuals relative to those collected from the flats. Through examination of hormone

concentrations in concert with gonadal histology, high-resolution data regarding the reproductive status were obtained.

METHODS

Initial sample collection and preservation

Female bonefish were opportunistically sampled from several locations along the southern shore of Grand Bahama Island, The Bahamas (Fig. 1), during the day (8:00 AM to 5:00 PM) of the full moon for February, March, April, and September 2017. Samples collected during September 2017 were considered to be from non-spawning fish

(Danylchuk et al. 2011). Fish were collected from two general sites: 1) a pre-spawn aggregation located in water greater than 4 m deep along the southeast coast of the island

(n = 1 site) and 2) shallow tidal flats less than 1 m deep (n = 6 sites), which are not used as a PSA site (Fig 1). Fish in the PSA were captured via hook and line using cut shrimp or white jigs and 50lb test line. Fish fight time did not exceed 2 min. Individuals on the tidal flats were captured using a 50 m x 1m beach seine with a 2.5 cm mesh. All individuals collected were kept for 3-5 minutes in plastic, floating containers modified with holes to allow adequate water exchange. During sampling, fish were held inverted to induce a state of tonic immobility. Bonefish are not sexually dimorphic so fish that did not readily release gametes (spawning-ready males frequently release sperm when abdominal pressure is applied) were cannulated for oocytes using a soft-tube catheter

(Bard 100% latex-free infant feeding tube, 8Fr (2.27 mm diameter, 26 cm length) attached to a 3 ml syringe barrel. This method was adapted from Rottman et al. (1991).

Captured females were first measured for fork length (FL) and cannulated oocytes

(volume of 1-2 ml) were expelled into a 2.0 ml capped vial filled with 1 ml of 10 %

neutral buffered formalin. During the September sampling, only bonefish that produced identifiable ovarian tissue during cannulation were identified as females. Following oocyte collection, blood was then drawn from the ventral side of the fish’s caudal vein using a heparinized syringe and deposited into a lithium heparin lined BD vacutainerTM.

Blood samples were placed in a cooler above wet ice for later processing. At the laboratory, plasma was separated from blood by centrifugation (2500 rpm for 20 min) and stored at -80 ˚C until specific assays could be performed.

In total, blood samples were collected from 37 female bonefish (456 – 649 mm

FL) at the flats (n =27) and PSA (n =10) sites during the full moons of February 2017

(n=13) and April 2017 (n=22), as well as two samples collected from a PSA during the full moon of March 2017. Blood samples were also collected from female bonefish at several flats habitats in a non-spawning month (September 2017, n=12). Oocytes from 20 bonefish females were collected and histologically prepared from flats sites, and 7 from the PSA sites during the full-moon of April 2017. Bonefish from the PSA was not sampled for either blood or eggs during February due to logistical difficulties that limited our ability to target the deeper water fish.

Histological preparation of oocytes

Oocytes stored in 10% neutral buffered formalin during field collection were transferred to 70% ethanol prior to preparation (Barber 1996, Wilson et al. 2005).

Oocytes were dehydrated through a series of ethanol solutions (70 – 100%) for 60 min, clarified in toluene, and embedded within paraffin wax. A microtome cut 8-10 μm thick sections from the embedded samples, which were stained with hematoxylin and eosin before being mounted on pre-labeled glass slides for examination. A subset of oocytes (n

≥ 60 oocytes) from each female was photographed from each histology sample using an

OLYMPUS SZX7 stereozoom microscope at magnifications of 30 - 200x. For each subset, developmental stages of observed oocytes were categorized as late vitellogenic

(LV), cortical alveolus (CA), and primary growth (PG) based on the classification scheme developed by Crabtree et al. (1997) (Fig. 2). In general, PG oocytes occur as the initial development phase and develop into CA oocytes during early reproductive development. The final stage of oocyte development prior to final maturation is LV and exhibit both substantial lipid accumulation and a relatively large size (Brown-Peterson et al. 2011). If fully mature (hydrated) oocytes were present, they were also categorized and enumerated. Oocytes in the LV stage were measured for diameters (µm) by bisecting the nucleus through the center of the oocyte using a calibrated ruler tool within the image processing software (West 1990). Only LV oocytes fully enclosed within the frame were included. In total, 2228 oocytes were categorized, and 1775 LV oocytes were measured.

Sex-hormone plasma level determination

17β-estradiol and testosterone concentration levels were quantified via enzyme- linked immunoassay (ELISA) kits (Cayman Chemical Company, USA). For extraction, a

100 l plasma sample was diluted with ELISA buffer based on the manufacturer specifications (Cayman Chemical Company, USA). Hormones were then extracted twice by vigorous vortex using dichloromethane for 17β-estradiol and diethyl ether for testosterone. The supernatant organic phase was removed via Pasteur pipette, evaporated under a gentle stream of nitrogen at 30 ˚C, and reconstituted using ELISA buffer prior to plating. This process was repeated according to the protocol provided by Cayman

Chemical Company, USA. Samples were run at two dilutions to fulfill manufacturer

requirements and minimize interference within wells. A control sample provided by the manufacturer was serially diluted according to manufacturer specifications and run as a standard with each plate of samples. Plates were analyzed via absorbance at a wavelength of 405 nm using a microplate reader (Biotek, Synergy H1, USA). Absorbance values were converted to concentration values (pg.ml-1) using software provided by Cayman

Chemical Company, USA.

Statistical analysis

Reproductive development was compared between sites (Flat vs. PSA) using mean oocyte diameter as a response variable (West 1990). This analysis was conducted using a non-parametric Kruskal Wallis test, since parametric assumptions of normality and homoscedasticity could not be met. Analyses were performed in R (R Core Team

2014).

Differences in the frequency of occurrence of developmental stages (PG, CA, and

LV) at PSA and flats sites were assessed using a permutational multivariate analysis of variance (PERMANOVA with 999 permutations), including a posteriori pair-wise comparisons with PRIMER 7 (v. 7.1.12) and PERMANOVA+ (v.1.0.2). Assumptions of homoscedasticity were verified with a PERMDISP test, and data were transformed (log or arcsine square root) when necessary.

Differences in sex hormone (17ß-estradiol and testosterone) concentrations between sampling months were analyzed using a one-way ANOVA for each hormone.

Tests were conducted following a square-root transformation of data to meet parametric assumptions of normality and homoscedasticity. Comparison by month included only bonefish females sampled on the flats in February, April, and September 2017.

Differences in sex hormone concentrations during the spawning season were analyzed across sites (Flat and PSA) using a two-sample T-test and specifically used samples collected during spawning months (February, March, and April 2017).

RESULTS

Histology

The diameter of late-vitellogenic oocytes sampled at the PSA sites during April

2017 (591 ± 134 µm) were significantly larger than those from the flats during the same period (556 ± 129 µm) (H = 345.21, p < 0.001). The frequency of LV oocytes ranged from a mean of 63 % (± 15%) in fish taken on the flats to a mean of 78 % (± 7%) in fish collected from the PSA. The overall frequency distribution of oocyte stages was significantly different between flats and PSA sites (Pseudo F (1, 24) = 4.97, p < 0.05; Fig.

3). An average dissimilarity of 12.9% existed between samples collected from these two sites and was driven by the relative percentages of oocytes at LV and PG stages. The occurrences of LV stages were higher in bonefish from PSA than the flats and contributed up to 41.5% of the dissimilarity. The occurrence of oocytes at PG stages was lower in bonefish from the PSA than the flats and contributed up to 36.4% of the dissimilarity. No hydrated oocytes were observed in any fish sampled regardless of capture site.

Sex hormone concentration

I. Seasonal differences

Mean 17ß-estradiol levels of 3000-4000 pg·ml-1 were detected during the spawning months of February and April combined, dropping to <1000 pg·ml-1 in the non- spawning month of September (Fig. 4). These levels were significantly different among

seasons (F (2, 35) = 12.920, p = <0.001). A post-hoc pairwise comparison (Tukey) indicated that this difference was driven by significantly higher levels of 17ß-estradiol during the reproductive season (February: q = 5.668, df = 1, p = <0.05; April: q = 6.721, df = 1, p = <0.05) compared to the non-reproductive season (September). 17β-estradiol levels did not significantly differ between February and April sampled bonefish (q =

4.247, df = 1, p = 0.825). Mean testosterone levels of 2000-3000 pg.ml-1 were not significantly different between females sampled during February, April, and September

(F (2, 32) = 1.947, p = 0.160) (Fig. 4).

II. Flats vs. PSA

Within the spawning season, mean 17ß-estradiol levels at the PSA were not statistically different from fish sampled on the flats (t = -1.632, df = 34, p = 0.112; Fig 5).

Testosterone levels were significantly higher in bonefish from PSA when compared to the fish sampled from flats (t = -5.021, df = 32, p = <0.001; Fig. 5).

DISCUSSION

This study revealed spatial variation in levels of testosterone in female bonefish from flats and pre-spawn aggregation locations. Furthermore, differences in 17ß-estradiol and testosterone levels between in-season and out-of-season bonefish females provide information regarding the spawning preparation of bonefish as they enter the reproductive season. The physiological significance of these results is examined below.

Seasonal differences

The increased levels of 17ß-estradiol in bonefish females sampled along the flats during the spawning season compared to the non-spawning season follow a trend seen in other marine teleosts as fish begin the process of vitellogenesis (Scott et al. 1983b,

Taghizadeh et al. 2013, Zupa et al. 2017). Increased plasma concentrations of 17ß- estradiol facilitate rapid oocyte growth through vitellogenesis as vitellogenin, a hepatically derived plasma precursor, is sequestered as yolk protein within the developing oocyte (Lubzen et al. 2010). In doing so, oocytes increase in size and nutritive quality, eventually reaching a developmental state when final enlargement and maturation through hydration occur (Wallace 1981). The significantly elevated levels of 17ß- estradiol found in sampled bonefish during February and April corresponds with previous findings of spawning behavior occurring in female bonefish around the full moons from

October to May (Danylchuk et al. 2011).

Bonefish sampled both prior to, and during the spawning season showed high levels in testosterone. In many spawning teleost fishes, testosterone is secreted around the same time as 17ß-estradiol (Scott et al. 1983a, Mandich et al. 2004, Zupa et al. 2017) and is thought to fulfill two major roles: the regulation of gonadotropin secretion by the pituitary (Scott et al. 1980, Bommelaer et al. 1981) and/or serve as the androgenic precursor in the formation of estrogens via an aromatase enzyme (Lambert et al. 1971,

Scott et al. 1980, Kagawa 2013). Our results indicate that gonadal production of testosterone occurs prior to 17ß-estradiol. The sampling month (September) for the non- spawning season occurred roughly one month before the winter spawning season historically begins (October) (Danylchuk et al. 2011) and may indicate that, just prior to vitellogenesis, bonefish increase testosterone production in preparation for synthesis of

17ß-estradiol. Future sampling should take place at the end of the spawning season to assess testosterone concentrations as bonefish proceed into non-spawning season.

Flats vs. pre-spawn aggregation

The high mean level of 17ß-estradiol found in bonefish females sampled on the flats during the spawning season relative to fish sampled in September (outside of the spawning season) indicates that vitellogenesis begins at these habitats before bonefish start their migration to the PSA sites. We expected to see slightly lower levels of 17ß- estradiol in fish sampled on flats, based on the high proportional occurrence of early development stage oocytes (PG and CA) in flats sampled fish compared to those at the

PSA. Visibly, mean concentration values of 17ß-estradiol seem lower in flats sampled fish compared to PSA fish and correlates well with the smaller diameters of late vitellogenic oocytes found in flats fish. However, statistically the differences in 17ß- estradiol between these two sites was insignificant, indicating that while levels may subtly differ by site, vitellogenic development is continuing while fish migrate too and aggregate at PSAs. In a study conducted on chum salmon (Oncorhynchus keta), levels of

17ß-estradiol found in females offshore (~15 ng/ml) were statistically similar to those at the pre-spawn staging sites in river mouths (~14 ng/ml) (Ueda et al. 1984), indicating that vitellogenesis had also not yet ceased. Similarly, our findings suggest that vitellogenesis continues to take place at the PSA, likely up until the moment bonefish move offshore to spawn. Generally, 17ß-estradiol values at the individual level are expected to remain elevated, falling once fish have completed vitellogenesis (Stuart-Kregor et al. 1981,

Barannikova et al. 2004). However, in this study we did not sample any fish with hormone levels indicating they had reached the end of vitellogenesis or recently spawned.

Therefore, it was not possible to determine whether 17ß-estradiol typically dissipates following spawning or remains elevated in preparation for the next spawning event.

Future analyses that incorporate post spawning fish hormone levels would be beneficial to our understanding of 17ß-estradiol dynamics in spawning female bonefish.

Comparisons of bonefish oocyte development between sites further supports the hypothesis that vitellogenic development is still occurring at the PSA. Bonefish sampled on the flats exhibited varied compositions of oocytes, likely a result of capturing both fish moving towards the PSA and those still preparing the next clutch. The measured increase in the occurrence of late vitellogenic oocytes, and conversely decreased occurrence of PG oocytes at the PSA compared to flats, suggests ovaries are more developed at the PSA.

Late vitellogenic oocytes at the PSA were also larger than those from flats fish, which is an indication of further advanced development (West 1990). The slightly elevated mean levels of 17ß-estradiol levels found in PSA fish compared to those from flats could explain this difference.

The absence of fully mature oocytes from bonefish collected in any of the sampling events raises the question of temporal proximity of bonefish females to the completion of vitellogenesis (i.e., final maturation and hydration). In many studies, the completion of vitellogenesis is marked by a sharp decline in 17ß-estradiol levels and typically occurs with the onset of full oocyte maturation (Whitehead et al. 1978, Idler et al. 1981, Zupa et al. 2017). In this study, 17ß-estradiol levels were sustained in sampled female bonefish at the PSA, indicating vitellogenesis was still occurring. However, several studies have documented maximum levels of testosterone occurring at the end of vitellogenesis in female fish (Campbell et al. 1976, Ueda et al. 1983, Ijiri et al. 1995).

These findings were explained by the occurrence of a holding phase, where testosterone continues to be released by the follicle after the aromatase enzyme is turned off (Kime

1998). Indeed, we have observed that while 17ß-estradiol levels seem to remain stable between the flats and PSA habitats, it is likely that vitellogenesis concludes at the PSA site based on the significant increase in testosterone levels observed in bonefish females at these sites. The continued production of testosterone might also suggest that this androgen could play a role in stimulating the synthesis of a maturation inducing hormone to cue hydration (Crim et al. 1981). For instance, increases in plasma gonadotropins that cue final oocyte development occurred in both female rainbow trout (Salmo gairdneri) and white suckers (Catostomus commersoni) after peaks in testosterone occurred (Scott et al. 1983a, Scott et al. 1983b). Therefore, the increase in testosterone found at the PSA sites likely indicates that female bonefish are indeed close to initiating final maturation.

Field observations from Danylychuk et al. (2011) suggest that this spawning migration takes place at night. Since sampling efforts had ceased at this point due to the inability to maintain visual contact with the aggregation, it is possible that full maturation may be occurring in the late evening at the PSA, during their journey offshore, or at the location where spawning occurs.

The hormonal levels of 17ß-estradiol and testosterone collected in this study provide baseline data regarding bonefish development useful for identifying the occurrence of reproductive dysfunction that may be occurring in highly impacted areas.

For example, the bonefish population in the Florida Keys has declined in the last 20 years despite being a predominantly catch-and-release fishery (Larkin et al. 2011, Santos et al.

2017, Rehage et al. 2018). The causes of this decline are likely complex given the series of anthropogenic disturbances within Florida Bay (USA) and the Florida Keys

(Fourqurean et al. 1999, Larkin 2011, Santos et al. 2017). Pre-spawn aggregations have

also not yet been documented in Florida, an interesting fact considering recently compiled traditional ecological knowledge indicates this spawning behavior is common globally where most Albula spp. exist (Johannes and Yeeting 2000, Adams et al. 2018).

In regions where anthropogenic impact is still relatively low (i.e., outer Bahamian islands), habitat, diet, and movement characteristics of bonefish are very similar to what is found in South Florida (Larkin et al. 2011). By sampling in these more remote areas, we have been able to provide baseline information regarding bonefish life history, particularly reproductive physiology, to aid our ability to identify and address factors influencing the survival of bonefish in impacted areas such as Florida.

CHAPTER 2

Large-Scale Analysis of Bonefish (Albula spp.) Spawning Physiology Across the Northwest Bahamas

INTRODUCTION

Marine fishes exhibit aggregating behavior for a variety of reasons, including safety (Cushing and Jones 1968), food acquisition (Pitcher 2001), migration (Humston et al. 2000), and/or spawning (Domeier 2012). These events have been documented in many species throughout the world’s oceans and are understood to play important roles in the survival of those species which exhibit them. Spawning aggregations, in particular, have gathered considerable interest in both the scientific community and in fisheries management due to their importance in sustaining valuable fish stocks (Erisman et al.

2017). Tropical marine fishes that aggregate for the purpose of spawning adaptively select for specific habitats that maximize larval survival and subsequent recruitment

(Johannes et al. 1978). Geographically isolated fishes of the same genus or species that share reproductive strategies and physiological requirements select for similar aggregation times and habitat characteristics. For instance, Nassau grouper (Epinephelus striatus), which are distributed throughout the tropical western Atlantic Ocean, have been historically observed spawning in aggregations throughout their range. These events have been shown to predominantly occur with temporal and spatial predictability at locations distinct and distant from habitats used by individuals outside of the spawning season

(Heyman et al. 2008, Kobara et al. 2010, Domeier 2012). However, variability in peak

spawning times seems to vary between site, even extending beyond management seasonal closure dates in some locations (Scharer-Umpierre et al. 2014) Some transient aggregate spawners do not necessarily aggregate at spawning locations, instead congregating at staging locations distinctly separate from spawning grounds. This behavior has been classified as pre-spawning and likely fulfils some other purpose in addition to condensing individuals that are ready to spawn. Studies on anguilliformes, including both the

Japanese eel (Anguilla japonica) and European eel (A. anguilla) suggest aggregations occur well before fish migrate to spawning grounds, often greater than 100 km away

(Schmidt 1992, Arai 2014). A similar behavior has been observed in both tarpon

(Megalops atlanticus; Crabtree et al. 1992) and bonefish (Albula spp.). In both cases, this behavior possibly ensures individuals collectively make it to spawning locations at the same times while also providing protection in numbers from the assumed elevated predation risks associated with pelagic spawning. However, relatively little is known regarding development, duration, and frequency of both pre-spawning and spawning events in species that aggregate. This lack in knowledge is largely due to the fact that many of these species travel long distances to reach spawning locations, often during the dark hours of dawn and dusk, in many cases finally spawning at restrictively deep location which prevent direct observation.

Bonefish (Albula spp.), a globally distributed tropical coastal fish classified within the same superorder as both tarpon and anguilliforms (elopomorpha), also exhibits a similar behavior during their spawning season (November to May; Crabtree et al. 1996,

Danylchuk et al. 2011, Donavan et al. 2015), aggregating with lunar periodicity (full and new moon; Johannes et al. 1978, Johannes and Yeeting 2000, Danylchuk et al. 2011).

Throughout the year, bonefish occur within shallow (< 2 m), nearshore environments called “flats”, which can include mangroves, sand flats, seagrass, or occasional rocky outcroppings (Murchie et al. 2010). The diversity of these environments provides sufficient nutrients for juvenile growth and predator avoidance as well as adequate foraging potential for bonefish adults (Colton and Alevizon 1983, Crabtree et al. 1998).

However, bonefish aggregation events occur in geomorphologically distinct deeper water areas (>4m) in close proximity to both the continental shelf and nearby flats (Adams et al.

2018) and are typically prefaced by large migratory events as bonefish leave flats for aggregation sites (Murchie et al. 2015). Similar to the aforementioned tarpon and eel species, these aggregation events do not occur at spawning locations and instead develop as staging events prior to their perceived offshore spawning runs (Danylchuk et al. 2011).

Since these aggregation events occur within close proximity to shore and are easily visible due to tropical water clarity, they are easily exploitable.

Throughout their range, bonefish are targeted by a variety of different fisheries

(commercial, recreational, subsistence) during these events and in some regions are relied upon as a predominant food fish (e.g., Kiribati; Johannes et al. 1978). The recreational fishery, however, is perhaps the most common form of targeting bonefish and is predominantly a catch-and-release (C&R) fishery. Due to the inherent difficulties associated with hooking bonefish, fishing guides and anglers also occasionally target aggregations since fish are inherently easier to catch when fish density is high. The consequences of the elevated C&R pressure that occurs at both PSAs and along migratory routes are not fully understood. In bonefish, release mortality is highly dependent on fight time, air exposure, and relative presence of predators such as sharks

(Cooke and Philipp 2003), which occur in higher numbers at PSAs (J. Lewis, pers. obs.).

During the spawning season, angling pressure in other species has also been shown to negatively influence complex mating behavior and sexual selection (mate choice, mate competition), possibly having an effect on reproductive success (Rowe and Hutchings

2003, Dean et al. 2012). Based on these implications, there is a clear need to understand the physiological role of these events in order to inform local and regional management action regarding closures and gear limitations.

To date, little research exists regarding the reproductive ecology of this species or the physiological role these pre-spawning events play in the spawning preparation of bonefish species (Ault et al. 2008). Recent work to identify PSAs in The Bahamas indicates at least one PSA occurs at each of the islands assessed (Andros, Abaco, Grand

Bahama, Long Island, etc.) and is therefore likely a critical step in bonefish reproductive success (Adams et al. 2018). Preliminary work in Grand Bahama showed fish at the PSA were more prepared for spawning when compared to those found along flats habitat based on the higher proportional occurrence of larger vitellogenic oocytes and hormonal evidence of vitellogenic completion in females at PSAs (Chapter 1). However, due to differences in habitat type, water current profiles, and the limited gene flow between regionally separate populations of bonefish, assessing the significance that PSAs play in facilitating bonefish reproductive success remains an important topic. Similarly, due to the commercial and subsistent fisheries that exploit these aggregation events and the increasing value of bonefish as a popular recreational C&R species, identifying any variability in the timing, duration, and location of PSAs throughout regions where they

occur will provide critic insight for designing management plans to protect specific habitats from fishing pressure.

No studies have assessed the reproductive disparities between different bonefish habitat across multiple locations where PSAs exist. Therefore, the objectives of this study were to: (1) compare reproductive maturity and sexual hormone variability between flats and PSA habitats, across geographically distinct locations, and (2) characterize reproductive phases for female bonefish at flats and PSAs during full moons of the spawning season. This study was conducted at three different locations within the

Bahamian Archipelago where bonefish are commonly found: the east end of Grand

Bahama, the eastern side of Central Andros, and the eastern side of South Andros. Each of these locations are relatively pristine, contain substantial flats habitat, and have a single identified PSA at each location.

METHODS

Sample collection

Female bonefish were sampled for oocytes and blood from Grand Bahama

(March and April 2018), Central Andros (December 2017), and South Andros (January

2018), the Bahamas, during the days immediately surrounding (± 2 days) full moons of known spawning months (Fig. 6). Samples were also collected from Grand Bahama in

June 2018, a sampling event considered to be well outside of the spawning season

(Danylchuk et al. 2011). At each island, fish were collected from two general habitat types: 1) a pre-spawn aggregation located in water greater than 4 m deep (n = 1 PSA per island) and 2) shallow tidal flats less than 1 m deep (n  1 flat per island; Fig. 6).

Details regarding methodology of fish capture, sample collection, and sample preservation can be found in the Methods section of Chapter 1. One significant improvement was made regarding oocyte collection via cannulation. Sampling conducted in Chapter 1 only included fish that showed obvious presence of yolked eggs. For this study, all samples that showed any evidence of oocyte material within the cannula were placed in 10% neutral buffered formalin for later analysis. This modification allowed for analyses to include a broader spectrum of reproductively developing females.

In total, 57 females (373 – 640 mm FL) were sampled for both blood and oocytes from flats (n =37 fish) and PSAs (n =20 fish) during the spawning season. Blood samples were also collected from 10 females at several flats habitats in a non-spawning month

(June 2018; Table 1).

Histological preparation of oocytes

Oocytes stored in 10% neutral buffered formalin during field collection were transferred to 70% ethanol prior to preparation (Barber 1996, Wilson et al. 2005).

Oocytes were dehydrated through a series of ethanol solutions (70 – 100%) for 60 min, clarified in toluene, and embedded within paraffin wax. A microtome was used to cut 8-

10 μm thick sections from the embedded samples, which were stained with hematoxylin and eosin before being mounted on pre-labeled glass slides for examination. A subset of oocytes (n ≥ 60 oocytes) from each female was photographed from each histology sample using an OLYMPUS SZX7 stereozoom microscope at magnifications of 30 - 200x. For each subset, developmental stages of oocytes were categorized as vitellogenic (Vtg), cortical alveolus (CA), and primary growth (PG) based on the classification scheme developed by Crabtree et al. (1997). In general, PG oocytes occur as the initial

development phase and develop into CA oocytes during early reproductive development.

Secondary development occurs through vitellogenesis and results in vitellogenic (Vtg) oocytes. Vitellogenic oocytes were identified by their relatively large size, yolk accumulation within the cytoplasm, and the presence of cytoplasmatic inclusions (i.e., oil droplets). Vtg oocytes were measured for diameters (µm) by bisecting the nucleus through the center of the oocyte using a calibrated ruler tool within the image processing software (West 1990). Only oocytes fully enclosed within the frame were included. In total, 9232 oocytes were categorized, and 1907 Vtg oocytes were measured.

Reproductive phase classification

Histologically prepared oocytes from individual fish were evaluated to classify the state of reproductive development at time of sampling using the method developed by

Brown-Peterson et al. (2011). This approach categorizes individuals along a reproductive development gradient based on the relative proportion and presence/absence of certain oocyte stages. To complete this analysis, Vtg oocytes were further separated into three sub-stages (primary [Vtg1], secondary [Vtg2], and tertiary [Vtg3] vitellogenesis) based on oocyte diameter, amount of yolk within cytoplasm, and the presence and appearance of oil droplets (Brown-Peterson et al. 2011; Fig. 7). Fish were then categorized as:

“immature”, “developing”, “spawning capable”, and “regressing”. The categorization of individuals in this way allowed for histological and hormonal metrics to be coupled for a finer scale evaluation of bonefish reproductive development.

Sex-hormone plasma level determination

17β-estradiol and testosterone concentration levels were quantified via enzyme- linked immunosorbent assay (ELISA) kits (Cayman Chemical Company, USA). For

extraction, a 100 l plasma sample was diluted with ELISA buffer based on the manufacturer specifications (Cayman Chemical Company, USA). Hormones were then extracted twice by vigorous vortex using dichloromethane for 17β-estradiol and diethyl ether for testosterone. The supernatant organic phase was removed via Pasteur pipette, evaporated under a gentle stream of nitrogen at 30 ˚C, and reconstituted using ELISA buffer prior to plating. This process was repeated according to the protocol provided by

Cayman Chemical Company, USA. Samples were run at two dilutions to fulfill manufacturer requirements and minimize interference within wells. A control sample provided by the manufacturer was serially diluted according to manufacturer specifications and run as a standard with each plate of samples. Plates were analyzed via absorbance at a wavelength of 405 nm using a microplate reader (Biotek, Synergy H1,

USA). Absorbance values were converted to concentration values (pg.ml-1) using software provided by Cayman Chemical Company, USA.

Luteinizing Hormones (LH) was quantified via enzyme-linked immunosorbent assay (ELISA) kits (MyBioSource, USA). A 50 μl plasma sample, 50 μl HRP conjugate, and 50 μl antibody were added to each well. These reagents were mixed and then incubated for 2 h at 37 °C. Following plate washing, any remaining wash buffer was aspirated or decanted off, and 50 μl each of substrates A and B were added to each well.

These substrate solutions were then incubated for 15 min at 37 °C in the dark, during which time they changed from colorless to dark blue. Following incubation, 50 μl stop solution was then added to each well, which changed the color from blue to yellow. The optical density of the solution in each well was then determined within 5 min using a microplate reader set to 450 nm.

Statistical analysis

Mean diameter of vitellogenic oocytes was compared across both island (Grand

Bahama, South Andros, and Central Andros, the Bahamas) and habitat (Flat and PSA) to test the null hypothesis that all sampled islands share the same spatial reproductive development relationship (Flat vs. PSA) previously identified by Luck et al. 2018. Data were found to meet parametric assumptions of normality and homoscedasticity and were therefore analyzed using a two-way nested ANOVA. “Habitat” and “Island” were treated as fixed factors with “Habitat” nested within “Island”. The significance threshold for all analyses were set at p < 0.05)

The frequency of occurrence of the three predominant oocyte stages (PG, CA, and

Vtg) was compared across islands (Grand Bahama, Central Andros, and South Andros) and habitats (Flats and PSA) using a nested permutational multivariate analysis of variance (PERMANOVA with 999 permutations), including a posteriori pair-wise comparisons with PRIMER 7 (v. 7.1.12) and PERMANOVA+ (v.1.0.2). “Island” and

“Habitat” were treated as fixed factors with “Habitat” nested within “Island”.

Assumptions of homoscedasticity were verified with a PERMDISP test, and data were transformed (arcsine square root) when necessary. Dissimilarity between both “island” and “habitat” was also evaluated using a SIMPER analysis. Variables with significant effect on dissimilarity among either island or habitat were individually analyzed following the PERMANOVA using two-way nested ANOVAs and a post hoc comparison (Tukey’s HSD).

Seasonality of 17ß-estradiol and testosterone concentrations were analyzed using a one-way ANOVA for each hormone. Analyses were conducted following a logarithmic

transformation of data to meet parametric assumptions of normality and homoscedasticity. Comparison by month included only bonefish females sampled in

March, April, and June 2018 as well as data collected from a non-spawning month the previous year (September 2017). Only bonefish sampled from flats habitat from Grand

Bahama were included in this comparison.

Spatial comparison (Island and habitat) of 17β-estradiol, testosterone, and LH concentrations were conducted via a two-way nested ANOVA. “Habitat” and “Island” were treated as fixed factors with “Habitat” nested within “Island”. Data was log- transformed to meet parametric assumptions of normality and homoscedasticity.

17β-estradiol and testosterone concentrations were compared across each reproductive phase using a one-way ANOVA following log transformation of hormone data. Analysis only included individuals sampled along flats of all three islands sampled

(Grand Bahama, South Andros, and Central Andros, The Bahamas). All ANOVA tests were performed using R software (R Core Team 2014).

RESULTS

Histology

During the spawning season, mean vitellogenic oocyte diameter across all islands

(Grand Bahamas, South Andros, and Central Andros) did not significantly differ (F (2, 31)

= 1.21, p = 0.31). However, mean vitellogenic oocyte diameter was significantly larger in fish sampled at the PSA compared to those sampled along the flats for both South Andros

(p = 0.01) and Grand Bahama (p = 0.03; Fig. 8). Mean vitellogenic oocyte diameter did not significantly differ between flats and the PSA at Central Andros (p = 0.60).

Composition of oocyte type present in female bonefish during spawning months differed

significantly by both island (pseudo-F (2, 44) = 3.99, p < 0.05) and habitat (pseudo-F (3,44) =

5.38, p < 0.001). Differences between islands were driven by a significantly higher proportional occurrence of PG oocytes in South Andros fish compared to those from

Grand Bahama (p = 0.01; Fig 9). Differences between flats and PSAs were driven by both a higher occurrence of PG in fish sampled along flats compared to those at the PSA

(F (3, 44) = 5.13, p < 0.01) and an inversely higher occurrence of Vtg oocytes in fish at

PSAs compared to those from flats (F (3, 44) = 7.20, p < 0.001; Table 2 and Fig. 9).

Sex hormone concentrations

I. Seasonal differences

17ß-estradiol and testosterone concentrations in bonefish sampled during the spawning months, March and April 2018, were statistically similar and combined prior to analysis (17ß-estradiol: t = 0.26, df = 1.095, p = 0.83; Testosterone: t = 0.03, df = 1.20, p

= 0.98). 17ß-estradiol values were significantly higher in fish sampled during March and

April 2018 (1792 ± 396 pg.ml-1) and September 2017 (821 ± 378 pg.ml-1) compared to

-1 those sampled during June 2018 (61 ± 20 pg.ml ) (F (2, 28) = 12.62, p < 0.001). 17ß- estradiol values from March and April 2018 were not significantly different from levels found in fish sampled in September 2017 (Fig. 10). Testosterone values were significantly higher in fish sampled during March and April 2018 (3163 ± 584 pg.ml-1) and September 2017 (3314 ± 523 pg.ml-1) compared to those sampled during June 2018

(1246 ± 256 pg.ml-1) (p < 0.001). Testosterone values from March and April 2018 were not significantly different from levels found in fish sampled in September 2018 (Fig. 10).

II. Spatial differences

Across all islands sampled during the spawning season, significant differences in mean concentration of 17ß-estradiol were observed (F (2, 43) = 4.13, p < 0.05).

Comparison between flats and PSAs within islands revealed mean concentrations of 17ß- estradiol were significantly higher at the PSA compared to flats habitat (F (3, 43) = 15.29, p

< 0.001) (Fig. 11). Testosterone levels were significantly different when compared across all islands (F (2, 28) = 3.80, p = 0.031). Within each island, testosterone concentrations were significantly higher in fish sampled from the PSA compared to those sampled along

flats (F (3, 28) = 7.18, p < 0.001). LH levels did not significantly differ when compared across all islands (F (2, 24) = 2.36, p = 0.115). Within each island, LH levels were significantly higher at PSAs compared to flats habitat (F (3, 24) = 3.43, p = 0.032; Fig. 11).

III. Reproductive Classification

Since mean 17β-estradiol and testosterone concentrations found in bonefish sampled along flats did not significantly differ by island, samples were pooled based on assigned reproductive classification (F (2, 27) = 0.92, p = 0.41). Significant differences in mean 17β-estradiol were found among reproductive phases exhibited by bonefish sampled along flats (F (3, 25) = 5.90, p < 0.01). A post hoc comparison revealed this difference was driven by significantly lower 17β-estradiol levels in “immature” and

“regressing” fish compared to those “developing” or “spawning capable” (Fig 12). Mean concentration for testosterone did not significantly differ by reproductive phase (F (3, 24) =

2.53, p = 0.08; Fig 12). All fish sampled at PSAs were spawning capable with 88.6% of spawning capable fish showing evidence of germinal vesicle migration (GVM) or breakdown (GVBD). Conversely, 29.0% of fish sampled along the flats were spawning capable with 3.1% exhibiting evidence of GVM or GVBD.

DISCUSSION

The findings of this study not only support the importance of habitat specificity for reproductively developing female bonefish but also indicate the potential for variability in this spatial relationship across other islands where bonefish thrive and actively spawn. The multi-island assessment of female bonefish reproductive development simultaneously provided a unique opportunity to assess the variability in spawning readiness within and across habitat types. These results provide valuable insight into the reproductive development of wild bonefish and are discussed below.

Seasonal development

The dramatically lower concentrations of both testosterone and 17β-estradiol found in fish sampled during June (non-spawning month) compared to those sampled in

March and April (spawning months) indicated significant decreases in sex hormone production outside of the spawning season. The previous chapter showed no significant differences in testosterone levels in bonefish sampled along Grand Bahama flats during spawning months (February and April 2017) compared to a non-spawning month

(September 2017). However, since September occurs one month prior to the first observed pre-spawning events (October), it was suggested that the lack in difference of testosterone between seasons was either due to sustained “elevated” concentrations, or that testosterone was acting as a preparatory precursor to the imminent spawning month.

The findings of this study support the latter suggestion based on the chronological increase of testosterone found in non-spawning females (September 2017) compared to the low levels found during June (a non-spawning month substantially outside the determined spawning season). The variability in concentrations of testosterone suggest

that as female bonefish pass through spawning and non-spawning months, testosterone is produced as a critical hormone for spawning preparation. Testosterone is thought to play a significant role in vitellogenesis through a catalyst reaction known as aromatization

(Kagawa 2013). During this reaction, testosterone is converted into 17β-estradiol, allowing levels of 17β-estradiol to quickly become elevated. Based on high levels of both testosterone and 17β-estradiol in female bonefish during the spawning season, the elevation of testosterone in September is likely followed shortly by increases in 17ß- estradiol concentrations. The lack of significant differences between levels of 17ß- estradiol between March/April 2018 and September 2017 contrasts with findings from

Chapter 1, which indicated significant differences between Feb/April 2017 and

September 2017. The lack in differences found in this present study is likely a product of the reproductive state of females sampled. In Chapter 1, females were identified based on the visible presence of yolked oocytes in the cannulas, evidence that all sampled individuals were likely either developing or spawning capable. Our analyses indicate developing and spawning capable females tend to have significantly higher levels of 17ß- estradiol when compared to immature or regressing fish. Sampling in this study included spawning capable, developing, immature, and regressing fish which increased the variance in observed 17ß-estradiol values and likely promoted lower mean values than what was found in 2017. However, it is important to note that the values found in

September 2017 were still relatively low with little variance compared to what was observed during March/April 2018 supporting the previous hypothesis that testosterone elevates prior to 17ß-estradiol in female bonefish just prior to the spawning season.

Spatial trends

The occurrence of vitellogenesis in female bonefish during secondary development of oocytes is heavily dependent on the presence of adequate levels of 17β- estradiol (Nagahama 1994). In bonefish, which exhibit ovarian characteristics of group synchronous batch spawners, this process occurs along a continuum as fish constantly prepare clutches of eggs prior to spawning (Mejri et al. 2018). Findings from Chapter 1 suggest that, based on levels of 17β-estradiol in fish sampled from Grand Bahama, vitellogenesis begins on the flats and continues through to the PSA where it is assumed to terminate. The findings here suggest this continuum is indeed occurring across all islands sampled given the continuously elevated levels of 17β-estradiol observed in females at both habitats. The significantly higher levels of both 17β-estradiol and testosterone found at the South Andros PSA may have been a consequence of the greater distance between the flats and PSA (~30 km) habitat compared to other islands (~12 km). Assuming the majority of vitellogenesis is occurring at the flats and during migration to PSAs, it is conceivable that fish are more likely to have significantly different levels of both 17β- estradiol and testosterone at their start (flats) and end (PSA) location given the greater distance, and therefore, more time available for hormone synthesis to occur. This same theory can also be applied to bonefish at Central Andros by simply examining the mean values presented. The differences in mean concentrations of both 17β-estradiol and testosterone at PSAs and flats habitats are noticeably greater at both Central and South

Andros islands compared to Grand Bahama, where flats were sampled in close proximity to the PSA (~12 km). Females may leave flats at the same time, undergoing the same timeline of oocyte development, and show up at the PSA earlier than fish making the same journey from flats further away. Another possible explanation is based on the

relative occurrence of GVM/GVBD at sampled PSAs. Bonefish sampled at PSAs in

Grand Bahama showed a lower proportion of spawning capable fish with GVM (~66 %) compared to South and Central Andros (~100%). In other words, while all fish at PSAs were spawning capable, all South and Central Andros spawning capable females sampled had reached the final stages of vitellogenesis compared to Grand Bahama. These contrasting levels of oocyte development between islands were likely driven by higher overall concentrations of LH observed in fish sampled at both Central and South Andros

Island PSAs compared to Grand Bahama. Since LH is widely known to be the primary gonadotropin which drives final maturation in fish, significantly higher levels found in

PSAs sampled fish from Andros Island and the subsequent occurrence of GVM indicates that fish at Andros have clearly begun final oocyte maturation (Nagahama and Yamashita

2008). Furthermore, several other studies have indicated that both testosterone and 17β- estradiol levels in fish can reach maximum concentration at the completion of vitellogenesis (Norberg et al. 2004, Kang et al. 2015). While it has become generally accepted that bonefish spawn with lunar periodicity (full and new moon; Danylchuk et al.

2011), peak development at the PSA just prior to offshore migration may actually vary by island. All three islands were sampled through the full moon of a known spawning month, assuming spawning events occur primarily during this time (Danylchuk et al.

2011, Adams et al. 2018). However, the lower mean 17β-estradiol and testosterone values from Grand Bahama PSAs sampled in 2017 (Luck et al. 2018) and 2018 (the current study) combined with the lower levels of LH compared to Central and South Andros indicate peak lunar development at PSA habitat in Grand Bahama may occur either earlier or later than other islands (before or after full moon, instead of during the full

moon). The ability to further address these hypotheses falls outside the scope of this study and would require monitoring of fish movement both to and from PSAs as well as offshore movement at several islands in concert with repeated hormonal and histological sampling. Such an approach could reveal a more specific timeline of what is occurring physiologically in bonefish during late stage development.

Within flats habitat, substantial variability was observed in the reproductive state of females during the spawning season at all three islands. The occurrence of four major reproductive phases (immature, developing, spawning capable, and regressing) commonly observed in reproductively active group synchronous marine spawners

(Lowerre-Barbierri et al 2011, Ganias and Lowerre-Barbieri 2017) suggests flats not only provide normal home range habitat year-round, but also expose individuals to the abiotic variables (temperature, salinity, photoperiod, etc.) that influence primary and secondary oocyte development (Hansen et al. 2001, Norberg et al. 2004, Holt et al. 2007, Mazzeo et al. 2014). The use of non-spawning habitat for initial oocyte development prior to spawning related movement has been observed in many species of batch spawning marine fishes including spotted seatrout (Cynoscion nebulosus), and tarpon (Megalops atlanticus) (Crabtree et al. 1997, Lowerre-Barbieri et al. 2009). Theoretically, this strategy allows energetically expensive processes such as vitellogenesis to occur where food is readily available and predatory threats are minimal prior to moving towards spawning habitat. The hormonal changes in 17β-estradiol and stability of elevated testosterone levels of fish ready to migrate to PSAs (spawning capable female bonefish) compared to those that are either rebuilding for the next spawn (regressing) or have just

begun initial development (immature) provides tremendous insight into our understanding of the vitellogenic process of wild bonefish.

The findings from this study not only expand upon our limited understanding of the seasonal reproductive dynamics of aggregate spawning marine fishes, but also provides a unique viewpoint on the physiological differences between two commonly used bonefish habitats during spawning months. In this study, I assessed hormonal and developmental variability along three geographically separate areas where bonefish occur and where PSAs have been identified. My results indicate that at each island sampled,

PSAs play a critical role in facilitating the final stages of vitellogenesis and onset of final maturation. The differences in testosterone, 17β-estradiol, and LH between Grand

Bahama and Andros PSAs suggests variability in peak spawning times may be occurring and requires significant further investigation into understanding aggregation periodicity.

My results have also clarified the importance of flats as critical habitats for reproductively developing females, which range from immature to spawning capable, suggesting that the majority of vitellogenic development occurs there. The implications of these findings are therefore extremely relevant to management, supporting the importance of protecting both flats and PSA habitats from anthropogenic disturbance.

CONCLUSIONS AND FUTURE DIRECTIONS

The findings of these two chapters highlight that vitellogenesis occurs in bonefish at both habitat types, beginning at the flats and concludes at the PSA. The elevated testosterone levels prior to the spawning season are perhaps preparatory reserves for aromatase but may facilitate some other process not yet understood. During the spawning season, the absence of hydrated oocytes at any sampling event indicates that final maturation is likely occurring at some point offshore at night, where spawning is presumed to take place. Initial oocyte maturation was observed exclusively at the PSA and, given the fact that no sampled fish showed evidence of hydrated eggs, likely occurs just prior to offshore migration. Further research regarding this period of development is critical to our understanding of bonefish reproductive physiology.

These migratory events from flats habitat to PSAs are still largely full understood in terms of the frequency of female visitation patterns and how long individuals remain at

PSAs. However, the findings from these studies indicate substantial differences between the two sites, particularly regarding the developmental status of females sampled at the

PSA. The occurrence of four major reproductive phases (immature, developing, spawning capable, regressing) present in flats fish across all three islands sampled compared to the singular occurrence of spawning capable females at PSAs also supports previous hypotheses that final oocyte development is only occurring at these PSAs.

Significantly higher quantities in all hormones (17β-estradiol, testosterone, and LH),

larger vitellogenic oocyte diameters, and the visible occurrence of germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) seen only in late vitellogenic/early final maturation stage oocytes at these aggregation sites confirm their occurrence as true prespawning aggregation sites.

While both of these studies provide significant information regarding bonefish reproductive ecology and physiology, conclusively understanding a species’ reproductive development should be done at the individual level through time. This is typically achieved in a controlled environment once the reproductive cycle has been “closed” and fish are successfully spawned, reared, and respawned captively. However, achieving this is tremendously challenging and requires an intense understanding of the hormonal profiles exhibited by fish in their natural spawning habitat. Given the current work underway at Harbor Branch Oceanographic Institute to successfully spawn bonefish in captivity, the data from this thesis should prove to be quite valuable in informing rearing protocols with baseline hormonal goals and expected developmental outcomes (e.g. Fig.

13). The benefits of successfully closing the reproductive cycle of this species are substantial. For one, critical information regarding duration of oocyte development, number of spawning events during a spawning season, and variability in fecundity among individuals can be gleaned from following individual females through time. Furthermore, the pelagic nature of bonefish leptocephalus and the inherent difficulties studying them makes captive rearing the only way to fully understand larval development, behavior, and feeding ecology. This information in turn, can be used to inform larval transport models which estimate both recruitment and expected abundances of juvenile bonefish as well as drive management decisions to conserve bonefish. It is therefore critical that further work

regarding bonefish reproduction is conducted, including spawning location identification and characterization, temporal and spatial spawning biotic and abiotic cues, and the estimating frequencies with which individuals spawn. These data gaps must be addressed to sustain declining populations of this economically important species

APPENDICES

Appendix A. Figures Appendix B. Tables

APPENDIX A. Figures

Figure 1: Map showing sampling locations for both flats and pre-spawn aggregation (PSA) sites during spawning (February, March, and April 2017) and non-spawning (September 2017) months of bonefish (Albula vulpes) in Grand Bahama Island, Bahamas. Exact PSA location has been generalized for conservation purposes (Adams et al. 2018).

Figure 2: Micrographs of histologically sectioned oocytes sample collected from a wild female bonefish (Albula vulpes) on a shallow water flat during full moon (April 2017). (a) three different developmental oocyte stages are present: (b) primary growth (PG), (c) cortical alveolar (CA), and (d) late vitellogenic (LV). Oocyte samples collected at the pre-spawn aggregation (PSA) were visually similar to flats samples

Figure 3: Proportional occurrence for each of the three oocyte stages: late vitellogenic (LV), cortical alveolar (CA), primary growth (PG) at the two sites (flats and pre-spawn aggregation [PSA]) sampled during the spawning month of April 2017. Oocytes were collected from sexually mature bonefish (Albula vulpes) females. Each bar indicates an individual fish. Table displaying mean  SD by oocyte stage has also been included.

Figure 4: Monthly plasma concentrations of 17β–estradiol and Testosterone (mean  SEM) from bonefish (Albula vulpes) females sampled in the flats during spawning (February and April 2017) and non-spawning (September 2017) months (mean  SEM). Letters indicate significant differences in levels of 17β –estradiol between seasons.

Figure 5: Plasma concentrations of 17β – estradiol and testosterone (mean  SEM) at flats and pre-spawn aggregation (PSA) sites in female bonefish (Albula vulpes) sampled during three spawning months (February, March, and April 2017) in Grand Bahama. Letters indicate significant differences in Testosterone levels between sites.

Figure 6. Map showing sampling locations across three islands in the Bahamas, and the respective flats and pre-spawn aggregation (PSA) habitat sampled for each. All islands were sampled during the full moon of a spawning month (Grand Bahama: March and April 2018; South Andros: December 2017; and Central Andros: January 2018) as well as one sampling event outside of the spawning season (Grand Bahama: June 2018) of bonefish (Albula spp.). Exact PSA locations have been generalized for conservation purposes (Adams et al. 2018).

Figure 7: Chronological schematic of different oocyte stages present during the reproductive development of bonefish (Albula spp.) including: primary growth (PG), cortical alveolar (CA), primary vitellogenic (Vtg 1), secondary vitellogenic (Vtg 2), tertiary vitellogenic (LV), germinal vesicle breakdown (GVBD), and germinal vesical migration (GVM).

Figure 8: Diameters of vitellogenic oocytes collected from female bonefish (Albula spp.) sampled at two different habitats (Flats and PSA) across three different islands (Grand Bahama, Central Andros, and South Andros, The Bahamas). Black line indicates median for each habitat type sampled. Letters indicate significant differences in mean oocyte diameters between habitat types across island.

Figure 9: Proportional distribution of primary growth (PG), cortical alveolar (CA), primary vitellogenic (Vtg 1), secondary vitellogenic (Vtg 2), tertiary vitellogenic (LV), and atretic oocytes found in female bonefish (Albula spp.) sampled from both the flats and PSAs of Grand Bahama, Central Andros, and South Andros, The Bahamas.

Figure 10: Seasonal variation in both 17β-estradiol and testosterone concentrations (mean ± SEM) for Bonefish (Albula spp.) sampled during both spawning (March/April 2018) and non-spawning months (June 2018, September 2017) along the flats of Grand Bahama. Black letters indicate significant differences in 17β-estradiol. Bolded black letters indicate significant differences in testosterone.

40000 14000

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2000 M 5000 M

0 0 Flats PSA Flats PSA Flats PSA Flats PSA Flats PSA Flats PSA Grand Bahamas Central Andros South Andros Grand Bahamas Central Andros South Andros

200 b

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M 20 0 Flats PSA Flats PSA Flats PSA Grand Bahamas Central Andros South Andros Figure 11: Spatial variation in 17β-estradiol, testosterone, and luteinizing hormone (LH) concentrations (mean ± SEM) for bonefish (Albula spp.) sampled across three islands (Grand Bahama, Central Andros, South Andros; The Bahamas) and two habitat types (Flats and PSA) within each island. Black letters indicate significant differences.

Figure 12: 17β-estradiol and testosterone concentrations (mean ± SEM) for each reproductive phase observed in flats from female bonefish (Albula spp.) across all Islands (Grand Bahama, South Andros, and Central Andros; The Bahamas). Letters indicate significant differences in mean concentration of 17β-estradiol across phases. No significant differences were observed for testosterone.

Figure 13: Non-linear regression (power) assessing correlation between vitellogenic oocyte diameter and 17β-Estradiol values from all mature female bonefish sampled from Grand Bahama (2017, 2018), Central Andros (2017), and South Andros (2018).

APPENDIX B. Tables Table 1: Sample sizes for all fish blood plasma (hormone) and oocyte (histology) collected at each Island (Grand Bahama, Central Andros, and South Andros, the Bahamas), habitat (Flats, PSA), and Month/Year.

Grand Bahama Central Andros South Andros March 2018 April 2018 June 2018 December 2017 January 2018 Flats PSA Flats PSA Flats Flats PSA Flats PSA Hormones 2 1 7 7 10 5 6 16 6 Histology 2 1 7 7 10 5 6 16 6

Table 2: Proportional distribution (mean ± SD) of each major oocyte stage (primary growth [PG], Cortical Alveolar [CA], and Vitellogenic [Vtg]) found in female bonefish (Albula spp.) across both Island (Grand Bahama, Central Andros, and South Andros, the Bahamas) and habitats (Flats and PSA). All vitellogenic stages (Vtg 1, Vtg2, Vtg3) have been combined for clarity. Letters have been applied to indicate significant differences among both islands and habitat for each oocyte stage.

Grand Bahama Central Andros South Andros Flats PSA Flats PSA Flats PSA PG 0.45 ± 0.27a 0.34 ± 0.19b 0.62 ± 0.13ac 0.54 ± 0.15bd 0.72 ± 0.22c 0.35 ± 0.16b CA 0.14 ± 0.17 0.06 ± 0.06 0.15 ± 0.07 0.06 ± 0.06 0.10 ± 0.08 0.05 ± 0.05 Vtg 0.42 ± 0.32b 0.60 ± 0.15a 0.22 ± 0.15b 0.40 ± 0.19a 0.18 ± 0.24b 0.59 ± 0.15a

Table 3: Statistical results for Chapter 2. Significance threshold for all analyses based on 95 % confidence, p < 0.05. Significant tests results have been bolded.

Test Item Source df F/Pseudo-F p Two-Way Island 2 1.2074 0.313 Oocyte Diameter ANOVA Habitat(Island) 3 8.7019 <0.001 Island 2 3.9978 0.019 PERMANOVA Oocyte Type Habitat(Island) 3 5.3845 0.001 Two-Way Island 2 4.1347 0.023 17ß-Estradiol ANOVA Habitat(Island) 3 6.5809 <0.001 Two-Way Island 2 3.8029 0.035 Testosterone ANOVA Habitat(Island) 3 3.0517 0.045 One-Way 17ß-Estradiol 2 12.619 0.001 Season (GB) ANOVA Testosterone 2 11.308 <0.001 60 One-Way 17ß-Estradiol 2 0.915 0.4126 Flats (GB, CA, SA) ANOVA Testosterone 2 1.677 0.2073 One-Way 17ß-Estradiol 3 5.9011 0.003 Repro Phase ANOVA Testosterone 3 2.5313 0.08102

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