REPRODUCTIVE POTENTIAL AND LIFE HISTORY OF SPOTTED GAR LEPISOSTEUS OCULATUS IN THE UPPER BARATARIA ESTUARY, LOUISIANA. A Thesis

Transcription

1 REPRODUCTIVE POTENTIAL AND LIFE HISTORY OF SPOTTED GAR LEPISOSTEUS OCULATUS IN THE UPPER BARATARIA ESTUARY, LOUISIANA A Thesis Submitted to the Graduate Faculty of Nicholls State University in Partial Fulfillment of the Requirements for the Degree Master of Science in Marine and Environmental Biology By Olivia Alpha Smith B. S., Nicholls State University, 2006 Spring 2008

2 CERTIFICATE This is to certify that the thesis entitled Reproductive potential and life history of spotted gar Lepisosteus oculatus in the upper Barataria Estuary, Louisiana submitted for the award of Master of Science to Nicholls State University is a record of authentic, original research conducted by Miss Olivia Alpha Smith under our supervision and guidance and that no part of this thesis has been submitted for the award of any other degree, diploma, fellowship, or other similar titles. APPROVED: SIGNATURE: DATE: Allyse Ferrara, Ph.D. Assistant Professor of Biological Sciences Committee Chair Quenton Fontenot, Ph.D. Assistant Professor of Biological Sciences Committee Member Gary LaFleur, Jr., Ph.D. Associate Professor of Biological Sciences Committee Member Enmin Zou, Ph.D. Associate Professor of Biological Sciences Committee Member i

3 ABSTRACT The spotted gar Lepisosteus oculatus is a physostomous fish that inhabits bayous, lakes, and backwater floodplains from the Great Lakes to the Gulf coast and from central Texas to western Florida. Although this species evolved over 150 million years ago, its reproductive potential is poorly understood. Gonad histology is useful for the identification and classification of gonad developmental phases of fish populations. The goal of this study was to characterize the reproductive potential of a spotted gar population in the upper Barataria Estuary in southeastern Louisiana using standard histological techniques. This study also focused on age and size distributions, total fecundity, egg sizes, and gonadosomatic index (GSI). From 5 October 2006 through 26 September 2007, spotted gar were collected weekly to biweekly from the upper Barataria Estuary, using monofilament gill nets, hook and line, and electrofishing. Histological samples were used to classify individuals into reproductive phases (immature, developing, spawning capable/actively spawning, regressing, and regenerating) based on gonad development. Based on histological analyses, males (N = 94) and most females (N = 123) in this population may be capable of spawning year round. However, because spawning did not occur year round, females most likely have a threshold egg size that is required for spawning. Females exhibited determinate fecundity and group-synchronous oocyte development. GSI peaked in spring and decreased through summer for both males (N = 215) and females (N = 253). Based on histological analyses and GSI values, spawning occurred from March through May. Mean egg diameter was 2.5 ± 0.3 mm (N = 131) for females collected from 9 February 2007 to 26 September Mean total fecundity was 6,493 ± 4,225 eggs per fish (N = 192; mean TL = 579 ± 44 mm). However, based on macroscopic observation of ovaries, the majority of spawned females did not spawn completely and, instead, retained and reabsorbed a portion of their eggs (atresia). Therefore, total fecundity estimates are probably overestimates of the ii

4 number of eggs annually spawned in the upper Barataria Estuary. Total length and age distributions were different between males and females. Females were longer than males of the same age for ages 2 through 5 and were heavier with greater girths than males of the same age for ages 3 through 5. More females were collected than males in the older age classes (3 to 6 years). The growth rate (k value from von Bertalanffy growth equation) was In our sample, male spotted gar matured by age 1 and 344 mm TL whereas females matured by age 2 and 410 mm TL. The life history strategy of spotted gar is most likely intermediate between periodic and equilibrium strategies with closer relation to the equilibrium strategy when compared to existing data from other gar populations. Reproductive characteristics and life history information from this study will be useful for understanding the reproductive potentials of gars and for formulating ecosystem-based management plans for the upper Barataria Estuary. iii

5 ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor, Dr. Allyse Ferrara, for her support and friendship during my entire educational career at Nicholls State University. She has been an amazing mentor during these years and has always opened many adventurous doors for me. I also want to sincerely thank the other members of my committee, Dr. Quenton Fontenot, Dr. Gary LaFleur, Jr., and Dr. Enmin Zou, for their never-ending assistance and guidance. I especially want to thank Dr. LaFleur for our many intriguing discussions on oogenesis. Gratitude is extended to the Department of Biological Sciences and the Bayousphere Research Laboratory at Nicholls State University for providing vessels, gear, and funding for my research. This study was also funded by a grant from Coastal Restoration and Enhancement through Science and Technology (CREST). I want to thank Ms. Dorinda Bearse, Ms. Anke Tonn, and all of the Nicholls faculty for their unending help and tolerance with me during my research. Thank you to all of the Nicholls students who assisted in field and lab work, especially Thomas Widgeon and Tim Clay for reading otoliths. I particularly want to thank Sean Jackson for his companionship and skills in our adventurous field excursions at night in the upper Barataria Estuary. Many thanks to Ms. Cheryl Crowder at the LSU School of Veterinary Medicine for processing my histology slides. Also, Ms. Nancy Brown-Peterson at the Gulf Coast Research Laboratory was a wealth of knowledge and continuous help with histology. Lastly, I want to deeply thank my parents, Denise and Dan Smith, for their continual love and support during my education. They are the reason I made it to where I am today. I also want to thank my brother, Andre, for his patience and use of his truck when the department s was unavailable and my sister, Madeleine, for her perpetual humor along the way. iv

6 TABLE OF CONTENTS CERTIFICATE...i ABSTRACT...ii ACKNOWLEDGEMENTS...iv TABLE OF CONTENTS...v LIST OF FIGURES...vi LIST OF TABLES...x INTRODUCTION...1 METHODS...16 RESULTS...26 DISCUSSION...57 FUTURE RECOMMENDATIONS...70 LITERATURE CITED...71 APPENDIX...79 BIOGRAPHICAL SKETCH...98 CURRICULUM VITAE...99 v

7 LIST OF FIGURES Figure 1. Spotted gar collected from the upper Barataria Estuary, Louisiana (photograph by Sean Jackson)...4 Figure 2. Location of the Barataria Estuary (dashed line) in southeastern Louisiana. Bar = kilometers...9 Figure 3. Boundaries, major waterways, and some of the major highways (dashed lines) of the upper Barataria Estuary. Bar = 7.7 kilometers...10 Figure 4. Oogenesis in fishes (as modified from West 1990; Brown-Peterson 2003). 2N diploid; 1N haploid; GVM germinal vesicle migration; GVBD germinal vesicle break down...13 Figure 5. Cystic spermatogenesis in fishes (as modified from Sadleir 1973). SG spermatogonium; 2N diploid; CY spermatocyst; SC spermatocytes; 1N haploid; ST spermatids; SZ spermatozoa...14 Figure 6. Percent of monthly catch of male (N = 215) and female (N = 253) spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January. Numbers above columns indicate the number of fish collected each month...29 Figure 7. Total length frequency distributions of male (N = 215) and female (N = 253) spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...31 Figure 8. Age frequency distributions of male (N = 207) and female (N = 246) spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...32 Figure 9. Relationship between log 10 weight and log 10 total length for male spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...34 Figure 10. Relationship between log 10 weight and log 10 total length for female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...35 vi

8 Figure 11. Histological section of a spawning capable/actively spawning male spotted gar (TL = 457 mm) testis with discontinuous/continuous germinal epithelia collected on 26 September 2007, in the upper Barataria Estuary. Bar = 0.1 mm. CY spermatocyst; SZ spermatozoa; GE germinal epithelium...36 Figure 12. Histological section of a spawning capable/actively spawning male spotted gar (TL = 485 mm) testis with discontinuous germinal epithelia collected on 10 March 2007, in the upper Barataria Estuary. Bar = 0.1 mm. SZ spermatozoa; GE germinal epithelium...37 Figure 13. Seasonal changes in germinal epithelia of male spotted gar (N = 94) collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January. Numbers above columns indicate the number of fish collected each month. C continuous germinal epithelia; DC discontinuous/continuous germinal epithelia; D discontinuous germinal epithelia...38 Figure 14. Histological section from the ovary of a spawning capable/actively spawning female spotted gar (TL = 652 mm) collected on 6 December 2006, in the upper Barataria Estuary. Bar = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte...39 Figure 15. Monthly reproductive phases for female spotted gar (N = 123) collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January. Numbers above columns indicate the number of fish collected each month. REGEN regenerating phase; DEV developing phase; SC/AS spawning capable/actively spawning phase...40 Figure 16. Histological section from the ovary of a developing female spotted gar (TL = 568 mm) collected on 30 June 2007, in the upper Barataria Estuary. Bar = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte...41 Figure 17. Histological section from the ovary of a regenerating female spotted gar (TL = 530 mm) collected on 23 March 2007, in the upper Barataria Estuary. Bar = 0.5 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte...42 vii

9 Figure 18. Ovaries from a female spotted gar (TL = 645 mm) collected on 31 May 2007, in the upper Barataria Estuary: (A) gross appearance of ovaries, (B) histological section of left portion of left ovary classified as regressing, and (C) histological section of right portion of left ovary classified as spawning capable/actively spawning. Overall, this female was classified as spawning capable/actively spawning. Bars = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte; POF post-ovulatory follicle...44 Figure 19. Histological section from the ovary of a developing female and potential virgin spotted gar (TL = 412 mm) collected on 31 August 2007, in the upper Barataria Estuary. Bar = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte...45 Figure 20. Histological section from the ovary of a spawning capable/actively spawning female spotted gar (TL = 652 mm) collected on 6 December 2006, in the upper Barataria Estuary. Bar = 0.1 mm. VTGO vitellogenic oocyte...46 Figure 21. Mean (± SD) gonadosomatic index (GSI) by sample date for male spotted gar (N = 215) collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January...47 Figure 22. Mean (± SD) gonadosomatic index (GSI) by sample date for female spotted gar (N = 253) collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January...48 Figure 23. Mean monthly egg diameter (± SD) for female spotted gar (N = 131) collected from 9 February 2007 to 26 September 2007, in the upper Barataria Estuary. Means with the same letter indicate no difference...49 Figure 24. Linear relationship between total fecundity and weight of female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...50 Figure 25. Linear relationship between total fecundity and total length of female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...51 Figure 26. Linear relationship between estimated count and whole count methods for determining total fecundity in female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...54 viii

10 Figure 27. von Bertalanffy growth curve, maximum theoretical total length (L ), von Bertalanffy growth coefficient (k), and time when total length would theoretically equal zero (t o ) for spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. L was derived from Suttkus (1963)...55 Figure 28. Catch-curve regression, total annual survival rate (S), total annual mortality rate (AM), instantaneous rate of total mortality (Z), and theoretical maximum age (Max age) for spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...56 ix

11 LIST OF TABLES Table 1. Processing procedure for histological preparation of spotted gar gonad samples (Histology Laboratory 2007a). Xylene (Thermo, Pittsburgh, Pennsylvania). P/V pressure/vacuum; abs absolute...19 Table 2. Staining procedure for histological preparation of spotted gar gonad samples (Histology Laboratory 2007b). Propar (Anatech, Ltd., Battle Creek, Michigan); Alcohol, absolute (AAPER Alcohol and Chemical Co., Shelbyville, Kentucky). N no; Y yes; abs absolute; W wash...20 Table 3. Reproductive classification system for male and female fishes according to histological characteristics of gonads (as modified from Brown-Peterson et al. 2007). Female regenerating phase was modified to include cortical alveolar oocytes. Information on indeterminate fecundity, hydration, and determining fecundity/spawning frequency was removed (This information either did not pertain to spotted gar or to this study s objectives.). PGO primary growth oocytes; CAO cortical alveolar oocytes; VTGO vitellogenic oocytes; POF post-ovulatory follicles; GVM germinal vesicle migration; GVBD germinal vesicle break down; SG spermatogonia; CY spermatocysts; SC spermatocytes; ST spermatids; SZ spermatozoa; GE germinal epithelia...21 Table 4. Description of reproductive classification system for male fishes according to histological characteristics of gonads (as modified from Brown-Peterson et al. 2007). SG spermatogonia; CY spermatocysts; SC spermatocytes; ST spermatids; SZ spermatozoa; GE germinal epithelia...22 Table 5. Description of reproductive classification system for female fishes according to histological characteristics of gonads (as modified from Brown-Peterson et al. 2007). PGO primary growth oocytes; CAO cortical alveolar oocytes; CA cortical alveoli; VTGO vitellogenic oocytes; POF post-ovulatory follicles...23 Table 6. Total number of each fish species collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...27 x

12 Table 7. Number (N), mean (± SD), and range of total length, pre-pelvic girth, weight, left gonad weight, right gonad weight, age, and egg diameter for male and female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...28 Table 8. Mean (± SD) and range (below mean) for total length (TL; mm), pre-pelvic girth (mm), and weight (g) of male (N = 207) and female (N = 246) spotted gar for each age class in which both sexes were collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. Differences between the sexes are marked with an asterisk...30 Table 9. Number (N), mean (± SD), and range of total fecundity for each age class of female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary...53 xi

13 INTRODUCTION The ancient garfish family Lepisosteidae consists of two genera (Atractosteus and Lepisosteus) and sixteen species (Wiley 1976). Only seven gar species are extant (alligator gar A. spatula, Cuban gar A. tristoechus, tropical gar A. tropicus, spotted gar L. oculatus, longnose gar L. osseus, shortnose gar L. platostomus, and Florida gar L. platyrhincus) and are confined to North America (Gilbert and Williams 2002). Lepisosteidae belongs to the Holostean group of fishes, which first evolved 290 million years ago (mya) during the Permian era and were very abundant during the Jurassic (206 mya) and Lower Cretaceous periods (146 mya; Rayner 1941). Extant Holosteans include the gars and bowfin Amia calva (Rayner 1941). Gars have elongated and cylindrical bodies that contain both bony and cartilaginous skeletons, posteriorly located dorsal fins, and rounded, abbreviate-heterocercal caudal fins (Eddy 1957; Suttkus 1963; Gilbert and Williams 2002). Members of Lepisosteidae are the exclusive fish group to possess ganoid scales, which are composed of layers of ganoin and isopedine (Ross 2001). Ganoid scales interlock, providing an armor-like covering that protects gars from predators (Gilbert and Williams 2002). Gars possess unique gamete transport systems. Unlike teleosts, male gars excrete urine and sperm through a single duct called the urogenital duct, and female gars possess a continuous oviduct that extends from the ovary to the vent (Pfieffer 1933; Sadleir 1973). Additionally, gars are the only freshwater fishes of North America to have toxic eggs (Brooks 1851; Goodger and Burns 1980). Gars and bowfin possess physostomous swim bladders, allowing them to respire at the water s surface (Potter 1927). When gulping oxygen at the water s surface, a gar transfers oxygen to its swim bladder via an open pneumatic duct that connects the dorsal region of the 1

14 esophagous to the anterior region of the swim bladder (Potter 1925, 1927). In the swim bladder, atmospheric oxygen is exchanged for carbon dioxide (Potter 1927). The ability to breathe air allows gars and bowfin to withstand hypoxic conditions (dissolved oxygen; DO < 2 mg/l), which are exacerbated at high temperatures, unlike many teleosts (Potter 1927; Eddy 1957; McCormack 1967; Renfro and Hill 1970; Hill et al. 1972; De Roth 1973). De Roth (1973) reported that the frequency of aerial respiration of spotted gar increases with increased temperature and is more common at night. The capacity to breathe air may help to explain why gars have somewhat reduced gill surface areas as compared to many teleosts (Landolt and Holt 1975). Smatresk (1986) demonstrated that aerial respiration in the longnose gar is controlled by external chemoreceptors in or near the gills and that gill respiration is controlled by internal chemoreceptors in or near the branchial circulation. The range of spotted gar includes the southern Great Lakes to the Gulf of Mexico and central Texas to western Florida (Douglas 1974). Spotted gar are commonly found in bayous, lakes, and backwater floodplains (Goodyear 1966; Douglas 1974; Snedden et al. 1999; Fontenot et al. 2001; Bonvillain 2006; Davis 2006). According to Goodyear (1966), spotted gar from the Mississippi Gulf coast are often found in shallow waters and prefer areas of thick vegetation or cover, such as fallen trees. In the Atchafalaya River Basin, Louisiana, Snedden et al. (1999) described the movement of spotted gar onto inundated floodplains during periods of high water in spring months and their association with shorelines during periods of low water in fall and winter months. Spotted gar prefer salinities ranging from 0 to 10 ppt although they have been observed in salinities of 18 ppt in Mississippi (Goodyear 1966). Spotted gar and Florida gar appear to be the least salt tolerant of the gar species (Suttkus 1963). In many areas, spotted gar 2

15 are top predators that control the abundance of lower trophic level species (Scarnecchia 1992; Ostrand et al. 2004). Adult spotted gar are brown to olive on their dorsal and upper lateral regions with lighter shades on their lower lateral and ventral regions (Figure 1; Ross 2001; Gilbert and Williams 2002). This species is often darker in color than the other gar species (Hoese and Moore 1998). The dorsal, anal, and pelvic fins possess brown bars, and all fins are spotted (Ross 2001). The signature brown and black spots on the mid-dorsal region appear when the fish is 100 to 150 mm total length (TL; Suttkus 1963). Spotted gar are distinct from other gar species by the presence of large spots on their heads (Ross 2001). Spotted gar living in darker colored and turbid waters are often darker in color than are those in clearer waters (Suttkus 1963). Spotted gar are sexually dimorphic in that females are typically longer and heavier than same age males (Tyler and Granger 1984; Ferrara 2001; Love 2002). Love (2002) reported that females collected in the Lake Pontchartrain Estuary, Louisiana, live longer than males. Additionally, females possess longer snouts than males; however, the ratio of snout length to head length changes with fish size and is, therefore, not an accurate identifier of sex (Suttkus 1963). Love (2002) reported that females in the Lake Pontchartrain Estuary have longer snouts than males when mass, snout width, body depth, and age are considered. Little information exists, however, on the snout morphology of different populations. Prey species of spotted gar include a variety of arthropods and smaller fish species. Goodyear (1967) documented blue crabs Callinectes sapidus and fiddler crabs Uca spp. as common prey items of spotted gar from the Mississippi Gulf coast. Smaller fish species that have been reported by stomach analyses include bluegill Lepomis macrochirus (Tyler and 3

16 Figure 1. Spotted gar collected from the upper Barataria Estuary, Louisiana (photograph by Sean Jackson). 4

17 Granger 1984), mosquitofish Gambusia affinis, pirate perch Aphredoderus sayanus, pygmy sunfish Elassoma zonatum (Dugas et al. 1976), and gizzard shad Dorosoma cepedianum (Bonham 1941). Dugas et al. (1976) also described spotted gar feeding on crayfish Procambarus spp. in the Atchafalaya River Basin. Spotted gar feed primarily at night (Snedden et al. 1999) or during incoming or high tides in coastal areas (Goodyear 1967). Spotted gar are lie-in-wait predators that remain motionless or swim very slowly when stalking prey before quickly snapping at their targets (Ostrand et al. 2004). According to Echelle and Riggs (1972), spotted gar are more abundant in shallow waters at night in Lake Texoma, Texas and Oklahoma, than during the day, and this abundance could indicate aggregations of feeding spotted gar. Spotted gar have few predators, but Valentine et al. (1972) reported that Lepisosteus spp. comprised 8 % of the diets of the American alligator Alligator mississippiensis in 1961 in southwestern Louisiana. Other predators of spotted gar include river otters Lontra canadensis and recreational fishermen (A. Ferrara and Q. Fontenot, Nicholls State University, personal communication). In the past, gars were often considered nuisance predators of game and commercial fishes (Gowanloch 1939, 1940; Suttkus 1963). Accordingly, some management programs for gar species emphasized eradication techniques (Sutton 1998), including electricity (Burr 1931) and traps (Gowanloch 1940). In more recent years, however, gars are enjoyed as game and food fish in the southeastern United States (Sutton 1998). In 2003, the value of Louisiana commercial fisheries landings for gars (alligator gar, longnose gar, shortnose gar, and spotted gar combined) was greater than $515,000 (LDWF 2003). Recently, research has been conducted on gar ecology (Snedden et al. 1999; Ferrara 2001; García de Leόn et al. 2001; Love 2004), and management 5

18 and conservation plans have been developed for some gar populations in the United States (Scarnecchia 1992; Todd 2005). Gars are not threats to game fish populations and sometimes act as scavengers (Eddy 1957; Suttkus 1963; García de Leόn et al. 2001). Spotted gar usually choose their prey by vulnerability and availability (Scott 1968) and more often feed on non-game fishes, such as gizzard shad, instead of game fishes, such as smallmouth bass Micropterus dolomieu and spotted bass Micropterus punctulatus (Bonham 1941). According to Echelle and Riggs (1972), the most abundant species in young gar (alligator gar, longnose gar, shortnose gar, and spotted gar) stomachs from Lake Texoma in 1965 was the Mississippi silverside Menidia audens, probably because this species has also been documented as the most abundant species in shallow waters of the lake. Dugas et al. (1976) also reported that although crayfish were a component (13 %) of spotted gar diets in the Atchafalaya River Basin in 1974 and 1975, spotted gar predation was not harmful to the crayfish harvest. Length and timing of spawning periods for spotted gar vary across the species range. Tyler and Granger (1984) reported that the earliest spotted gar spawning event in Lake Lawtonka, Oklahoma, was 22 April 1981, and the latest was 10 June Peak spawning time for this population was mid-may (Tyler and Granger 1984). Echelle and Riggs (1972) reported that spotted gar spawned in dead vegetation in calm waters in Lake Texoma and that spawning occurred between mid-april through May (temperature range: C). Spotted gar collected in Lake Seminole, Georgia, spawned from late spring to early summer (Ferrara 2001). The spawning period of a spotted gar population in the Lake Pontchartrain Estuary was February to June in 1999 (Love 2004). A population of Florida gar, a species of similar size to spotted 6

19 gar, from north central Florida was reported to spawn from February to March of 1998 (Orlando et al. 2003, 2007). Fertilization in spotted gar is external (Suttkus 1963). When spawning, a single female is followed by three to five males in shallow, vegetated water (Tyler and Granger 1984). Love (2004) described a spotted gar spawning event in April 1997, where six to eight fish were sighted near vegetation in water that was approximately 1.5 m in depth. Two of the fish were larger than the others and were assumed to be females (Love 2004). After spawning, gars typically leave the spawning site (Suttkus 1963). Tyler and Granger (1984) reported that a spawning event in Lake Lawtonka was interrupted by the onset of cooler temperatures and turbidity as a result of precipitation. In 2005, during induced spawning of spotted gar in the Bayousphere Research Laboratory at Nicholls State University, Louisiana, spawned eggs adhered to the sides and bottom of the spawning tank and to artificial vegetation (mean water temperature = 20.6 C; Boudreaux 2005). Fish were injected with Ovaprim on 23 April, spawning began on 25 April, and hatching was first observed on 30 April (Boudreaux 2005). After hatching, larvae attached to the walls of the holding tank and artificial vegetation via their anterior suctoral discs and began swimming 5 days later (Boudreaux 2005). Echelle and Riggs (1972) also noted that larval gars will attach to a film on the water s surface in aquaria. Spotted gar adults do not exhibit parental care after spawning (Suttkus 1963). According to studies in Lake Texoma, spotted gar are approximately 8 mm TL at hatching (Echelle and Riggs 1972). Yolk sac larval gars aggregate near their spawning sites, usually attached to vegetation or debris (Simon and Wallus 1989). If larvae become unattached from their substrates, they will 7

20 sink (Echelle and Riggs 1972) or will swim to re-attach to available substrates (A. Ferrara, Nicholls State University, personal communication). Simon and Wallus (1989) reported that the majority of larval gar (longnose gar and spotted gar) were collected from the top meter of the water column in the Ohio and Tennessee River Basins and were collected during the day. Larval spotted gar can grow at a rate of 1.7 mm per day (range: mm per day; Simon and Wallus 1989). The suctoral disc in spotted gar disappears at approximately 17.6 mm TL, and the yolk sac is completely absorbed at greater lengths (Simon and Wallus 1989). After absorption of the yolk sac, gars disperse and begin aerial respiration and feeding (Echelle and Riggs 1972). In spotted gar, flexion commences at 21.9 mm TL, and all of the fin rays have begun development by 35.9 mm TL (Simon and Wallus 1989). This study was conducted in the upper reaches of the Barataria Estuary, Louisiana. The Barataria Estuary is bordered by the Mississippi River to the east and Bayou Lafourche to the west (Figure 2) and contains cypress swamps, freshwater marsh, intermediate marsh, brackish marsh, and saltwater marsh. The upper Barataria Estuary is a cypress-tupelo swamp that includes the following major waterways: Grand Bayou, Bayou Citamon, Bayou Chevreuil, the St. James Canal, and Lac Des Allemands, which drain in an east-southeast direction (Figure 3). Overall, 41.5 % of the upper Barataria Estuary is forested wetlands (Braud et al. 2006). Agricultural lands comprise 38.0 % of land use in the upper Barataria Estuary (Braud et al. 2006), and many of these lands drain into the St. James Canal. The upper Barataria Estuary once received an annual floodpulse from the Mississippi River. However, due to levee construction, the upper Barataria Estuary is no longer annually inundated by a predictable floodpulse. Presently, inundation of the upper Barataria Estuary floodplain results from heavy, local precipitation (Sklar and Conner 1979). 8

21 N Figure 2. Location of the Barataria Estuary (dashed line) in southeastern Louisiana. Bar = kilometers. 9

22 N Mississippi River Highway 70 Bayou Citamon Bayou Lafourche Highway 20 St. James Canal Grand Bayou Bayou Chevreuil Lac Des Allemands Highway 3127 Lake Beouf Highway 90 Figure 3. Boundaries, major waterways, and some of the major highways (dashed lines) of the upper Barataria Estuary. Bar = 7.7 kilometers. 10

23 The timing and duration of a river-driven floodpulse correspond with the spawning periods of many fish species in large-river floodplains (Junk et al. 1989). During periods of high water, many species of fish (e.g., spotted gar and bowfin) move onto inundated floodplains to feed and spawn in the shallow, vegetated waters (Snedden et al. 1999; Bonvillain 2006; Davis 2006). Therefore, the lack of an annual, river-driven, predictable floodpulse may have negative impacts on the reproductive success of floodplain-dependent fish species. When floodplaindependent species are denied access to suitable spawning habitat, the reproductive output of the populations may decline. Additionally, when the floodpulse is absent, primary and secondary production decrease in floodplain systems, reducing food availability for fish species that forage on the inundated floodplain (Bayley 1995). In 2006, macroscopic examination of bowfin ovaries from the upper Barataria Estuary revealed egg atresia (retention and reabsorption of eggs) in 96 % of females sampled from February to May (N = 136; Davis 2006). Apparently, in 2006, the majority of bowfin did not spawn in this system. Bowfin typically move onto inundated floodplains during periods of high water to spawn and forage (Davis 2006). Water levels in the upper Barataria Estuary were below that needed for inundation of the adjacent floodplain during the bowfin s spawning season (February through March) in 2006 (Davis 2006; Estay 2007). However, based on gonadosomatic indices (GSI), the gizzard shad population in the upper Barataria Estuary spawned from late March through May 2006 (Fontenot 2006). Additionally, GSI, age distributions, and size distributions have been determined for bowfin (Davis 2006) and gizzard shad (Fontenot 2006) populations in the upper Barataria Estuary. Unlike the bowfin and gizzard shad populations, there is little information on the life history and reproduction of spotted gar in the upper Barataria Estuary. Before the current thesis, only GSI, gross examination of gonads, 11

24 and egg counts have been used to describe the reproduction of spotted gar (Tyler and Granger 1984; Ferrara 2001; Love 2004). Therefore, a detailed analysis is needed to better understand the reproductive cycle of spotted gar in this system. Gonad histology is the most accurate method for assessing gonad development (West 1990) and involves microscopically examining a portion of gonads to classify individuals into reproductive phases. As male and female fishes progress through their reproductive cycles, they undergo phases that are identifiable with the use of gonad histological techniques. Individuals can be identified as immature (not capable of spawning), developing (active gametogenesis and not capable of spawning), mature (capable of or actively spawning), regressing (retention and reabsorption of gametes), and regenerating (preparation of new generation of gametes; Brown- Peterson et al. 2007). By quantifying and categorizing individual males and females into reproductive phases, a population s reproductive cycle can be better analyzed. Gonad histological techniques are typically used on fish species of high economic value and have been successfully applied to a variety of species, including common snook Centropomus undecimalis (Lowerre-Barbieri et al. 2003), spotted seatrout Cynoscion nebulosus (Brown-Peterson et al. 1988), cobia Rachycentron canadum (Brown-Peterson et al. 2002), and northern anchovy Engraulis mordax (Hunter and Macewicz 1984). However, gonad histology has been used to describe the reproductive cycle of only two gar species, Florida gar (Orlando et al. 2003, 2007) and tropical gar (A. Hernández-Franyutti, Universidad Juárez Autόnoma de Tabasco, personal communication). By accurately defining different stages of oogenesis (Figure 4) and spermatogenesis (Figure 5), individual spotted gar can be classified into reproductive phases based on gonad development. Histological techniques can be used to more specifically describe the reproductive biology of the spotted gar population in the upper Barataria Estuary. 12

25 Primary Growth Oocytes Perinucleolar Oocyte (2N) Chromatin Nucleolar Oocyte (2N) Oogonium (2N) Germinal Vesicle Nucleolus/ Nucleoli Vitellogenic Oocyte (2N) CA Oocyte (2N) Vitelline Envelope Follicle Cell Yolk Vesicle Thecal Cell Cortical Alveoli Final Oocyte Maturation (species-specific): Lipid Coalescence, GVM, GVBD, Yolk Coalescence, Hydration, Meiosis I (release of first polar body), Ovulation Ripe Oocyte (2N) Ova (1N) Figure 4. Oogenesis in fishes (as modified from West 1990; Brown-Peterson 2003). 2N diploid; 1N haploid; GVM germinal vesicle migration; GVBD germinal vesicle break down. 13 Spawning and Meiosis II (release of second polar body)

26 Remain as Primary SG or Stem Cells Mitosis CY with Primary SC (2N) Primary SG (2N) Mitosis Secondary SG (2N) Meiosis I CY with Secondary SC (1N) CY with SZ (1N) CY with ST (1N) Meiosis II Spermiogenesis Spermiation (released into lumens of lobules) SZ Travel to Sperm Ducts Spawning Figure 5. Cystic spermatogenesis in fishes (as modified from Sadleir 1973). SG spermatogonium; 2N diploid; CY spermatocyst; SC spermatocytes; 1N haploid; ST spermatids; SZ spermatozoa. 14

27 Additionally, gonad histology can verify macroscopic observations of spawning and egg atresia in spotted gar. When combined with GSI, fecundity, and age and size distribution data, histological analyses of gonads can produce a detailed reproductive characterization of this spotted gar population. There is a lack of life history information on spotted gar populations due to the notion that spotted gar are a limitless, non-game species. Population models designed for the population in the upper Barataria Estuary could be developed and modified for spotted gar populations elsewhere. Specifically, information from this study will be useful for regions, such as the northern United States and southern Canada, that are interested in spotted gar management and conservation. The goal of this study was to describe reproductive phases and to determine the life history of spotted gar in the upper Barataria Estuary. This study included histological analyses of gonad development and assessment of life history characteristics. The specific objectives of this project included the following: 1.) Document and quantify reproductive phases of male and female spotted gar in the upper Barataria Estuary for a year using standard histological techniques, 2.) Determine sex-specific age and size distributions of spotted gar in the upper Barataria Estuary, 3.) Quantify sex-specific, seasonal changes in GSI of spotted gar in the upper Barataria Estuary, 4.) Quantify age-specific fecundity of female spotted gar in the upper Barataria Estuary, and 5.) Quantify seasonal changes in egg size of female spotted gar in the upper Barataria Estuary. 15

28 METHODS Field Sampling Spotted gar were collected weekly to biweekly from 5 October 2006 to 26 September 2007 (except for January 2007) in the upper Barataria Estuary, using monofilament gill nets, hook and line, and electrofishing. Monofilament gill nets were either 28 or 50 m long and 1.8 m deep and contained one of three different bar mesh combinations (38 mm, 95 mm, or 25.4 mm/38 mm experimental bar mesh). Gill nets were placed parallel to the bank, either near small channels with floodplain access or large beds of floating (e.g., water hyacinth Eichhornia spp.) and/or submerged (e.g., coontail Ceratophyllum demersum) aquatic vegetation. Electrofishing was conducted with a 5.0kW Smith-Root (Generator Powered Pulsator) Electrofisher System. Spotted gar were stored in an ice chest until being processed in the Bayousphere Research Laboratory at Nicholls State University. All fish were processed within 17 hours of collection. At each sample location, dissolved oxygen (mg/l), temperature (ºC), specific conductance (µs), and salinity (ppt) were measured with a handheld YSI 85 meter (Yellow Springs Instruments, Yellow Springs, Ohio). If sampling occurred between 1000 and 1600 hours central standard time (CST) and when cloud cover was minimal, Secchi disk depth (cm) was measured to determine water clarity. At the intersection of Bayou Citamon, Bayou Chevreuil, and the man-made canal that connects to Grand Bayou, a Louisiana Department of Natural Resources (LDNR) staff gauge was used to measure relative water level (cm). Laboratory Processing In the Bayousphere Research Laboratory, each individual was assigned a unique identification number. Total length (mm), pre-pelvic girth (mm), and body weight (g) were 16

29 measured for each spotted gar. To retrieve the gonads, spotted gar were cut from the vent to the head using tin snips. Sex determination was based on the gross examination of gonads and gamete release pathways (Ferrara and Irwin 2001). Photographs were taken of whole ovaries for macroscopic examination. Left and right gonad weights (g) were measured. GSI was calculated according to the equation derived by Snyder (1983): GSI = (gonad weight) / (total body weight) x 100. Each month (except for January 2007), up to fifteen male and fifteen female spotted gar were used for gonad histology. Using a scalpel, a small portion (approximately 1 g) of one gonad from each individual was removed and preserved in a labeled vial containing 10 % neutral buffered formalin (NBF; Fisher Scientific, Kalamazoo, Michigan). Ten fresh eggs, prior to preservation, were randomly selected from the ovaries of each female spotted gar, and egg diameters (mm) were measured using digital calipers (Davis 2006). Egg diameters were only measured for large, visible eggs sampled from 9 February 2007 to 26 September The remaining portions of whole gonads were preserved in labeled jars containing 10 % non-buffered formalin (Fisher Scientific, Fair Lawn, New Jersey). For each spotted gar, sagittal otoliths were removed, washed, dried, and placed in labeled, plastic vials for age determination (Ferrara 2001). Gonad Histology, Fecundity, and Age Determination Gonad histology samples were cut (approximately 5 mm thick), placed in labeled tissue cassettes, and preserved in 75% ethyl alcohol (StatLab, Lewisville, Texas) for 1 to 6 days before being sent to Louisiana State University (LSU). Samples were processed onto microscope slides by the Histology Laboratory in the Department of Pathobiological Sciences at the LSU School of Veterinary Medicine. Samples were subjected to a dehydration series and embedded in paraffin 17

30 (McCormick Scientific, St. Louis, Missouri; Table 1). Samples were then sliced at approximately 5 µm and subjected to staining with hematoxylin and eosin (Anatech, Ltd., Battle Creek, Michigan; Table 2). Slides were viewed using compound and/or dissecting microscopes, and digital photographs were taken of each slide. Male and female samples were classified into corresponding reproductive phases based on a modification of the system developed by Brown- Peterson et al. (2007; Table 3). Descriptions of the modified reproductive classification system were established for males (Table 4) and females (Table 5) to provide physical/visual descriptions of spotted gar gonad histology. For histological analyses of both sexes, the spawning capable and actively spawning phases were combined. In males, the distinguishing factor for these two phases is the gross observation of free flowing milt, which was not observed in this study. The distinguishing factor for females is the ability to age postovulatory follicles, which has not yet been determined. Total fecundity, the number of advanced vitellogenic eggs in an ovary at a particular time (Hunter et al. 1992), was determined by counting all visible eggs in a 10 % (by weight) subsample of each ovary (Ladonski 1998). Total number of eggs in each ovary was extrapolated by multiplying the number of eggs in the 10 % subsample by 10 (estimated count). Total fecundity estimates did not include females that showed macroscopic evidence of recent spawning (N = 61). Each month, whole counts of both ovaries were determined for two randomly selected female spotted gar (whole count). Multiple readers (N = 3) determined ages of individual spotted gar by examining annuli on whole sagittal otoliths submerged in water using a dissecting microscope (Ferrara 2001). 18

31 Table 1. Processing procedure for histological preparation of spotted gar gonad samples (Histology Laboratory 2007a). Xylene (Thermo, Pittsburgh, Pennsylvania). P/V pressure/vacuum; abs absolute. Reagent Laboratory Station Time (minutes) Temperature ( C) P/V Stir Alcohol, 70 % 1 Until start Ambient No On Alcohol, 80 % 2 30 Ambient No On Alcohol, 95 % 3 30 Ambient No On Alcohol, abs 4 30 Ambient No On Alcohol, abs 5 30 Ambient No On Xylene 6 30 Ambient No On Xylene 7 40 Ambient No On Xylene 8 50 Ambient No On Paraffin Left Yes On Paraffin Middle Yes On Paraffin Right Yes On 19

32 Table 2. Staining procedure for histological preparation of spotted gar gonad samples (Histology Laboratory 2007b). Propar (Anatech, Ltd., Battle Creek, Michigan); Alcohol, absolute (AAPER Alcohol and Chemical Co., Shelbyville, Kentucky). N no; Y yes; abs absolute; W wash. Event Laboratory Station Reagent Time (minutes) Exact 1 Oven Oven 65 C 8:00 N 2 1 Propar 2:00 N 3 2 Propar 2:00 N 4 3 Propar 1:00 N 5 4 Alcohol, abs 0:30 N 6 5 Alcohol, 90 % 0:30 N 7 6 Alcohol, 80 % 0:30 N 8 W5 Wash 0:30 N 9 9 Hematoxylin 2:30 Y 10 W4 Wash 1:00 N Acid Alcohol 0:05 Y 12 W3 Wash 0:30 N Ammonia Water 1:00 Y 14 W2 Wash 0:30 N Alcohol, 95 % 1:00 N Eosin 1:00 Y Alcohol, 95 % 0:30 N Alcohol, abs 0:30 N Alcohol, abs 0:30 N Alcohol, abs 0:30 N Xylene 1:00 N 22 Exit Xylene 0:30-15:00 N 20

33 Table 3. Reproductive classification system for male and female fishes according to histological characteristics of gonads (as modified from Brown-Peterson et al. 2007). Female regenerating phase was modified to include cortical alveolar oocytes. Information on indeterminate fecundity, hydration, and determining fecundity/spawning frequency was removed (This information either did not pertain to spotted gar or to this study s objectives.). PGO primary growth oocytes; CAO cortical alveolar oocytes; VTGO vitellogenic oocytes; POF post-ovulatory follicles; GVM germinal vesicle migration; GVBD germinal vesicle break down; SG spermatogonia; CY spermatocysts; SC spermatocytes; ST spermatids; SZ spermatozoa; GE germinal epithelia. Phase Male Female Immature Small testes, only primary SG, no lumens in lobules. Only oogonia and PGO present. Usually no atresia. Developing Initiation of spermatogenesis and formation of CY. Secondary SG, primary SC, secondary SC, ST, and SZ can be present in CY. No SZ in lumens of lobules or sperm ducts. GE continuous. PGO, CAO, early VTGO, and mid VTGO may be present. No POF. Some atresia can be present. Spawning capable SZ in lumens of loblues and/or sperm ducts. All stages of spermatogenesis (SG, SC, and ST) can be present. CY throughout testis. GE continuous or discontinuous. Histologically undistinguishable from actively pawning phase. VTGO predominant. Some atresia and old POF may be present. Less-developed oocytes often present. Actively spawning SZ in lumens of lobules and/or sperm ducts. All stages of spermatogenesis (SG, SC, and ST) can be present. CY throughout testis. GE continuous or discontinuous. Histologically undistinguishable from spawning capable phase. Ovulating (spawning) or approximately 12 hours prior to or after spawning as indicated by either GVM, GVBD/hydrated oocytes, or POF <~12 hours old. Atresia of late VTGO may be present. Regressing Residual SZ in lumens of lobules and sperm ducts. Widely scattered CY near periphery containing ST. SG proliferation and GE regeneration common in periphery of testis. Atresia present (any stage). Majority of VTGO undergoing early atresia. Less-developed oocytes often present. POF may be present. Regenerating No CY. Lumens of lobules small or nonexistent. Proliferation of primary, occasionally secondary, SG throughout testis. Residual SZ may be present in lumens of lobules and sperm ducts. Only oogonia, PGO, and CAO present. Muscle bundles, enlarged blood vessels, thick ovarian wall and/or late atresia may be present.

34 Table 4. Description of reproductive classification system for male fishes according to histological characteristics of gonads (as modified from Brown-Peterson et al. 2007). SG spermatogonia; CY spermatocysts; SC spermatocytes; ST spermatids; SZ spermatozoa; GE germinal epithelia. Phase Immature Developing Spawning capable Actively spawning Regressing Regenerating Description Only primary SG present along edges of lobules. Primary SG are large and stained light purple. Lobules present with no lumens inside (Each lobule is an individual circle with its own germ cells.). Secondary SG (smaller and darker than primary SG) give rise to CY that form along edges of lobules. CY are clusters of cells in the same stage of spermatogenesis. Secondary SG, primary SC, secondary SC, ST, and SZ may be present in CY. As spermatogenesis proceeds from SG to SZ, cells become smaller, are more abundant, and are more darkly stained. ST and SZ are similar in appearance except that SZ possess bright pink tails. No SZ are present in lumens of lobules. Throughout testis, GE is continuous, indicating that lobules are completely lined with CY. SZ have been released into lumens (empty space in middle of lobules) and sperm ducts. Sperm ducts are stained bright pink and are a series of tubes that eventually lead to the vas efferentia of the testis. SZ are scattered in lumens and not in tight clusters as in CY. SG, SC, and ST may also be present in CY. GE can be continuous or discontinuous (lobules are not completely lined by CY) throughout testis. Histologically undistinguishable from actively spawning phase. SZ released into lumens of lobules and sperm ducts. SG, SC, and ST may also be present in CY. GE may be continuous or discontinuous throughout testis. Histologically undistinguishable from spawning capable phase except for macroscopic examination of free flowing milt (with gentle pressure) from fish s vent. Majority of lumens are empty except for a few with residual SZ. Some residual SZ in sperm ducts. Scattered CY containing ST near edge of testis. Formation of primary SG and regeneration of GE near edge of testis. No CY present. Lumens are small and difficult to see. Formation of primary and secondary SG throughout entire testis. Sometimes, residual SZ in lumens and sperm ducts. 22

35 Table 5. Description of reproductive classification system for female fishes according to histological characteristics of gonads (as modified from Brown-Peterson et al. 2007). PGO primary growth oocytes; CAO cortical alveolar oocytes; CA cortical alveoli; VTGO vitellogenic oocytes; POF post-ovulatory follicles. Phase Immature Developing Spawning capable Actively spawning Regressing Regenerating Description Oogonia typically not visible. PGO are small and stained dark purple. PGO nuclei are large and stained light pink. Tissue and cells are tightly associated and not scattered. PGO present. CAO are slightly larger and stained light purple. CA are small, light purple spheres that form a circle inside CAO. Early VTGO are similar to CAO (in size) but possess small, bright pink yolk vesicles that form a circle inside VTGO. Mid VTGO have substantially more yolk vesicles and are larger in size. Mid VTGO possess a thin, pink, striated vitelline envelope. PGO, CAO, early VTGO, and mid VTGO possess follicle and thecal cells (thin purple layers surrounding oocyte) that may be difficult to distinguish. Atresia includes degraded structures. Early atresia of late VTGO are degraded VTGO with loss of yolk vesicles. Late atresia are light purple structures with several empty holes, indicating previous location of fatty tissue. Atresia may also occur on CAO, early VTGO, mid VTGO, and late VTGO. Late VTGO are prominent and are more than twice the size of mid VTGO. Late VTGO possess a wide, pink vitelline envelope and a thin outer layer of purple follicle and thecal cells. PGO and CAO also present. Old POF are thick, convoluted strands of light purple follicle cells. Early and late atresia may be present. Few late VTGO present. PGO and CAO also present. New POF are prominent and are thin, dark purple convoluted strands. Some early atresia of late VTGO may be present. Early and late atresia present. Majority of cells are degraded. PGO, CAO, and sometimes old POF present. Many scattered cells from old POF and atretic cells are present. Only PGO and CAO present. Muscle bundles are scattered and thick. Blood vessels often enlarged. Is similar to immature in appearance but oocytes are more scattered and tissues are loose or used in appearance. Late atresia may be present. 23

36 Statistical Analyses A chi-square test was used to compare sex-specific differences in catch throughout the sampling year (SAS 2003). Two-sample student s t-tests (assuming equal variance) were used to determine if males and females differed in TL, girth, and weight for each age class (in which both sexes were collected) and to determine whether left and right gonad weights were different for each sex. Kolmogorov-Smirnov two-sample tests were used to compare the distributions of TL and age between the sexes. Total length and weight were log 10 -transformed, and linear regressions were used to quantify the relationships between the two measurements for each sex (SAS 2003). Seasonality of reproductive phases was plotted separately for male and female spotted gar to identify the spawning season. Mean GSI was plotted separately for males and females for each sample date and was used with histological analyses to identify the spawning season. Linear regressions were used to quantify the relationships between total fecundity and weight and between total fecundity and TL for female spotted gar (SAS 2003). Mean fecundity was calculated for each age class. A linear regression was used to quantify the relationship between the estimated count and the whole count methods for estimating total fecundity (SAS 2003). Mean egg diameter was plotted by month. Mean egg diameter was log 10 -transformed and subjected to a two-way analysis of variance (ANOVA) followed by Tukey s post hoc comparison to determine monthly differences (SAS 2003). Mean TLs at age were calculated for each sex. Even though TL of females differed from males in the same age classes, a single von Bertalanffy growth curve was developed for both sexes (FAST Version 3.0; Slipke and Maceina 2001) due to the absence of individuals in some age classes (e.g., age 1 females). The L was forced to 819 mm, the maximum TL reported by Suttkus (1963). Maximum theoretical TL (L ), von Bertalanffy growth coefficient (k), and time when TL would theoretically equal zero (t o ) were determined (Slipke and Maceina 2001). A catch-curve regression was used to determine 24

37 instantaneous rate of total mortality (Z), total annual mortality rate (AM), total annual survival rate (S), and theoretical maximum age of spotted gar (Slipke and Maceina 2001). All tests were based on α =

38 RESULTS Field Data A total of 615 spotted gar were collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. Four-hundred and sixty-eight of these individuals were used for this study, and the remainder were released. Eighteen additional fish species were collected during this study (Table 6). Overall, more female spotted gar (N = 253) were collected than males (N = 215; Table 7). The sex ratio of females to males was 1.2 : 1. Females dominated the catch throughout the sampling period except in February, March, and April (Figure 6). In July, the number of females collected equaled number of males collected (Figure 6). In February, more males were collected than females (chi-square, P < ). In October, more females were collected than males (chi-square, P < ). Dissolved oxygen ranged from 0.13 to mg/l with an average of 2.33 ± 2.09 mg/l (± standard deviation; SD). Temperature ranged from 8.4 to 32.6 C with an average of 20.9 ± 7.5 C. Specific conductance ranged from 99.0 to 1,136.0 µs with an average of ± µs. Secchi disk depth ranged from 0 to 100 cm with an average of 35 ± 18 cm. Salinity ranged from 0.0 to 0.6 ppt with an average of 0.1 ± 0.1 ppt. Water level ranged from to cm with an average of ± cm. Laboratory Data Females were longer than males for all age classes in which both sexes were collected (Table 8). Females were heavier and had greater girths than males in age classes 3, 4, and 5 but not age class 2 (Table 8). Left ovaries were heavier than right ovaries (P < ), but no difference was observed between left and right testes weights (P = ; Table 7). Total length (Figure 7) and age (Figure 8) frequency distributions were different for males and females. 26

39 Table 6. Total number of each fish species collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. Species Common Name Number Lepisosteus oculatus Spotted gar 615 Dorosoma cepedianum Gizzard shad 226 Ictalurus furcatus Blue catfish 39 Pomoxis nigromaculatus Black crappie 35 Amia calva Bowfin 34 Ictalurus punctatus Channel catfish 27 Mugil cephalus Striped mullet 10 Ictiobus bubalus Smallmouth buffalo 8 Lepomis macrochirus Bluegill 6 Micropterus salmoides Largemouth bass 5 Morone mississippiensis Yellow bass 5 Dorosoma petenense Threadfin shad 4 Lepomis microlophus Redear sunfish 4 Aplodinotus grunniens Freshwater drum 3 Chaenobryttus gulosus Warmouth 3 Ameiurus spp. Bullhead 2 Micropogonias undulatus Atlantic croaker 2 Atractosteus spatula Alligator gar 1 Cyprinus carpio Common carp 1 Total 1,030 27

40 Table 7. Number (N), mean (± SD), and range of total length, pre-pelvic girth, weight, left gonad weight, right gonad weight, age, and egg diameter for male and female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. Variable N Mean ± SD Range Males Total length (mm) ± Girth (mm) ± Weight (g) ± ,050.0 Left gonad weight (g) ± Right gonad weight (g) ± Age (years) ± Females Total length (mm) ± Girth (mm) ± Weight (g) ± ,710.0 Left gonad weight (g) ± Right gonad weight (g) ± Age (years) ± Egg diameter (mm) ±

41 Figure 6. Percent of monthly catch of male (N = 215) and female (N = 253) spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January. Numbers above columns indicate the number of fish collected each month. 29

42 Table 8. Mean (± SD) and range (below mean) for total length (TL; mm), pre-pelvic girth (mm), and weight (g) of male (N = 207) and female (N = 246) spotted gar for each age class in which both sexes were collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. Differences between the sexes are marked with an asterisk. Age (years) Measurement Male Mean ± SD (Range) Female Mean ± SD (Range) 2 TL* 3 TL* 4 TL* 5 TL* 2 Girth 3 Girth* 4 Girth* 5 Girth* 2 Weight 3 Weight* 4 Weight* 504 ± 29 ( ) 524 ± 25 ( ) 540 ± 24 ( ) 543 ± 26 ( ) 162 ± 13 ( ) 167 ± 11 ( ) 172 ± 11 ( ) 175 ± 11 ( ) ± ( ) ± 96.5 ( ) ± ( ,050.0) 532 ± 47 ( ) 571 ± 43 ( ) 590 ± 46 ( ) 628 ± 46 ( ) 167 ± 20 ( ) 181 ± 16 ( ) 189 ± 18 ( ) 205 ± 19 ( ) ± ( ) ± ( ,710.0) ± ( ,500.0) 5 Weight* ± ( ) 1,074.6 ± ( ,610.0) 30

43 Figure 7. Total length frequency distributions of male (N = 215) and female (N = 253) spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. 31

44 Figure 8. Age frequency distributions of male (N = 207) and female (N = 246) spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. 32

45 Weight increased with increased total length for males (Figure 9) and females (Figure 10). All male spotted gar used for histological analyses (N = 94) were placed in the spawning capable/actively spawning phase. Therefore, males in the spawning capable/actively spawning phase were separated into groups based on the presence of purely continuous germinal epithelia, discontinuous/continuous germinal epithelia (Figure 11), or purely discontinuous germinal epithelia (Figure 12). Active spermatogenesis is indicated by numerous spermatocysts and continuous germinal epithelia (Brown-Peterson et al. 2002), which appear after the spawning season when males are preparing for the next spawning season. Less active spermatogenesis can be indicated by few spermatocysts and discontinuous germinal epithelia (Brown-Peterson et al. 2002). Testes undergoing little spermatogenesis that possess large amounts of spermatozoa in the lumens of the lobules are primarily used for sperm storage instead of sperm production (Grier et al. 1987). Discontinuous germinal epithelia were prominent from October through April and also in June and August, and discontinuous/continuous germinal epithelia became prominent in March and remained present through September (Figure 13). The only occurrence of purely continuous germinal epithelia was in September (Figure 13). Of all females used for histological analyses (N = 123), the majority were placed in the spawning capable/actively spawning phase (N = 107; Figure 14). During each month of the sampling period, females classified as spawning capable/actively spawning were more prevalent than females of any other phases (Figure 15). Females classified as developing (Figure 16) were collected during October, November, March, May, June, and August (Figure 15), and females classified as regenerating (Figure 17) were collected during February, March, and May (Figure 15). On 31 May 2007, a female spotted gar was collected in which half of her ovaries was classified as spawning capable/actively spawning while the other half was 33

46 Figure 9. Relationship between log 10 weight and log 10 total length for male spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. 34

47 Figure 10. Relationship between log 10 weight and log 10 total length for female spotted gar collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. 35

48 Lobule with a continuous GE SZ in lumen CY Figure 11. Histological section of a spawning capable/actively spawning male spotted gar (TL = 457 mm) testis with discontinuous/continuous germinal epithelia collected on 26 September 2007, in the upper Barataria Estuary. Bar = 0.1 mm. CY spermatocyst; SZ spermatozoa; GE germinal epithelium. 36

49 Lobule with a discontinuous GE SZ in lumen Figure 12. Histological section of a spawning capable/actively spawning male spotted gar (TL = 485 mm) testis with discontinuous germinal epithelia collected on 10 March 2007, in the upper Barataria Estuary. Bar = 0.1 mm. SZ spermatozoa; GE germinal epithelium. 37

50 Figure 13. Seasonal changes in germinal epithelia of male spotted gar (N = 94) collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January. Numbers above columns indicate the number of fish collected each month. C continuous germinal epithelia; DC discontinuous/continuous germinal epithelia; D discontinuous germinal epithelia. 38

51 PGO Late VTGO Atretic egg CAO Figure 14. Histological section from the ovary of a spawning capable/actively spawning female spotted gar (TL = 652 mm) collected on 6 December 2006, in the upper Barataria Estuary. Bar = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte. 39

52 Figure 15. Monthly reproductive phases for female spotted gar (N = 123) collected from 5 October 2006 to 26 September 2007, in the upper Barataria Estuary. No fish were collected in January. Numbers above columns indicate the number of fish collected each month. REGEN regenerating phase; DEV developing phase; SC/AS spawning capable/actively spawning phase. 40

53 CAO PGO Early VTGO Figure 16. Histological section from the ovary of a developing female spotted gar (TL = 568 mm) collected on 30 June 2007, in the upper Barataria Estuary. Bar = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte. 41

54 CAO PGO Figure 17. Histological section from the ovary of a regenerating female spotted gar (TL = 530 mm) collected on 23 March 2007, in the upper Barataria Estuary. Bar = 0.5 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte. 42

55 regressing (Figure 18). Therefore, the overall phase selected for this female was spawning capable/actively spawning. No immature females were collected during this study; however, two females classified as developing possessed closely associated primary growth oocytes and cortical alveolar oocytes, which is a characteristic of fish that have never spawned (N. Brown- Peterson, University of Southern Mississippi, personal communication; Figure 19). Both of these females were collected on 31 August Atretic eggs were observed throughout the year in the alpha and beta stages (early atresia) and in the gamma and delta stages (late atresia; Figure 20). These stages were defined by Hunter and Macewicz (1984) and were based on work by Bretschneider and Duyvene de Wit (1947) and Lambert (1970). Additionally, post-ovulatory follicles (Figure 18B) were observed every month throughout the year except for January (no fish collected during this month), October, and June. Post-ovulatory follicles were typically observed individually and not in clusters. Mean GSI by sample date increased in spring and decreased through late summer for males (Figure 21) and females (Figure 22). Based on mean GSI values and histological analyses, spawning occurred from March through May. Mean egg diameter ranged from 1.5 mm in August to 2.9 mm in March and averaged 2.5 ± 0.3 mm (N = 131; Figure 23). Total fecundity ranged from 1,200 to 21,350 eggs per fish with an average of 6,493 ± 4,225 eggs per fish (mean TL = 579 ± 44 mm). Mean number of eggs per gram of ovary-free body weight was 9 ± 5 eggs/g of ovary-free body weight. Only females collected during and just prior to the spawning season (February through May) were used to determine the mean number of eggs per gram of ovary-free body weight (N = 89). Total fecundity was more closely related to weight (Figure 24) than total length (Figure 25). On average, mean total fecundity 43

56 Regressing Spawning capable/ actively spawning A PGO CAO Atretic egg POF B Late VTGO PGO Atretic egg CAO C Figure 18. Ovaries from a female spotted gar (TL = 645 mm) collected on 31 May 2007, in the upper Barataria Estuary: (A) gross appearance of ovaries, (B) histological section of left portion of left ovary classified as regressing, and (C) histological section of right portion of left ovary classified as spawning capable/actively spawning. Overall, this female was classified as spawning capable/actively spawning. Bars = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte; POF post-ovulatory follicle. 44

57 Mid VTGO PGO Early VTGO CAO Figure 19. Histological section from the ovary of a developing female and potential virgin spotted gar (TL = 412 mm) collected on 31 August 2007, in the upper Barataria Estuary. Bar = 1.0 mm. PGO primary growth oocyte; CAO cortical alveolar oocyte; VTGO vitellogenic oocyte. 45

58 Early atretic egg Late atretic egg Late VTGO Figure 20. Histological section from the ovary of a spawning capable/actively spawning female spotted gar (TL = 652 mm) collected on 6 December 2006, in the upper Barataria Estuary. Bar = 0.1 mm. VTGO vitellogenic oocyte. 46