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Environmental Influences on Mosquitofish Reproduction

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ENVIRONMENTAL INFLUENCES ON MOSQUITOFISH REPRODUCTION By THEA M. EDWARDS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Thea M. Edwards

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To my parents, Margaret and Peter

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iv ACKNOWLEDGMENTS During the process of creating my dissert ation, I depended on the help and support of a large number of people. First and foremost, I thank my advisor, Lou Guillette for sharing with me his wealth of experience and knowledge. I also thank my supervisory committee, who offered me their time and access to their labs: Lauren Chapman guided me in developing my statistical abilities; Da ve Evans and his graduate students offered me insight and ideas on nitrate physiology a nd gave me free access to their wonderful plate reader; Tom Frazer gave me access to hi s lab for nitrate measurements of countless water samples; Taisen Iguchi read my manus cripts and gave opinions and suggestions. Frank Nordlie was an honorary member of my committee by attending my qualifying exam, lending me his hematocrit reader, and helping me think about Gambusia I also thank Dr. Mari Carmen Uribe for helping me interpret Gambusia histology, Neal Benson and Melissa Chen for their assistance with my sperm assay, and Stephanie Keller for measuring the nitrate in my water samples. I was accompanied on this journey through graduate school by my fellow graduate students in the Guillette lab. They are: Br andon Moore, my inspiration; Tam Barbeau and Teresa Bryan, who helped me hang up the estrogen molecule; D. Bermudez, who threw some of the most memorable partie s in my graduate school career; Mark Gunderson, who taught me how to do RIAs a nd draw blood from alligators; Heather Hamlin, whose enthusiasm for research know s no bounds; Krista McCoy, to whom I owe all that I know about frogs; Matt Milnes, with whom I shared many scientific discussions

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v and many beers, sometimes together; Ed Or lando, who showed me how to dissect my first mosquitofish and enthusiastically engages in Gambusia conversations that would be impossible with anyone else; and G unnar Toft, whose motivation got my Gambusia projects started. Although I worked hard at my research, it s completion would have been impossible without the spectacular help of the 22 underg raduate students who joined me at different times during my projects. In particular, I thank Lisa Choe, Rebecca Emrich, Annie Heffernan, Hilary (Thompson) Miller, Mari a Paredes, and John Matt Thro, who formed the long-term core of “Team Gambusia. ” In addition, I thank Kelly April, Tricia Bardis, Donnell Bowen, Koo Chung, Chi Chi Echeazu, Jaime Joyce, Elisabeth Kuehlem, Gina Long, Rich Lufkin, Chad Mackman, Fred No rris, Laura Patterson, Scott Schultz, Paree Taslimi, Sarah Tynes, and Scott Watson. Finally, I thank my family for their love encouragement, faith, comfort, advice, and willingness to learn about the science that is such an important part of my life.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xi v CHAPTER 1 OVERVIEW OF Gambusia BIOLOGY, STUDY SITES, AND CONTAMINANTS OF INTEREST............................................................................1 Study Overview............................................................................................................1 Ga mbusia Life History.................................................................................................3 Taxonomy and Distribution of Gambusia holbrooki ............................................3 Habitat and Diet.....................................................................................................4 Female Reproductive Cycle..................................................................................5 Male Reproductive Cycle......................................................................................6 Gambusia holbrooki as a Sentinel Species...................................................................9 Overview of Reproductive Endocrinology in Fishes..................................................11 Lakes.......................................................................................................................... .12 Lake Apopka.......................................................................................................13 Lake Woodruff....................................................................................................14 Florida Springs and Nitrate.........................................................................................15 Overview.............................................................................................................15 Nitrogen Cycling, In Vivo Nitrogen Metabolism, and Effect of Nitrate on Steroidogenesis................................................................................................16 Effects of Nitrates on Sperm Motility and Viability...........................................18 Summary of Nitrate’s Effects on Reproduction..................................................18 Review of Endocrine Disruption in Fishes.................................................................19 Hypotheses and Goals.................................................................................................19 2 TEMPORAL REPRODUCTIVE PATTER NS FOR FEMALE MOSQUITOFISH CAPTURED FROM TWO FLORIDA LAKES.........................................................24 Introduction.................................................................................................................24 Methods......................................................................................................................27

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vii Field and Tissue Collections...............................................................................27 Muscle Estradiol Measurements..........................................................................29 Statistics...............................................................................................................30 Gonadosomatic Index..........................................................................................31 Results........................................................................................................................ .31 Environmental Differenc es between Lakes.........................................................31 Temporal and Lake-Associated Variati on in Response Variables Related to Reproduction....................................................................................................32 Body size......................................................................................................32 Temporal changes in reproductive activity..................................................32 Embryo number, size, and stage of development.........................................33 Hepatosomatic index....................................................................................34 Estradiol.......................................................................................................34 Discussion...................................................................................................................35 3 SEASONAL SPERM QUALITY IN MALE Gambusia Holbrooki (EASTERN MOSQUITOFISH) COLLECTED FR OM TWO FLORIDA LAKES......................48 Introduction.................................................................................................................48 Methods......................................................................................................................51 Field Collections..................................................................................................51 Testicular Histology............................................................................................52 Sperm Collection.................................................................................................52 Sperm Staining....................................................................................................53 Sperm Counts and Viability................................................................................53 Calculations.........................................................................................................54 Statistics...............................................................................................................55 Results........................................................................................................................ .55 Sperm Viability...................................................................................................56 Sperm Counts......................................................................................................57 Discussion...................................................................................................................58 Seasonal Variation in Sperm Counts...................................................................58 Lake-Associated Variation in Sperm Counts and Quality..................................60 Conclusions.........................................................................................................63 4 SEASONAL VARIATION IN BODY SIZE, MUSCLE ANDROGEN CONCENTRATIONS, AND TESTICULAR AND HEPATIC WEIGHTS AMONG MALE MOSQUITOFISH FROM TWO LAKES IN CENTRAL FLORIDA...................................................................................................................72 Introduction.................................................................................................................72 Methods......................................................................................................................74 Field Collections..................................................................................................74 Muscle Androgen Measurements........................................................................76 Statistics...............................................................................................................77 Results........................................................................................................................ .78 Abiotic Factors....................................................................................................78

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viii Body Size.............................................................................................................78 Gonopodium Length............................................................................................79 Androgens............................................................................................................79 Testicular Weight................................................................................................80 Hepatic Weight....................................................................................................81 Discussion...................................................................................................................82 Body Size.............................................................................................................82 Gonopodium Length............................................................................................84 Androgens............................................................................................................84 Testicular Weight................................................................................................85 Hepatic Weight....................................................................................................86 Summary..............................................................................................................87 5 WATER QUALITY INFLUENCES REPRODUCTION IN FEMALE MOSQUITOFISH ( Gambusia Holbrooki ) FROM EIGHT FLORIDA SPRINGS....95 Introduction.................................................................................................................95 Methods......................................................................................................................98 Field Collections and Water Quality...................................................................98 Body Size and Dissections..................................................................................99 Estradiol Concentration.....................................................................................100 Statistics.............................................................................................................101 Relationships among reproductive variables..............................................101 Relationships among water quality parameters and reproductive variables..................................................................................................102 Outliers.......................................................................................................103 Results.......................................................................................................................1 03 Relationships among Repr oductive Variables...................................................103 Water Quality....................................................................................................104 Relationships between Wate r Quality and Reproduction..................................105 Discussion.................................................................................................................106 Matrotrophy.......................................................................................................108 Conclusion.........................................................................................................109 6 WATER QUALITY INFLUENCES REPRODUCTION IN MALE MOSQUITOFISH ( Gambusia Holbrooki ) FROM EIGHT FLORIDA SPRINGS..118 Introduction...............................................................................................................118 Methods....................................................................................................................120 Field Collections and Water Quality.................................................................120 Body Size and Dissections................................................................................121 Muscle Androgen Measurements......................................................................122 Sperm Counts and Sperm Viability...................................................................123 Statistical Analysis............................................................................................125 Relationships among reproductive and morphometric variables...............125 Relationships among water quality parameters and reproductive variables..................................................................................................126

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ix Results.......................................................................................................................1 27 Water Quality....................................................................................................127 Relationships among Reproductive and Morphometric Variables....................128 Relationships among Water Quality Para meters and Reproductive Variables.128 Discussion.................................................................................................................130 Relationships between Nitrate and Reproduction.............................................130 Gonopodium Length..................................................................................131 Testicular Hypertrophy and Mu scle 11-KT Concentrations.............................131 Spermatogenesis................................................................................................132 Conclusions.......................................................................................................133 7 SUMMARY AND FUTURE DIRECTIONS...........................................................142 Overview...................................................................................................................142 Summary...................................................................................................................142 New Hypotheses and Development of Gambusia as a Model Species....................143 LIST OF REFERENCES.................................................................................................151 BIOGRAPHICAL SKETCH...........................................................................................171

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x LIST OF TABLES Table page 1-1 Endocrine disruption in fishes..................................................................................21 2-1 Additional water quality information for Lake Apopka and Lake Woodruff..........38 5-1 Relationships of reproductive response variables measured in adult female Gambusia holbrooki collected from eight Florida springs.....................................110 5-2 Florida collection sites for female Gambusia holbrooki .......................................110 5-3 Relationships of water quality parame ters and response vari ables measured in adult female Gambusia holbrooki collected from eight Florida springs................111 6-1 Florida collection sites for male Gambusia holbrooki .........................................134 6-2 Male mosquitofish sample sizes.............................................................................134 6-3 Significant relationships among water qua lity parameters from fish-collection sites and response variables measured in adult male Gambusia holbrooki collected from eight Florida springs.......................................................................135 7-1 Results, integrated with other studi es of endocrine disruption in fishes................147 7-2 Summary of new hypotheses sugge sted by dissertation results.............................150

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xi LIST OF FIGURES Figure page 2-1 Stages of embryonic development for eastern mosquitofish...................................39 2-2 Percentage of female mosquitofish w ith broods at different stages of embryonic development.............................................................................................................40 2-3 Seasonal changes in water temperatur e for Lake Apopka and Lake Woodruff.......41 2-4 Temporal variation in body size of adult female mosquitofish from Lake Apopka and Lake Woodruff.....................................................................................42 2-5 Temporal variation in mean embryo number and percentage of sampled females from each lake..........................................................................................................43 2-6 Embryo number observed for female mosqu itofish of different standard lengths...44 2-7 Mosquitofish embryonic wet weight at different developmental stages..................45 2-8 Mean adjusted hepatic weight of mosqu itofish with embryos at different stages...46 2-9 Temporal variation in muscle estr adiol concentrations of adult female mosquitofish from Lake Apopka and Lake Woodruff.............................................47 3-1 Testicular histology of Gambusia holbrooki ............................................................64 3-2 Sperm methods for Gambusia holbrooki .................................................................66 3-3 Gambusia sperm counts flow cytometry printout....................................................67 3-4 Water temperature and daylengt h data for the collection period.............................68 3-5 Mean percent live sperm observed among adult male Gambusia holbrooki collected from two lakes in central Florida..............................................................69 3-6 Mean sperm count per spermatozeugma observed among adult male Gambusia holbrooki collected from two lake s in central Florida.............................................70 3-7 Mean live sperm count observed among adult male Gambusia holbrooki from Lake Apopka and Lake Woodruff............................................................................71

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xii 4-1 Water temperature for Lake Apopka and Lake Woodruff, shown with ambient photoperiod for each collection date........................................................................88 4-2 Mean standard length of adult male mosquitofish from Lake Apopka and Lake Woodruff..................................................................................................................89 4-3 Body weight of adult male mosquitofish from Lake Apopka and Lake Woodruff.90 4-4 Mean gonopodium length adjusted for standard length, of adult male mosquitofish from Lake Apopka and Lake Woodruff.............................................91 4-5 Mean muscle androgen concentrations for adult male mosquitofish from Lake Apopka and Lake Woodruff.....................................................................................92 4-6 Adjusted testicular weight of adult male mosquitofish from Lake Apopka and Lake Woodruff.........................................................................................................93 4-7 Adjusted hepatic weights of adult ma le mosquitofish from Lake Apopka and Lake Woodruff.........................................................................................................94 5-1 Adjusted hepatic weight, embryo wet weight; and embryo dry weight plotted by embryonic stage......................................................................................................112 5-2 Percentage of non-reproductive, mature females sampled from Florida springs with varying nitrate concentrations........................................................................113 5-3 Adjusted mean embryo number for females captured in Florida springs with varying temperatures..............................................................................................114 5-4 Embryo dry weight for embryos taken fr om females captured in Florida springs with varying concentrations of nitrate....................................................................115 5-5 Adjusted hepatic weight for females captured in Florida springs with varying dissolved oxygen concentrations............................................................................116 5-6 Muscle estradiol concentrations for females from each spring..............................117 6-1 Relationship between water nitrate co ncentrations and water pH among eight Florida springs........................................................................................................136 6-2 Muscle androgen concentrations for a dult male mosquitofish collected from eight Florida springs...............................................................................................137 6-3 Linear relationships between wate r nitrate concentra tions and several reproductive response variable s of adult male mosquitofish captured from eight Florida springs........................................................................................................138

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xiii 6-4 Linear relationships between wate r temperature and several reproductive response variables of adult male mosqu itofish captured from eight Florida springs....................................................................................................................139 6-5 Linear relationship between muscle 11-KT concentrations and water pH for adult male mosquitofish captured from eight Florida springs................................140 6-6 Mean muscle 11-KT concentrations of adult male mosquitofish captured from high or low nitrate springs......................................................................................141

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENVIROMENTAL INFLUENCES ON MOSQUITOFISH REPRODUCTION By Thea M. Edwards August 2005 Chair: Louis J. Guillette, Jr. Major Department: Zoology Reproduction in all organisms is regulat ed by a wide range of environmental factors. In fishes, these include (among other factors): temp erature, photoperiod, nutrition, and social interactions. In the past 50 years, an a dditional factor has emerged: anthropogenic pollution. Many widely used cont aminants (PCBs, pesticides, fertilizers, plasticizers) are now distribut ed throughout our environment, particularly in aquatic systems. Several of these have been show n to disturb normal development, growth, and reproduction of vertebrates through disruptive in teractions with the endocrine system. In this extended seasonal study, we eval uated reproductive parameters in adult Gambusia holbrooki captured from two central Florid a lakes: Lake Apopka (with a documented history of organochlorine cont amination) and Lake Woodruff Wildlife Refuge (reference site). Relative to the Lake Woodruff population, males and females from Lake Apopka exhibited increased hepato somatic indices; females also exhibited altered estradiol patterns and an unexpected increase in fecundity. Males from Lake

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xv Apopka exhibited increased testicular si ze, but decreased sp erm counts and sperm viability in some months, particularly at the end of the reproductive season. In a second suite of studies, we assess ed reproduction, at one point during the breeding season, among adult Gambusia captured from eight Florida springs with varying concentrations of nitr ate. Nitrate contamination of Florida springs is a growing concern. In fact, nitrate concentrations in some springs exceed the EPA-established concentration limit for drinking water (10 mg/L NO3-N). Nitrate exposure is associated with altered development, reduced steroidoge nesis, and diminished reproductive success in a number of species. In male mosquitofish exposed to elevated nitrate concentrations (4 to 5 mg/L NO3-N), we observed increased gonadosomatic index, reduced 11ketotestosterone concentrations, and reduced sperm counts. Females from springs with elevated nitrate concentrations exhibited reduced embryo dry weights and a decreased rate of reproductive activity, based on presen ce or absence of vitellogenic oocytes. Taken together, our results suggest that long-term exposure to environmental contaminants, specifically organochlorines a nd nitrate, is associated with altered reproductive outcomes. In addition, the temporal nature of our rese arch greatly expands and integrates our knowledge of Gambusia reproduction in terms of seasonal variation and basic life history.

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1 CHAPTER 1 OVERVIEW OF Gambusia BIOLOGY, STUDY SITES, AND CONTAMINANTS OF INTEREST Study Overview Aquatic environments are inherently variable and often exhibit pronounced temporal changes that influence the biology a nd ecology of their resident flora and fauna. Many changes are natural in that they are nonanthropogenic. Some ar e also repetitively cyclical and represent phenomena to which the animals in the system are adapted. Mosquitofish, for example, become reproductively active in the spring, when water temperatures exceed 20 to 22 C, and they end activity in the fall when daylength shortens to less than 12 h (Chapters 1 and 2 of our study; Fraile et al., 1994; Koya and Kamiya, 2000). It is likely that this seasonal pattern evolved around practical considerations of prey abundance, mate readiness, and larval su rvivorship; factors that affect fitness, and that generally vary with seasonal envi ronmental change (Winemiller, 1993). In addition to the natural factors that regulate reproduction, aquatic animals are increasingly subject to “unnatural” regulation by anthr opogenic endocri ne-disrupting contaminants (EDCs). Widespread use of pe sticides, fertilizers, plastics, and other industrial chemicals has increa sed exponentially over the past 50 years (Danielopol et al., 2003). The effect is so dramatic that epidem iologists can track the related rise in human reproductive disorders that are causally linked to contaminant exposure (Carlsen et al., 1992; Skakkebaek et al., 1998).

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2 Contaminant-induced reproductive change s in the human population probably occur after similar changes in wildlife. This is because most wildlife species are smaller than humans and potentially sensitive to lower doses. Those with shorter generation times could be more susceptible to cumulativ e, cross-generational ch anges. In addition, some species possess a degree of phenotypic plas ticity that makes them more likely to exhibit overt symptoms. For example, in a wi de variety of fishes, sexual differentiation is highly labile, and exposure of differen tiating fishes to estrogens (regardless of genotypic sex) can produce morphologically female monocultures (Piferrer, 2001). Therefore, studies of wild animals are useful in risk assessment and should be used to guide environmental policy to improve c onservation, ecological sustainability, and human health and welfare. Our study investigated temporal patter ns of reproducti on in wild adult Gambusia holbrooki (eastern mosquitofish) captured from Lake Woodruff and Lake Apopka in central Florida. These lakes were sampled because other biological data on fishes (Gallagher et al., 2001; Toft et al., 2003) and alligators (Gui llette et al., 2000) from these lakes suggest that they provide a comparativ e system in which to study the impact of EDCs on reproductive variables. In addition to sampling these lakes, we conducted a single-month reproductive study of Gambusia holbrooki captured from eight artesian springs, located along the Suwannee and Santa Fe Rivers in north Florida. These springs represent (at present) a gradient of ni trate contamination and provided a natural experimental opportunity to assess the potential relationship between nitrate concentration and variation in mosquitofish reproduction. The remainder of this chapter details mosquitofish life hist ory, clarifies why I chose to work with mosquitofish, and

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3 presents an overview of reproductive endocri nology in fishes. This is followed by descriptions of the two lakes and eight springs sampled in our study, along with a review of the effects of pesticides and nitrate on fish reproduc tion. Finally, I present the hypotheses that guided our study. Gambusia Life History Taxonomy and Distribution of Gambusia holbrooki Within the genus Gambusia (family Poeciliidae), th ere are approximately 45 species with a native range that extends from New Jersey, west to Iowa and the Mississippi River drainage; and south to the Gulf coast, the Caribbean, and Mexico. From Mexico, the native range continues s outh through Central America, to Columbia (reviewed by Parenti and Rauchenbe rger, 1989). Of these 45 species, Gambusia holbrooki (eastern mosquitofish) and Gambusia affinis (western mosquitofish) are the most widely studied. In a ddition to their native ranges in the southern United States, these two species have been introduced for biol ogical control of mosquito larvae and are now found on all of the con tinents except Antarctica (rev iewed by Courtenay and Meffe, 1989). Thus, their functional ra nge is substantially larger than their native range. The focus of our study is Gambusia holbrooki which is distinguished from its western sister species ( Gambusia affinis ), by a thin geographic a nd biological line, across which hybridization does occur (Walters a nd Freeman, 2000). Morphologically, the two species are differentiate d by fin ray counts. Gambusia holbrooki possess 8 dorsal and 11 anal fin rays, whereas G. affinis have 7 dorsal and 10 anal fin rays (Walters and Freeman, 2000). In their native range, G. affinis are found west of Mobile Bay in Alabama; whereas G. holbrooki occur to the east, and the zone of sy mpatry lies within the Mobile River

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4 Basin (Wooten et al., 1988). Successful inte rspecific hybridization is possible between G. holbrooki females and G. affinis males, while the alternative hybridization of G. affinis females and G. holbrooki males does not result in viable offspring (Black and Howell, 1979). This one-way incompatibility is probably caused by species differences in the sex chromosomes (Black and Howell, 1979). Gambusia holbrooki do not exhibit heteromorphic sex chromosomes in either sex, although inheritan ce patterns of malelinked melanism suggest an XY system of sex determination (Angus, 1989; Black and Howell, 1979). Conversely, among G. affinis, females possess heteromorphic sex chromosomes and males possess homomorphic sex chromosomes indicative of ZW sex determination (Black and Howell, 1979). A ccording to Scribner and Avise (1994), in areas where the two species co-exist, G. holbrooki will quickly (within 4 generations) out-compete G. affinis in terms of mitochondrial and nuclear allele frequency, an outcome that is likely to be related to the one-way chromosomal incompatibility described above. Scribner et al. ( 1999) concluded that offspring from G. holbrooki females also have some selective advantage. Because G. holbrooki and G. affinis do hybridize where they co-occur, they were originally cons idered two subspecies of Gambusia affinis (thus G. affinis holbrooki and G. affinis affinis ). For the reasons above, the two are now considered separate species (Wooten et al., 1988), but pre-1988 literature often refers to Gambusia affinis without indicating the subspecies. Habitat and Diet Gambusia holbrooki are small, sexually dimorphic, viviparous omnivores that inhabit fresh and brackish waterways ranging from ephemeral ponds and ditches, to rice fields; to streams, ponds, rivers, lakes, sp rings, estuaries, and marshes (Daniels and Felley, 1992; McKinsey and Chapman, 1998; Porte et al., 1992; Toft et al., 2003; Vargas

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5 and de Sostoa, 1996). Adult sizes range fr om 13 to 32 mm for males and 17 to 63 mm for females (Chapters 2 to 6 of our study; Vargas and de Sostoa, 1996). Although they will eat algae and detritus, mosquitofish prefer animal prey, including insects, anuran eggs, crustaceans, rotifers, Daphnia and round worms (Meffe and Snelson, 1989). The life expectancy of mosquitofish is generally no more than 2 years (Vargas and de Sostoa, 1996). Although females mature about 20 days later than males (Koya et al., 2003), they typically grow faster, achieve a larger size, and outlive males (Vargas and de Sostoa, 1996). Female Reproductive Cycle Mosquitofish are reproductively active in the spring, summer, and fall in Florida (our study), Japan (Koya et al., 1998; Koya and Iwase, 2004) and Spain (Vargas and de Sostoa, 1996); or all year round in Hawa ii (Haynes and Cashner, 1995). Female Gambusia mature about 110 days after they ar e born (Koya et al ., 2003), and produce sequential, synchronized broods throughout th eir breeding season: as one brood nears parturition, the next cohort of oocytes are accumulating yolk (Koya and Kamiya, 2000). Females store sperm and thus can produce multiple broods, even in isolation from males, assuming they have mated once before (Hubbs, 1999). This feature contributes to their success as founder species. Fe rtilization of oocytes occurs after yolk accumulation, when eggs exceed 1.7 mm in diameter (Koya et al., 2000). Yolked oocytes become atretic if they do not reach maturity with their cohort (K oya et al., 2000). Litter size ranges from 1 to 245 precocious offspring, depending on female size and time of year (Chapters 1 and 4 of our study; Vargas and de Sostoa, 1996). Gestation (which ta kes place within the single, fused ovary) lasts 22 to 39 days, depending on temperature and photoperiod (reviewed by Koya et al., 1998). After birth, th e larvae live independently of the parents.

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6 Koya and Kamiya (2000) showed experimentally that (in Gambusia affinis collected from an irrigation canal in central Japan) ovarian recrudescence begins with the rise in springtime temperature, regardless of daylength. In th at population, they found that vitellogenesis occurred at 14C and pre gnancy at 18C. In th e fall, vitellogenesis ended when daylength was less than 12.5 h, rega rdless of temperature. The final brood finished its development at a temperature-dependent rate and ovarian regression occurred after final parturition. Mosquitofish are typically classified as lecithotrophs because they produce large yolky eggs (Haynes, 1995). However, lec ithotrophy implies that embryo nutrition is limited to the yolk placed in the oocyte before fertilization. By de finition, lecithotrophs lose weight during gestation because of respiratory losses. However, several studies, including ours, show that embryos gain in diameter and wet and dry weight during gestation (Chapters 1 and 4 of our study; Varg as and de Sostoa, 1996). This process is supported by appropriate changes in liver size that are presumably related to vitellogenesis (Chapters 1 and 4 of our study). Furthermore, (using Gambusia geiseri ) Marsh-Matthews et al. (2001) showed that ma ternal transfer of tritiated leucine to embryos was measurable within 2 hours of injection. Their findings suggest that (in addition to yolk provisioning) mosquitofish provide directly for their embryos during gestation via matrotrophy. Male Reproductive Cycle Mature poeciliid males ar e readily identified by th eir gonopodium (a grooved bony modification of the anal fin used to transf er spermatozeugmata to the genital opening of the female). The gonopodium forms duri ng puberty by elongation of anal fin lepidotrichia and fusion of pterygiophor es 3, 4, and 5, with a species-specific

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7 arrangement of hooks at the tip (Rosa-Molinar et al., 1994). This is in contrast with females, which have a fan-shaped anal fi n without fusion of th e pterygiophores. The structure and mechanical contro l of the gonopodium are supported by the internal skeleton, whic h (like the anal fin) becomes masculinized in response to androgens during puberty (Ogino et al., 2004; Rosa-Molinar et al., 1994, 1996). Specifically, in males (but not females), the hemal spine at vertebra 13 is resorbed; and the hemal spines at vertebrae 14, 15, and 16 thicken, elongate, and swing anteriorally (swing caudally in females). Spines 14, 15, and 16 are connected by interosseal ligaments (poorly developed in females) to the fused proximal pterygiophores of the anal fin. Therefore, as the hemal spines bend forw ard, they pull the anal fin in an anterior direction. This movement aligns the gonopodi um with the male’s urogenital opening so that sperm can be delivered via the g onopodium during mating. The movement also places the anal fin and its appendicular suppor t at the fish’s center of gravity (RosaMolinar et al., 1996). This improves fine -motor control of the gonopodium, which the male must abduct across his body with enough precision to insert the hooked tip briefly into the female. The entire copulatory ev ent takes less than a second and is performed while both fish are in motion (Rosa-Molinar et al., 1996). Lastly, the mature male skeleton provides support for the large muscle used to maneuver the gonopodium (RosaMolinar et al., 1996). Males copulate with females frequently, ma king attempts even before they are fully mature (Bisazza et al., 1996). Males possess mature spermatozoa about 90 days after birth (Koya et al., 2003), a lthough some variation among indi viduals or populations is likely. In mosquitofish, the testes are fused into a single, round, white-colored organ that

PAGE 23

8 is located centrally in the abdomen, dorsal to the origin of the gonopodium (Fraile et al., 1992). A single vas deferens connects the gono podium to the efferent ducts that coalesce within the central lumen of each testis (Fraile et al., 1992). The outer wall of the testis is lined with spermatogonia (Fraile et al., 1992). In spring through fall, spermatogonia proliferate in successive waves of mitosis, forming nests (cysts) of primary spermatocytes bounded by Sertoli cells (Fraile et al., 1992). In a process that takes approximately 30 days, spermatocytes within a single cyst undergo synchronized meiosis and differentiation to pr oduce spermatids and ultimately tailed spermatozoa (Fraile et al., 1992; Koya and Iwase, 2004). As the cysts mature, they move from the periphery of the testis to the cente r, where they are released to the efferent sperm ducts as spherical aggregates of sperm (spermatozeugmata), with tails in the center and heads on the periphery (Fraile et al., 1992 ). At this point, Sertoli cells no longer surround the spermatozeugmata; instead, they hypertrophy and become part of the efferent duct tubule (Fraile et al., 1992). The tubules secr ete a gelatinous matrix that holds the spherical structure of the spermato zeugma together until it reaches the oviduct of a female (reviewed by Meffe and Snelson, 1989). As winter approaches, production of new spermatocytes ceases. Through the wint er, stored cysts of mature spermatozoa occupy most of the testicular volume, and will be used during early spring copulation, which occurs before the first wave of sp ring spermatogenesis is complete (Koya and Iwase, 2004). Like most teleosts, male mosquitofish exhibit seasonal variation in sperm production that is regulated by changes in temperature and photope riod (Fraile et al., 1994). However, factors that initiate mosquitofish spermatogenesis are not yet fully

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9 understood. Males that have re cently entered testicular qu iescence cannot be stimulated to produce new sperm by increasing temper ature or photoperiod (Fraile et al., 1993). However, fish captured at the end of their quiescent period will proliferate spermatogonia, even if temperatures remain lo w and days are short (Fraile et al., 1994). Increasing ambient temperatures are require d for differentiation of spermatogonia to spermatocytes, and photoperiod must lengthen for spermatocytes to enter meiosis (Fraile et al., 1994; De Miguel et al., 1994). Gambusia holbrooki as a Sentinel Species I chose this prolific species for their sm all size, wide availability, rapid time to maturity (about 3 months)(Koya et al., 2003), and viviparity. In addition, endocrinedisrupting contaminants (EDCs) that im pact reproduction are often lipophilic and therefore bioaccumulate in the fatty tissues of exposed animals (Porte et al., 1992). Mosquitofish are intermediate in the food web, and are thus likely to bioaccumulate contaminants absorbed by their prey. Mate rnal provisioning of the egg, and later the embryo (in viviparous species like Gambusia ) involves transfer of stored lipids from the mother to the offspring (Meffe and Snels on, 1993). For individuals with high body loads of accumulated contaminants, this provisioni ng process can result in an exceptionally high dose of contaminants (relative to ambien t concentrations) to the offspring during the period of sexual differentiati on (Harding et al., 1997). Developmental exposure to EDCs can pe rmanently alter the organization of the reproductive system (Guillette et al., 1995). Th is could result in significant reproductive problems at the population level. According to Nakamura (1978) and Sone et al. (2005), Gambusia affinis larvae are sexually differentiated by the time they are born. Although in another study, Koya et al. (2003) repor t that, at two days before birth, all Gambusia

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10 affinis embryos are female, with oocytes present in their gonads. At birth, some of the offspring had developed testes instead. Thus, Koya et al. (2003) suggest that G. affinis exhibit embryonic protogyny; however, afte r birth, these fish are gonochoristic. In addition to certain convenient aspects of their biology (noted above), I chose to study Gambusia because they are already the subj ect of a few endocrine disruption studies. For example, abnormal vitellogenin pr oduction occurs in males after exposure to estrogenic compounds (Tolar et al., 2001). Angus et al. (2002) observed enlarged testes and livers among male Gambusia inhabiting water contaminat ed with treated sewage effluent. Note that the presence of excreted ethynylestradiol, deri ved from birth control pills gives sewage effluent an estrogenic ch aracter (Schultz et al., 2003). Dreze et al. (2000) observed skewed sex ratios that favored females, and/or delayed sexual development in male Gambusia exposed to estrogenic 4-nonylphenol. Orlando et al. (2002) showed increased ovarian and brain aromatase (converts androgens to estrogens) activity among adult female mosquitofish caught from the Fenholloway River in Florida. The Fenholloway River is polluted with paper mill effluent, which in other studies has been shown to masculinize the anal fins of female mosquitofish (Bortone and Cody, 1999; Howell et al., 1980; Parks et al., 2001). This last example of masculinized anal fins is one of the more famous early cases of endocrine disruption in fishes and is described in greater detail below. In 1978, masculinized female mosquitofish, Gambusia affinis holbrooki were discovered in Elevenmile Creek (Escambia County, Florida), downstream from a paper mill (Howell et al., 1980). The females possessed partially to fully formed gonopodia, complete with the hooks, spines, and fusion of fin rays 3, 4, and 5. There was no

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11 evidence of gonadal masculini zation: masculinized fema les possessed only ovarian tissue. In addition, Howell et al. (1980) obs erved the masculinized sexual behavior of these females, who pursued normal and masculinized females while swinging and thrusting their gonopodia. Immature males (12 to 13 mm standard length) from the same collection sites (downstream from the paper mill) displayed precocious gonopodial development and more aggressive courtship behavior compared to normal males (Howell et al., 1980). Howell et al. (1980) hypothesized that the masculinizing effect of paper-mill effluent was due to androgenic chemicals in the effluent. This hypothesis is supported by Turner (1941) and Angus et al. (200 1), who induced gonopodial development in immature female Gambusia affinis with ethynyl testosterone and 11-ketotestosterone, respectively. Androstenedione, a precursor to testosterone, was later identified in Fenholloway water (Durhan et al., 2002; Jenkins et al., 2001). However, after running in vitro tests of androgenic activity, Durhan et al. (2002) concluded that the isolated androstenedione was not responsible for the a ndrogenic character of Fenholloway water. Howell et al. (1980) made the inte resting observation that female Gambusia are genetically capable of gonopodial development, but the potential remains dormant in the normal absence of male androgen levels. Th is suggestion makes sense in light of the sexual bipotentiality of mosquitofish embryos described above. Overview of Reproductive Endocrinology in Fishes Although gonadal activity is ofte n the focus of teleostean fish reproduction studies, reproduction really begins in the hypothalamu s. In response to environmental stimuli such as photoperiod, the hypothalamus secr etes gonadotropin releasing hormone (GnRH), which causes the anterior pituita ry to release two gonadotropic hormones

PAGE 27

12 (GTH) (Norris, 1997). Thes e are sometimes called GTH-I, similar to mammalian follicle-stimulating hormone (FSH); and GT H-II, similar to mammalian luteinizing hormone (LH). FSH stimulates spermatogene sis and oogenesis; LH causes final gamete maturation and ovulation or sperm release (Norri s, 1997). In both ovaries and testes, LH affects gametes by stimulating the sy nthesis of progesterone-derived 17,20 -dihydroxy4-pregnen-3-one (17,20 -P) (Kobayashi et al., 1993; 2002). In addition to gamete production, LH and FSH also stimulate gonadal steroidogenesis in teleosts (Norris, 1997; Sc hulz and Miura, 2002). The three steroids relevant to our study are estradiol-17 testosterone, and 11-keto testosterone (11-KT). Although all three hormones occur in both sexe s, we focused on estradiol in females and testosterone and 11-KT in males. In fe males, estradiol promotes sexual maturation, gonadal growth, hepatic vitellogenesis (yol k precursor producti on), and oogenesis (Kobayashi et al., 2002; Norris, 1997). In males, testosterone promotes sexual maturation, development of secondary sex ch aracters, spermatogenesis (particularly toward the end), sperm quality, spawning, a nd sexual behavior (Norris, 1997, Ogino et al., 2004; Toft et al., 2003, Wu et al., 2003). It is likely that 11-KT also participates in these processes; it is best known for its ab ility to induce spermatogonial proliferation, which usually is not accomplished by test osterone (Schulz and Miura, 2002). Lakes For the studies described in Chapters 1 through 3, we sampled fish from Lake Apopka and Lake Woodruff in central Florida. These lakes differ in terms of their ecology and contaminant loads; some of the ma in differences are highlighted below. We selected these lakes because they are geographi cally close, and thus subject to the same photoperiod. Furthermore, our lab has previ ously published detailed contaminant data

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13 coupled with comparative reproduction studies of alligators from these two lakes (Guillette et al., 1999; Guillet te et al., 2000). These earl ier findings stimulated the hypotheses tested in our study. Lake Apopka Lake Apopka is impacted by a nearby (1 mile away) EPA-designated Superfund site. Superfund status indicates that the area is contam inated with uncontrolled hazardous waste and poses a recognized risk to the e nvironment or human health. Main pollutants in Lake Apopka include polychlorinated bi phenyls (PCBs) and se veral organochlorine pesticides, including Dicofol, DDT and its metabolites p,p’-DDD and p,p’-DDE, Dieldrin, Endrin, Mirex, Me thoxychlor, Chlordane, Toxaphene, and trans-Nonachlor (Guillette et al., 2000). Thes e chemicals were washed into the lake from agricultural lands, or the Superfund site, where a Dico fol (15% DDT) spill occurred in 1980. The chemicals listed here have been identified and measured in both alligator eggs and alligator serum taken from animals living in La ke Apopka (Guillette et al., 2000; Heinz et al., 1991). In addition, elevated concentrations of several of these compounds have been measured in the tissues of mosquitofish (U .S. Fish and Wildlife Service, unpubl. data) and brown bullheads (Gallagher et al., 2001) from Lake Apopka. In mosquitofish, contaminant concentr ations are around 0.17 mg/kg for DDT, 9.2 mg/kg for Toxaphene, 1.1 mg/kg for p,p ’-DDD and 0.54 mg/kg for trans-Nonachlor (Greg Masson, U.S. Fish and Wildlife Serv ice, pers. comm.). Alligator egg dosing studies of 0.1 to 10 mg/kg of DDE, DDD, or trans-Nonachlor have caused alterations in sex determination, endocrine function, s econdary sex character istics and/or gonadal anatomy (Crain, 1997; Matter et al., 1998; Rooney, 1998). Compared to cohorts from Lake Woodruff, female alligators from La ke Apopka exhibit above-normal plasma

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14 estradiol concentrations and abnormal ovarian morphology (with large numbers of polyovular follicles and polynuclear oocytes). Male alligators have lower-than-normal plasma testosterone concentrations, poorly organized testes, and abnormally small phalli (Guillette et al., 1994). In addition, allig ator eggs from Lake Apopka exhibit low hatchability; and neonates show increased ra tes of mortality, poor motor coordination, changes in metabolism (particularly in liver and steroidogenic enzymes), and altered gene expression (reviewed in Guillette et al., 2000; Guillette and Gunderson, 2001). Given these previous studies, it is reasonable to expect that Gambusia in Lake Apopka are subject to reproductive alterations in a ssociation with cont aminant exposure. Lake Woodruff Lake Woodruff is a National Wildlife Refuge. While not contaminant-free, alligators captured from Lake Woodruff (re lative to Lake Apopka) have fewer chemicals in their body tissues; and those chemicals occur at lower concentrations (Guillette et al., 1999). For this reason, our laboratory has traditionally used Lake Woodruff as a reference site. In addition to contaminant concentrations, other water-quality measures also distinguish the two lakes. Based on our data (in conjunction with average data for 2000 to 2003, taken from EPA’s STORET public-access database, http://www.epa.gov/storet/dbtop.html ), Lake Woodruff has a different seasonal temperature profile that favor s cooler temperatures in fa ll and spring, lower nitrogen and phosphorus concentrations, greater water clar ity (Secchi depth), and lower turbidity and total suspended solids relative to Lake Apopka. In evaluating the results of our study, we considered these ecological differences, and al so considered lake-associated variation in contaminant concentrations.

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15 Florida Springs and Nitrate Overview Over the past 40 years, concentrations of nitrate (NO3-N) in several of Florida’s artesian springs have increased from less th an 0.1 mg/L to more than 5 mg/L (Katz, 2004). The highest measured concentration was 38 mg/L NO3-N in a small spring along the Suwannee River in northern Florida (Katz et al., 1999). This is almost four times the EPA drinking-water standard of 10 mg/L NO3-N. Most of the nitrate comes from inorganic fertilizers applied to land, ultimat ely leaching through the ground to recharge Florida’s aquifer (Katz, 2004). A potentially important ecological problem often caused by increased nitrates is eutrophication, which can increase algal and plant growth. The excessive flora can cause fluctuations in aquatic light levels and dissolved oxygen concentrations, thus affecti ng survival and diversity of aquatic organisms and overall community structure (Attayde and Hansson, 1999; Capriulo et al ., 2002; Irfanullah and Moss, 2004). Apart from the negative ecological eff ects of eutrophication, nitrate can also directly harm animals living in affected aqua tic systems. These effects range from gross toxicity to subtle, but eq ually alarming, changes in physiology and development (Guillette and Edwards, 2005). For example, mortality of the larvae of cutthroat trout, Chinook salmon, and rainbow trout occurs at NO3-N concentrations ranging from 2.3 to 7.6 mg/L (Kincheloe et al., 1979). Survival of chorus frog and leopard frog tadpoles decreased significantly after exposure to 10 mg/L NO3-N (Hecnar, 1995). This concentration is considered the upper limit fo r safety in drinking water (EPA, 1996). On the other hand, the 96 h median lethal concentration (LC50) for fathead minnow larvae is 1,341 mg/L NO3-N; while it is 462 mg/L NO3-N for adult Daphnia magna (an

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16 invertebrate) (Scott and Crunkilton, 2000). Th ese examples show that sensitivity to nitrate varies greatly among species, and ofte n depends on the stage of development at the time of exposure. The best-known human health effect of nitrate is methemoglobinemia (blue-baby syndrome) (Gatseva et al., 1996; Scott a nd Crunkilton, 2000). This condition occurs when nitrate interacts with hemoglobin in the blood, causing it to crystallize. A similar condition, called brown blood disease, occurs in fishes exposed to high nitrite levels. Tissue hypoxia and cyanosis result because th e crystallized hemoglobin cannot function as an oxygen carrier. Risks associated with methemoglobinemia are the reason maximum nitrate concentration in dri nking water is regulated at 10 mg/L NO3-N (EPA, 1996). In addition to methemoglobinemia, nitr ate and nitrite have been implicated in mild hepatic degeneration in ra ts (Gatseva et al., 1999); redu ced steroidogenesis in rats (Panesar, 1999; Panesar and Chan, 2000), frogs (Barbeau, 2004), and alligators (Guillette and Edwards, 2005); decreased human sper m motility; and incr eased human sperm mortality (Rosselli et al., 1995). Nitrogen Cycling, In Vivo Nitrogen Metabolism, a nd Effect of Nitrate on Steroidogenesis Nitrogen is naturally cycled in terrestria l and aquatic ecosystems. For example, ammonia excreted by fishes is converted to nitrite (NO2) by aerobic nitrifying bacteria ( Nitrosomonas sp.), and then oxidized to more stable nitrate (NO3) by Nitrobacter bacteria. Nitrate is assimilate d by plants as a nutrient, or can be converted back to nitrite and then atmospheric nitrogen (N2) by anaerobic denitrifying bacteria. In anoxic environments, or when the nitrifying or deni trifying activity of bact erial populations is

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17 overwhelmed (as can result from overfeeding in aquaculture systems) nitrite levels can spike, placing fish populations at imme diate risk for brown blood disease. In vivo conversions between nitrate and nitrite also occur. Nitrate and nitrite enter the bodies of freshwater animals by cro ssing the gill epithelia and accumulating in extracellular fluid (Jensen, 1995). In crusta ceans, nitrate and nitr ite are transported against the concentration gradient by substitu ting for chloride in th e bicarbonate-chloride exchange mechanism that normally participat es in the osmoregulat ory and respiratory functions of the gill ( Jensen, 1995; Lee and Pr itchard, 1985). In cray fish, nitrate uptake is pH dependent with upta ke increasing as water pH declines (Jensen, 1995). In vivo NO3 can be converted to nitrite (NO2), and then nitric oxide (NO) (Panesar and Chan, 2000; Samouilov et al., 1998). Nitrite is c onverted to NO in various ways, including endogenous nitrite reductase (N R), nitric oxide synthase (NOS), various non-NOS enzymatic and non-enzymatic mechanisms, and low pH (Cadenas et al., 2000; Doblander and Lackner, 1996; Kozlov et al., 1999; Le pore, 2000; Meyer, 1995; Nohl et al., 2001; Panesar and Chan, 2000; Samouilov et al., 1998; Stuehr and Marletta, 1985; Vanin et al., 1993; Weitzberg and Lundberg, 1998; Zweier et al., 1999). Nitric oxide is a gas that diffuses through tissues, playing diverse role s in vasodilation, cell-to-cell signaling, neurotransmission, and immunity. One specific action of NO is the i nhibition of steroid hormone synthesis (DelPunta et al., 1996; Kostic et al., 1998; Panesar, 1999; Panesar and Chan, 2000; Vanvoorhis et al., 1994; Weitzberg and Lundberg, 1998). In normal steroidogenesis, free cholestero l is taken into th e mitochondria and converted to progesterone (the precursor of testosterone, 11-ketotestosterone, and estradiol). This involves st eroidogenic acute regulatory pr otein (StAR) and cytochrome

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18 P450 enzymes such as P450-sidech ain cleavage enzyme (SCC) and 3 -hydroxysteroid dehydrogenase (3 HSD). Nitric oxide inhibits these en zymes, with the result that steroid hormone production is reduced (Panesar and Chan, 2000). This has serious implications for reproduction and development, since both directly depend on appropriate hormone levels. Effects of Nitrates on Sperm Motility and Viability Effects of nitrate or nitric oxide (NO) on sperm motility and viability have been investigated only recently, and results are c onflicting. Certainly, if steroidogenesis is inhibited by nitrate, n itrite, or NO, then spermatogenesis could be affected since both androgens and estrogens are required for sp ermatogenesis (Cochran, 1992; Hess et al., 1997; Miura et al., 1991; Vizziano et al ., 1996). Additionally, NOS and NO are associated with activation of eggs and sperm (acrosome reaction) (Kuo et al., 2000; Revelli et al., 2001). Rosselli et al. (1995) found that human sperm incubated with NO have decreased motility and increased mortalit y. The percentage of immotile and dead sperm also correlated positively with nitrite-nitrate levels in the seminal plasma of the sperm donors (Rosselli et al., 1995). Furthermore, bulls exposed orally to nitrates (100 to 250 g/day/animal) showed reduced sperm motility, increased sperm abnormalities, and degenerative lesions in the spermatocyte and sper matid germ layers of the testis (Zraly et al., 1997). Summary of Nitrate’s E ffects on Reproduction Evidence in the literature shows that n itrate can affect sperm quality and the synthesis of sex steroid hormones, with possi ble deleterious down-stream effects on other

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19 reproductive variables, which rely on appropr iate hormone concentrations. Hypotheses based on this evidence are investigated in the 4th and 5th chapters of our study. Review of Endocrine Disruption in Fishes Table 1-1 shows known effects of endocrine disruption in fishes. To date, more data are available regarding effects on ma les than on females. Although numerous studies of nitrate-mediated endocrine disrup tion are published for mamm als, this area is understudied in fishes. Hypotheses and Goals Based on the review presented here, we hypothesized that adult female and male Gambusia holbrooki captured from sites with high or ganochlorine concentrations (Lake Apopka) or high nitrate (springs), would exhibit altered repr oductive parameters compared with reference or low nitrate sites. Based on previous studies (Table 1-1), we expected to observe increased hepatoso matic index, reduced gonadosomatic index, altered steroid hormone profiles (estradi ol in females; te stosterone and 11ketotestosterone in males), reduced embr yo number, decreased em bryo weights; poor sperm quality; and diminished gonopodial length among fish from contaminated sites. In addition to our assessment of possible endoc rine disruption in the sampled populations, we expected to observe seasonal variati on in reproduction related to changes in temperature and photoperiod. Overall, our goal was to understand the pot ential for endocrine disruption in our study systems in context of the seasonal reproductive cycle a nd some aspects of ecological variation. We measured variable s that are informativ e in terms of basic reproductive biology of Gambusia are likely targets of endocri ne disruption (Table 1-1: note parameters highlighted in gray), and that if disrupted, could a ffect fitness. Field

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20 studies such as mine are useful because th ey measure reproductive ch aracteristics in the wild, under natural conditions that are impossibl e to replicate in the lab. Thus, they can be used to generate hypotheses regarding any observed variati on in reproduction, and suggest causal mechanisms. However, it is understood that, without complementary experimental studies that test field-generated hypotheses, fi eld studies are correlative: they suggest, but do not show cause and effect.

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21 Table 1-1. Endocrine disruption in fishes Class of Endocrine Disruptors† Sample Compounds Observed Alterations in Reproduction Caused By or Associated With Endocrine Disruptor Exposure References Estrogen Estradiolc ghrstx 4-Tertpentylphenolfgh 4-Tert-octylphenolsx Octylphenolt P-nonylphenola 4-Nonylphenold e Nonylphenolw Endosulfanw Keponeb DDDbyz Bisphenol Ai PCBsjyz Treated sewage effluentklmn o p Chlordaneqyz Chronic hypoxia (1 0.2 mg/L)v Toxapheney z DDEyz Organochlorine mixtureyz* Plasma vitellogeninghlmnps Hepatosomatic indexlt Ovotestes/intersexaghkmnqs Oviduct formationgfkm Delayed puberty/persistent immature testesd Gonadosomatic indextux Sertoli cell structuret Gonadal developmentv Diameter of seminiferous tubulesh Atrophy of germinal epitheliumh Primordial germ cell numberfg PCG distribution in developing gonadw Malformed germ cellsn (intersex fish) Delayed spermatogenesisghn Loss of spermatogenic cystsh Sperm Countsikn z Milt volumeknt (intersex fishes) Sperm motilitykv (intersex fishes) Occluded reproductive ductsnt (intersex fishes) Delayed gonopodial developmentcdo Genital papilla lengthr Adult colorationx Courtship behaviorc Fertilization successkv (intersex fishes) Embryonic/larval survivalejuv Oocyte maturationb Oocyte atresian Embryo growths Ca++, amino acid availability to fetuses during gestations Delayed hatchingjv E2 binding in livers Serum E2, Tv Plasma T, 11-KTy Plasma E2,Tn (intersex fish) Serum E2 vy Serum T, 11-KTpuv 17, 20-DHPu aBarnhoorn et al., 2004 bDas and Thomas, 1999 cDoyle and Lim, 2002 dDreze et al., 2000 eFairchild et al., 1999 fGimeno et al., 1997 gGimeno et al., 1998a hGimeno et al., 1998b iHaubruge et al., 2000 jNakayama et al., 2005 kJobling et al., 2002b lLye et al., 1997 mRodgers-Gray et al., 2001 nJobling et al., 2002a oBatty and Lim, 1999 pFolmar et al., 1996 qHarshbarger et al., 2000 rKirby et al., 2003 sRasmussen et al., 2002 tRasmussen and Korsgaard, 2004 uSchultz et al., 2003 vWu et al., 2003 wWilley and Krone, 2001 xToft and Baatrup, 2001 yGallagher et al., 2001 zToft et al., 2003

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22 Table 1-1 Continued Class of Endocrine Disruptors† Sample Compounds Observed Alterations in Reproduction Caused By or Associated With Endocrine Disruptor Exposure References AntiAndrogen Vinclozolinabc DDEabd e Flutamideab Sperm countabc e Fertilization successc Adult colorationabc GSIa Courtship behaviorabc Delayed maturationb Gonopodial developmentb Serum E2 d Plasma T, 11-KTd aBaatrup and Junge, 2001 bBayley et al., 2002 cBayley et al., 2003 dGallagher et al., 2001 eToft et al., 2003 Androgen 11-ketotestosteroneb Paper mill effluentacd ef Methyl-testosteronefg Gonopodial developmentabcd f Male biased sex ratioe Male colorationf Number of reproductive femalesf Intersexg aHowell et al., 1980 bAngus et al., 2001 cBortone and Cody, 1999 dJenkins et al., 2001 eLarsson and Forlin, 2002 fLarsson et al., 2002 gHahlbeck et al., 2004 Aromatase** Inhibition Tributyltinabcd Male-biased sex ratiod Sperm countsa Sperm lacking flagellad ATP content of spermb Lactate dehydrogenase activity in spermb Sperm motilitybd Fertilization successc Hatchabilityc Embryo survivorshipc aHaubruge et al., 2000 bRurangwa et al., 2002 cNakayama et al., 2005 dMcAllister and Kime, 2003 Landfill leachateab Male-biased sex ratioab GSIab Brain aromatase activitya Plasma T, E2 a Delayed vitellogenesisb aNoaksson et al., 2001 bNoaksson et al., 2004 Other Nitratec Spawningc Egg numberc Fertilization ratec Delayed hatching timec Hatching rate of the eggsc cShimura et al., 2002

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23 Table 1-1 Continued †Endocrine disruptor classification is general and non -exclusive. Several ch emicals operate through a number of mechanisms that vary with context. For example, DDE has been called an estrogen, an anti-estrogen, and an anti-androgen. Classifications presented here depend on the papers cited. *Organochlorine mixture – refers to the chemical mixt ure detected in the plasma of juvenile alligators from Lake Apopka. The mix includes PCBs, DDE, DDD, mirex, endrin, dieldrin, trans-nonachlor, and oxychlordane. These chemicals cause male to fe male sex reversal of re ptile embryos (reviewed by Guillette et al., 2000) **Aromatase catalyzes the conversion of testosterone to estradiol, and andr ostenedione to estrone (Johnson and Everitt, 1995). Italicized descriptors refer to papers on Gambusia species. Superscripts match sample compounds, observed effects, and author citations. Highlighted descriptors are related to hypotheses tested in our study. = female. = male. (Intersex fish(es)) = the sex of the fish(es) in the cited study was an abnormal mix of female and male. = increase. = decrease or inhibition. = altered. 11-KT = 11ketotestosterone. ATP = adenos ine triphosphate. DDD, DDE = metabolites of the insecticide DDT. GSI = gonadosomatic index. E2 = estradiol. HSI = hepatosomatic index. PCBs = polychlorinated biphenyls. PGC = primordial germ cell. T = testosterone. T3 = tri-iodothyronine. 17, 20-DHP = 17 20 -dihydroxy-progesterone.

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24 CHAPTER 2 TEMPORAL REPRODUCTIVE PATTERNS FOR FEMALE MOSQUITOFISH CAPTURED FROM TWO FLORIDA LAKES Introduction In the field of endocrine disruption, Howell et al. (1980) first recognized mosquitofish as a model species when they reported that females living downstream from paper mills exhibited masculinized anal fin de velopment. Since then, a handful of other studies have been published (i.e., Dreze et al., 2000; Porte et al., 1992; Toft et al., 2003; Toft and Guillette, 2005), suggesting that mo squitofish are a valuable model. In part, the value of mosquitofish ( Gambusia holbrooki and their western sister, Gambusia affinis ) as sentinel species is associated with their ubiquitous global distribution, which has resulted from their wi despread use as a bi ological control agent for mosquitoes (Courtenay and Meffe, 1989). Th is practice continues today (based on a May 2005 internet search of government pest control programs), de spite the fact that most countries with introduced populations have found them to be ineffective, undesirable, or both (Courten ay and Meffe, 1989). The “ undesirability” is due to Gambusia’s extraordinary ability to adapt and proliferate in new environments (Courtenay and Meffe, 1989). Introduced Gambusia can have broad negative impacts on the biota of aquatic ecosystems. They harass or out-compete native fishes, and eat a wide variety of aquatic invertebrates, as well as the eggs, larvae, or adults of many anuran and fish species, including food and sport fish es (reviewed by Arthington and Lloyd, 1989). Outside their native range, these activiti es disturb habitats, cause economic losses, and,

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25 ironically, can eliminate other native mos quito predators, which are often more efficacious (Courtenay and Meffe 1989; Danielson, 1968). Mosquitofish are successful because they breed continuously during the reproductive season, producing seve ral sequential and slightly overlapping broods of 1 to 245 precocious offspring at approximat ely 22-39 day intervals, depending on environmental temperature (reviewed by Koya et al., 1998). A second reason for their success is their ability to tolerate poor water quality, hi gh levels of pollution, and ongoing habitat disturbance by humans (Courtenay and Meffe, 1989). In addition, their generalist diets (which expose mosquitofish to a vari ety of potential contaminant sources) and intermediate position in the food web s uggest that they bioaccumulate high concentrations of lipophilic contaminants (like PCBs) relative to their environment. This aspect of their biology is useful in biomon itoring programs that assess biological effects of anthropogenic contaminants. For exampl e, Porte et al. (1992) detected seasonal variation in PCBs and organophos phate (OP) pesticides in Gambusia muscle tissue that reflected seasonal inputs of these chemicals to the Ebro Delta in Spain, an area that is heavily used for rice farming. In that study, the authors noted that PCB concentrations were low in females sampled bi-monthly dur ing the April – November breeding season, but increased significantly in December and February, when females are reproductively quiescent. Given that mosquitofish produce large (1.5 to 2 mm (Vargas and de Sostoa, 1996)), yolky oocytes; this indirect evidence suggests that female mosquitofish can lighten their body contaminant loads by offloading contaminants as they lose fat reserves to yolk production. Moreover, during gestation, female Gambusia have been shown to transfer radiolabeled leucine and 4-nonylphe nol (a xenoestrogen) to their offspring

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26 following maternal exposure (Marsh-Matthews et al., 2001; Thibaut et al., 2002). In other fish species, when females bioaccumula te contaminants, they typically pass those contaminants to their offspring in doses that are concentrated, relative to ambient concentrations (Harding et al ., 1997; Nakayama et al., 2005). This can affect maternal fitness by impacting offspring survival, develo pment, or fertility (Black et al., 1998; Nakayama et al., 2005; Rasmussen et al., 2002; Saiki and Ogle, 1995). Given the global potential of Gambusia as model organisms for examining the effects of environmental contaminants on re production, coupled with their ecological and economic impacts, as described above, our study was undertaken to learn more about seasonal/temporal patterns of reproduction among female Gambusia holbrooki in their native range. We collected mosquitofish from two lakes in central Florida: Lake Apopka is eutrophic, with a 50-year history of agricultural, municipal, and industrial contamination, whereas Lake Woodruff National Wildlife Refuge is oligotrophic, is less impacted by human activities, and served as a reference site for a number of related studies (e.g. Guillette et al., 2000). Mosquitofi sh exhibit great genetic diversity coupled with a potential for phenotypic plasticity in life history characters (Downhower et al., 2000; Haynes and Cashner, 1995; Meffe et al., 1995; Stockwell and Vinyard, 2000). These features contribut e to the success of Gambusia as an introduced species (Greene and Brown, 1991). Therefore, the second goa l of our study was to assess reproductive variation between the two lake populations in context of lake-associated environmental differences.

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27 Methods Field and Tissue Collections Between March 2001 and June 2002, 16 mont hly collections of adult female Gambusia holbrooki were made from Lake Apopka (nor th shore, near Beauclair Canal) and Lake Woodruff Wildlife Ref uge (northwest shore, Spring Garden Lake) in central Florida, USA. Fish of mature size (> 1.7 cm, based on our experience) were captured using a 3-mm mesh dip net. Fish maturity was verified in the laboratory from the presence of clearly differentiated white or yolked oocytes in the ovary (Haynes, 1995). Fish were considered reproduc tively quiescent (mature, but not pregnant) at stage 0, when no yolk was visible in the oocytes (Fig. 2-1). Each lake collection took 1 day, plus additional days to process fish. Thus mont hly collections from the two lakes were made on different days (average of 4 days apart). Each monthly sample, ranging from 39 – 60 fish per lake, was divided into two subsets. The first subset was used to obt ain ovarian and hepatic weight, embryo number, and embryo stage. These fish (n = 23 – 31 pe r month, per lake) were held live in aerated coolers filled with lake water, fed flake fi sh food ad libitum, and processed within 1-2 days of capture. Before necropsy, fish were over-anesthetized in r oom-temperature water containing 0.1% MS222 (3-aminobenzoic acid et hyl ester, methanesulfonate salt, Sigma #A5040). The ovary (0.1 to 322 mg) and liver (0.1 to 31.5 mg) were removed and weighed to the nearest 0.1 mg. Embryos (sta ge 3 and greater) were separated from the ovarian stroma, counted, and staged accordi ng to Haynes (1995) (Fig. 2-1). Embryos were staged at half stages (e.g., 4.5) if they were tran sitional between two stages. Unfertilized oocytes that were younger than st age 3 were staged, but not counted. Note that, within a brood, embryos are generally s ynchronized in their development. Average

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28 embryo wet weight for each brood was calcula ted as ovarian weight divided by embryo number. Note that this value is exaggerated sl ightly for larvae just before birth (stage 9 – 10) because, at that time, oocytes for the next brood have started to accumulate yolk, thus adding weight to the ovary that is no t related to the counted embryos. The second subset of captured fish was us ed for measurement of muscle estradiol concentrations. These fish (n = 7 – 12 per month, per lake; mean = 10) were frozen immediately at capture and held on ice while in the field. U pon return to the laboratory, the caudal fin was removed, and all caudal pe duncle tissue posterior to the gonad was cut from the fish, weighed (average = 71 mg), and frozen (-80 C). Estradiol was measured on lipid extracts of these pe duncle tissues, as described below. Because the caudal peduncle is primarily muscle, we will refer to it as muscle for the remainder of the chapter. Heppell and Sullivan (2000) showed that seasonal patterns of muscle and plasma estradiol concentrations were compar able in female gag grouper, although actual concentrations in muscle (measured as pg/g) were an order of magnitude lower than plasma concentrations (measured as ng/ml). For all fish (both subsets), standard le ngth (SL) was measured to the nearest 0.01 cm from the snout tip to the caudal peduncle using calipers. Fish were blotted dry and weighed with an electronic balance to the nearest milligram. Water temperature, pH, and conductivity data were obtained at the time and location where fish were sampled, using a handheld Ultrameter (Model 6P, Myron L Company, Carlsbad, CA). To describe addi tional water quality parameters for the two lakes, we referred to STORET, a public -access EPA database of environmental measurements ( http://www.epa.gov/storet/dbtop.html ). We gathered data from 2000 –

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29 2003 on nitrogen and phosphorus concentratio ns, secchi depth, turbidity, and total suspended solids. It should be noted that th e usefulness of this database is limited by the relatively few sampling dates recorded each year: Lake Apopka was sampled 5 times in 2000, and once in each of 2001-2003; Lake Woodruff was sampled 6 times in 2000, twice in 2001, once in 2002, and 3 times in 2003. Our lake comparison based on STORET data reflects the average values for measurements made in 2000 – 2003 and thus provides a helpful, but generalized view of water quality in each lake. Muscle Estradiol Measurements For measurement of muscle concentrations of 17-estradiol, frozen peduncle tissues were thawed in glass tubes, on ice, and homogenized in 1 ml 65mM borate buffer (pH 8.0). Homogenate was extracted twice with 5 ml diethyl ether. For each extraction, ether and homogenate were mixed together for two minutes using a multi-tube vortex mixer. For the first extraction, tubes were allo wed to settle for three minutes to separate phases. For the second extraction, phases were separated by centrifugation for two minutes. The aqueous phase was frozen in a methanol bath chilled to -25C with dry ice. The ether from both extractions was combined in a second glass tube and evaporated under dry forced air. Hormone concentrations were determined using validated enzyme immunoassay (EIA) kits (Cat No. 582251) purchased from Cayman Chemical Company (Ann Arbor, Michigan). Dry extract was reconstituted in 500 l EIA buffer so that samples would fall within the range of the standa rd curve. EIAs were run as recommended by Cayman with an 18 h refrigerated incubation to increase se nsitivity. Data were quantified against a standard curve linearized using a logit tr ansformation of B/Bo (bound sample/maximum

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30 bound). Duplicate or triplicate interassay vari ance (IAV) samples at two dilutions were included with each plate. The coefficient of variance among all plates, averaged for the two dilutions was 22.6%. To normalize sample estradiol concentrations across assays, we multiplied by a correction factor derived from the relationship between individual plate IAV values and the mean IA V values for all plates. Statistics Estradiol data points lying more than thr ee standard deviations from the mean for all samples (n = 9, 3%) were excluded from the data set. Using a correlation matrix, data were screened for correlations among sta ndard length (SL), body weight, ovarian and hepatic weight, embryo number, embryo wet we ight, and embryo stage. We also looked for correlations between mate rnal body size and muscle estradiol concentrations. Apparent correlations were visualized using linear regression following log10 transformation of the variables. Embryo number was positively related to maternal SL ( r2 = 0.44, p < 0.0001). Likewise, hepatic weight wa s positively related to maternal body weight ( r2 = 0.57, p < 0.0001). Therefore, mean embryo number and hepatic weight were adjusted for body size (using ANCOVA) when appropriate. We used ANOVA or ANCOVA to compar e mean response variable values between consecutive months within a lake a nd to compare mean res ponse variable values between lakes in each month. Changes in embryo wet-weight and adjusted hepatic weight during gestation were similarly comp ared between stages within a lake, and between lakes at each stage. Correla tion, regression, and ANOVA analyses were completed using Statview 5.0. ANCOVA analys es and calculations of adjusted means were completed using SPSS 13.0. Results were considered significant at p 0.05.

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31 Gonadosomatic Index Gonadosomatic index (GSI) is a traditiona l measurement of fish ovarian weight relative to body size. M ean GSI is often used to indicate temporal changes in female (or male) reproductive status. However, in viviparous mosquitofish, GSI is not an accurate measure, particularly in a seasonal study th at invokes mean GSI values to describe reproductive trends. This is because female s are not reproductively synchronized as a group: in any given month dur ing the reproductive season, fema les exhibit all stages of embryonic development (Fig. 2-2) (alt hough within a female, the embryos are synchronized). Furthermore, ovarian weight is determined in part by embryo number, which varies with maternal body size. For th ese reasons, we do not consider GSI a valid measurement for female mosquitofish, particul arly when evaluated as an average value, and thus have not included it as a response variable. Results Environmental Differences between Lakes Water temperature, which exhibited a s easonal pattern, was similar between the two lakes, except at the end of fall and beginni ng of spring, when the temperature of Lake Apopka was about 5C warmer (Fig. 2-3). Mean conductivity, which differed slightly between lakes, ranged from 430 to 766 S (mean = 591 S) in Lake Apopka and from 700 to 1800 S (mean = 1231 S) in Lake Woodruff. However, this difference is unlikely to be biologically rele vant to osmotic balance (1800 S 4% seawater). Lake pH fluctuated monthly at both sites, ra nging from 6.78 to 8.73 (mean = 7.62) in Lake Apopka and 6.47 to 8.47 (mean = 7.22) in Lake W oodruff. Fluctuations in pH did not follow a predictable pattern in either lake, a nd thus we could not de tect any particular effect of pH on the measured variables re levant to our study. Additional descriptive

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32 information on lake water quality is shown in Table 2-1. On average (2000 to 2003), Lake Woodruff has lower nitrogen and phos phorus concentrations than the more eutrophic Lake Apopka. Lake Woodruff also ha s greater water clarit y (Secchi depth) and lower turbidity and total suspended solids. Ou r visual observations of water clarity in the two lakes corroborate these data. Temporal and Lake-Associated Variatio n in Response Variables Related to Reproduction Body size In general, the adult female mosquitofish collected from Lake Apopka were bigger than those from Lake Woodruff (Fig. 2-4) Mean body size of captured females from both lakes was higher at the be ginning of the reproductive seas on, and lower at the end of the year. This change could reflect fall recr uitment of newly matured fish born earlier in the year (which, although mature, will not fi nish growing until the next spring). We observed that 1.7 cm was the smallest size for mature females from either lake, suggesting that this is the minimum size fo r female reproductive activity. However, all fish at this size need not be mature, as we did observe immature fish that were up to 2.0 cm in standard length. Temporal changes in reproductive activity Females collected from both lakes were reproductively active ( 90% pregnant) during the spring summer and fall, and quies cent for at least two months during the winter (Figs. 2-2 and 2-5). The timing of spring recrudescence was different between the two sample populations, with females from Lake Woodruff being delayed by 2 to 3 months (Fig. 2-5). In addition, spri ng reproductive recrudescence was more synchronized in females from Lake Apopka compared to those from Lake Woodruff,

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33 which were more variable in their timi ng of reproductive onset (Fig. 2-5). This difference in timing is probably due to spri ng temperature differences between the lakes (Fig. 2-3), as Lake Apopka warms up more rapidly in the spring. The observed pattern suggests that temperature, rather than photope riod, is the cue respons ible for spring onset of reproductive activity in mosquitofish. Embryo number, size, and stage of development In sample populations from both lakes, embryo number was significantly ( p < 0.0001) and positively related to female standa rd length (SL), although the strength and slope of this relationship was different be tween females from the two lakes (Apopka: r2 = 0.54; Woodruff: r2 = 0.14; ANCOVA indicated a signifi cant interaction between lake and SL, p < 0.0005 ) (Fig. 2-6). In Lake Apopka, large females produced many more offspring than small females, a trend that was not as strong among females from Lake Woodruff (Fig. 2-6). On average, fema les from Lake Apopka produced 12.5 embryos per brood, whereas females form Lake Woodr uff produced only 5.1 embryos per brood. Mean litter size (adjusted for maternal st andard length) was typically larger among females from Lake Apopka, except at the beginning and end of the reproductive season, when litter sizes were similar in the two lakes (Fig. 2-5). Because litter sizes were larger among fe males from Lake Apopka, it is reasonable to hypothesize that a tradeoff between embryo number and embryo size exists such that Apopka embryos might be smaller than Woodr uff embryos. This, however, was not the case. Although all embryos gained wet wei ght as they developed, there was no lakeassociated difference in embryo wet-weight at birth, based on ANOVA (Fig. 2-7). We did observe lake related differences in embryo wet weight between stages 9 – 10,

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34 although this could be an artifact of the me thod of wet weight calculation, as explained above in Methods. Hepatosomatic index We observed that mean hepatic weight (adjusted for female body weight) was dependent on embryonic stage, an association th at is probably relate d to vitellogenesis. For maximum sample size, we combined fish from both lakes and noted that adjusted hepatic weight increased from stage 0 to st age 2.5, declined gra dually through stage 6 – 8, and rose again between stages 8 and 10.5 (Fig 2-8A). Note that females in transition between broods do not exhibit stage 0 (reproductive quiescence, no yolked oocytes present) as broods overlap slightly (Fig. 21, see photographic panel for stage 2). When HSI data were split between lakes, we observe d a similar gestation-related pattern in HSI among females from both lakes, although adju sted hepatic weight was often higher among females from Lake Apopka (Fig. 2-8B). Estradiol Temporal variation in female muscle estradiol (E2) concentrations was evident and often differed between the two lake populations (Fig. 2-9). Females from Lake Apopka exhibited a significant decline in E2 from September through December, followed by three significant peaks in February, April, a nd June. Females from Lake Woodruff also exhibited significant fluctuations in E2 throughout the year, but the amplitude of the fluctuation was less than for females from La ke Apopka. In addition, females from Lake Woodruff exhibited a fall peak in muscle E2 that was significantly higher than E2 concentrations in Apopka cohorts.

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35 Discussion Female mosquitofish in central Florida begin reproductive activity in spring when water temperatures exceed 22 C, and end activity in fall when daylength shortens to between 12 and 11 h, after which females no longer produce new broods, but presumably complete gestation of broods already in pr ogress. Interestingl y the influence of temperature in spring overrides short photope riods, because we observed reproductive females in the late January collection from Lake Apopka, when daylength was 10.5 h, but water temperature reached 27 C. Similarly, decreasing photo period in the fall overrides temperature, because fewer than 25% of Apopk a females were pregnant in late October (daylength = 11 h), even though daytime wa ter temperatures could still exceed 25 C. These observations are similar to those of Koya and Kamiya (2000), who observed that, in a Japanese population of Gambusia affinis spring vitellogenesis occurred when temperature exceeded 14 C, and pregnancy proceeded when temperature exceeded 18 C. In the same study, Koya and Kamiya (2000) repo rted that sexually active females ceased vitellogenesis when photoperiod shortened to 12.5 h. Previously published data, and data presen ted here, indicate th at Lake Apopka is more eutrophic in terms of nitrogen and phosphor us content and contains more estrogenic or antiandrogenic endocrine-disrupting co mpounds when compared to Lake Woodruff (EPA STORET database, http://www.epa.gov/storet/dbtop.html ; Guillette et al., 1999). Moreover, Lake Apopka has lower water clar ity as described by Secchi depth, turbidity, and total suspended solids. Exposure to estr ogenic chemicals is ofte n related to reduced fecundity. For example, dosing of female me daka or fathead minnows with estradiol or bisphenol A (weakly estrogenic) has been show n to increase vitellogenesis and, at higher

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36 doses, reduce or abolish egg production (Palac e et al., 2002; Patyna et al., 1999; Scholz, and Gutzeit, 2000; Sohoni et al., 2001). Li kewise, estradiol exposure arrests embryo development in zebra Danios (Kime and Nash, 1999). In addition, reduced water clarity in Lake Apopka might be expected to reduce reproductive output of females because of the lower light availability, as shown experimentally for G. affinis by Hubbs (1999). Despite these data, which would predict lo wer fecundity among Apopka females relative to females from Lake Woodruff, we found th e opposite. In our study, females from Lake Apopka were significantly larger and more f ecund, even when fecundity is adjusted for maternal body size. Moreover, their increa se in fecundity was not accompanied by a decrease in embryo size, suggesting that Apopka females genuinely have greater reproductive output compared to Woodruff female s. The larger size of Apopka females suggests that they grow faster and/or live longer than female s from Lake Woodruff. This may be due to higher primary productivity in Lake Apopka, driven by increased nutrient loads, which results in greater food availability with respect to mosquitofish. In a field study of Poeciliid growth rates, Grether et al. (2001) reported that guppy females and juveniles grew faster in rainforest streams with higher primary productivity. As with fecundity, adjusted hepatic weight was higher among Apopka females relative to females from Lake Woodruff. It is likely that fecundity and hepatic weight are causally related, but unclear in which directi on. For example, with their higher fecundity, Apopka females probably require increased yolk production. In mosquitofish, yolk is derived, in part, from vitellogenin produced in the liver in res ponse to circulating estrogens (Tolar et al., 2001). Thus, the in creased need for vitellogenesis could explain the increased hepatic weight observed among Ap opka fish. Alternatively, the estrogenic

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37 contaminants that characterize Lake Apopka (Guillette et al., 1999) could stimulate vitellogenesis (Palace et al ., 2002; Sohoni et al., 2001). In an unexpected twist, contaminant-stimulated vitellogenesis could actually permit the increased fecundity of Apopka females. However, offspring survivorship remains to be tested. In the first half of 2002, we noted a significant rise in muscle estradiol concentrations among females from Lake Apopka compared to females from Lake Woodruff. Gallagher et al. ( 2001) reported estradiol concentr ations for female bullheads caught in January and July from Lake Apopka and Lake Woodruff, but they observed no lake-associated differences. However, thei r limited number of collection dates could explain their non-detection of changes in e ndogenous estradiol. In other studies of female fishes captured from sites affected by estrogenic pollution, like Lake Apopka, increased estradiol concentrations have been reported. This is true, for example, of female walleye, captured from water cont aminated with estrogenic treated sewage effluent (Folmar et al., 2001). Conclusions and additional hypotheses. Data presented here indicate that female mosquitofish in central Florida exhibit a well-d efined reproductive cycl e. In addition, we observed lake-associated vari ation in hepatosomatic index and muscle estradiol concentrations that suggest a subtle level of estrogen ic endocrine disruption among Apopka females, consistent with other studie s. However, in conjunction with these observations, we also report that females from Lake Apopka exhibit greater reproductive output in terms of embryo number, compared to females from Lake Woodruff. Although relative survivorship of larval and juvenile fish remains to be established, our present data

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38 suggest that chemical pollution in Lake Apopka might not disrupt resident mosquitofish at the population level. In comparison to other species, mosquitofi sh are recognized for their ability to adapt to variable or polluted environments (Courtenay and Meffe, 1989). This ability is marked by increased heterozygosity and overall genetic diversity among exposed individuals (Downhower et al., 2000; Stockwell and Vinya rd, 2000; Theodorakis and Shugart, 1997). Genetic diversity is supported by Gambusia ’s mating system, in which females often mate with more than one male, resulting in broods characterized by multiple paternity (Zane et al., 1999). Gr eene and Brown (1991) observed that larger female Gambusia affinis were more likely than small females to mate with multiple males. They also observed that multiply inseminated females were more heterozygous than singly mated females, and, that the offspring of multiply inseminated females exhibited greater genetic diversity. Females from Lake Apopka are larger than females from Lake Woodruff, and it would be in teresting to know if their degrees of heterozygosity and genetic di versity were also greater. If that was the case, Gambusia could provide an informative model for the ev olution of characters that are adaptive in polluted environments. Table 2-1. Additional water quality information for Lake Apopka and Lake Woodruff Water Parameter Apopka* Woodruff* Phosphorus as P (mg/L) 0.11 0.03 0.07 0.01 Nitrite (NO2) + Nitrate (NO3) as N (mg/L) 1.15 0.71 0.05 0.02 Nitrogen, Kjeldahl (sum of free ammonia and organic nitrogen) (mg/L) 2.57 0.42 1.27 0.06 Secchi disk depth (m) 0.39 0.09 0.76 0.06 Turbidity (NTU) 12.24 2.66 3.27 0.69 Total suspended solids (TSS) (mg/L) 50.50 7.12 8.53 3.45 *Mean value for lake taken from sampling date s in 2000 to 2003; data retrieved from EPA’s public-access STORET database ( http://www.epa.gov/storet/dbtop.html ).

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39 Figure 2-1. Stages of embryonic deve lopment for eastern mosquitofish ( Gambusia holbrooki ). Stage 0 consists of small, white oocytes (black arrows), indicating a mature, but reproductively quiescent ovary. Stages 1 and 2 indicate progressive yolking of oocytes (black arrows). Note that broods overlap. Stage 3 is the first stage at which all yolked oocytes are similar in size. Stage 4 is distinguished by presen ce of the blastodisk (black arrow). Stage 5 embryos have elongated, and optic discs (black arrow) are visible but unpigmented. Stage 6 embryos exhibit pi gmented optic discs (black arrow). Stage 7 embryos are enlarged, have so me skin pigmentation and advanced, but incomplete eye development. Stage 8 embryos exhibit fully formed and pigmented eyes, pigmented skin, but ta il does not overlap head. Stage 9 embryos retain a large yolk sac but tail overlaps head. Stage 10 embryos have absorbed most their yolk sacs a nd often survive if removed from the ovary. Stage 11 embryos have absorbed their yolk sacs and are ready for birth. Staging is based on Haynes (1995). 0 4 5 7 6 10 8 3 2 1 11 9

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40 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 J-02 F-02 M-02 A-02 M-02 M-02 J-02Collection Date Lake ApopkaPercentage of Females Not Pregnant Stage 0.5 3.5 Stage 4 6.5 Stage 7 8.5 Stage 9 11 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 J-02 F-02 M-02 A-02 M-02 M-02 J-02Collection Date Lake Woodruf f Percentage of Females Not Pregnant Stage 0.5 3.5 Stage 4 6.5 Stage 7 8.5 Stage 9 11B A 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 J-02 F-02 M-02 A-02 M-02 M-02 J-02Collection Date Lake ApopkaPercentage of Females Not Pregnant Stage 0.5 3.5 Stage 4 6.5 Stage 7 8.5 Stage 9 11 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 J-02 F-02 M-02 A-02 M-02 M-02 J-02Collection Date Lake Woodruf f Percentage of Females Not Pregnant Stage 0.5 3.5 Stage 4 6.5 Stage 7 8.5 Stage 9 11B A Figure 2-2. Percentage of female mosquitofish with br oods at the indicated stages of embryonic development. A) Females from Lake Apopka. B) Females from Lake Woodruff. Embryos are developmentally synchronized within a brood, but, as this graph shows, they are not synchronized across broods. Females presenting as “not pregnant” were ma ture, but reproductively quiescent. Note that the first Ma y 2002 collection is missing from Lake Apopka: drought conditions on the lake prevented boat access for two weeks.

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41 10 15 20 25 30 35 40M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateWater Temperature (C ) 10:00 10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00Daylength (hours :mins) Apopka Woodruff Daylength Figure 2-3. Seasonal changes in water temper ature for Lake Apopka and Lake Woodruff, shown with ambient photoperiod for each collection date

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42 2 2.2 2.4 2.6 2.8 3 3.2M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateSL (cm) Apopka Woodruff * * * * * 100 200 300 400 500 600 700M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateBody Weight (mg) Apopka Woodruff * * * * ** *A B 2 2.2 2.4 2.6 2.8 3 3.2M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateSL (cm) Apopka Woodruff * * * * * 100 200 300 400 500 600 700M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateBody Weight (mg) Apopka Woodruff * * * * ** *A B Figure 2-4. Temporal variation in body size of adult female mosquitofish from Lake Apopka and Lake Woodruff. A) Standa rd length (SL). B) Body weight. Graphs show means 1 SE. *Months in which the mean body size of fish from the two lakes are signi ficantly different (ANOVA, p 0.05). A heavier line between data points indi cates a significant temporal change within a lake (ANOVA, p 0.05).

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43 0 2 4 6 8 10 12 14 16M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Embryo Number 0 10 20 30 40 50 60 70 80 90 100% Pregnan t Apopka Woodruff AP % preg WR % preg * * * * Figure 2-5. The right hand y-axis shows th e percentage of sampled females from each lake that were pregnant (contained yo lked oocytes) at each collection date. The left hand y-axis quantifies tempor al variation in mean embryo number (litter size), adjusted for standard length, of adult female mosquitofish from Lake Apopka and Lake Woodruff. Graph shows means 1 SE. *Months in which the mean adjusted embryo number of fish from the two lakes are significantly different (ANCOVA, p 0.05). A heavier line between data points indicates a significant seasonal change in embryo number within a lake (ANCOVA, p 0.05).

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44 0 0.1 MM Apopka Woodruff 0 10 20 30 40 50 60Embryo Number 1.5 2 2.5 3 3.5 4 4.5 5 SL cm 1.5 2 2.5 3 3.5 4 4.5 5 60 50 40 30 20 10 0 Female Standard Length (cm) Embryo Number r2 Apopka= 0.54 r2 Woodruff= 0.14 0 0.1 MM Apopka Woodruff 0 10 20 30 40 50 60Embryo Number 1.5 2 2.5 3 3.5 4 4.5 5 SL cm 1.5 2 2.5 3 3.5 4 4.5 5 60 50 40 30 20 10 0 Female Standard Length (cm) Embryo Number 0 0.1 MM Apopka Woodruff 0 10 20 30 40 50 60Embryo Number 1.5 2 2.5 3 3.5 4 4.5 5 SL cm 1.5 2 2.5 3 3.5 4 4.5 5 60 50 40 30 20 10 0 Female Standard Length (cm) Embryo Number r2 Apopka= 0.54 r2 Woodruff= 0.14 Figure 2-6. Embryo number (li tter size) observed for female mosquitofish of different standard lengths. Data for fish fr om Lake Apopka and Lake Woodruff are shown separately. Regression is significant for both populations (p < 0.0001).

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45 2 3 4 5 6 7 8 9 10 11 12 01234567891011 Embryonic StageEmbryo Wet Weight (mg) Apopka Woodruff * Figure 2-7. Mosquitofish embryonic wet weight at different developm ental stages. Data for fish from Lake Apopka and Lake Wo odruff are shown separately. Graph shows means 1 SE. *Stages at which the mean embryo wet weight was significantly different between fe males from the two lakes (ANOVA, p 0.05). A heavier line between data po ints indicates significant stage-tostage variation within a lake (ANOVA, p 0.05).

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46 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.00 0.5 1 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11Embryonic StageAdj. Hepatic Weight (mg) 1 2 3 4 5 6 7 8 9 100 0.5 1 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11Embryonic StageAdj. Hepatic Weight (mg) Apopka Woodruff * * * * *A B 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.00 0.5 1 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11Embryonic StageAdj. Hepatic Weight (mg) 1 2 3 4 5 6 7 8 9 100 0.5 1 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11Embryonic StageAdj. Hepatic Weight (mg) Apopka Woodruff * * * * *A B Figure 2-8. Mean hepatic weight (1 SE ), adjusted for female body weight of mosquitofish with embryos at different stages. A) Fish from Lake Woodruff and Lake Apopka combined. B) Fish fr om Lake Woodruff and Lake Apopka shown separately. Curved lines in (A) indicate significant changes in hepatic weight across stages (ANCOVA, p 0.05). Similar significant trends were observed in (B) (not shown for graph clar ity). *Indicates that adjusted mean hepatic weight at a given stage of embryonic development was different between lake populations (ANCOVA, p 0.05).

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47 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMuscle Estradiol (pg/mg) Apopka Woodruff * * Figure 2-9. Temporal variation in muscle estradiol concentrations of adult female mosquitofish from Lake Apopka and La ke Woodruff. Graph shows means 1 SE. *Months in which the mean estradio l concentrations of fish from the two lakes are significantly different (ANOVA, p 0.05). A heavier line between data points indicates a significant seas onal change within a lake (ANOVA, p 0.05).

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48 CHAPTER 3 SEASONAL SPERM QUALITY IN MALE Gambusia holbrooki (EASTERN MOSQUITOFISH) COLLECTED FR OM TWO FLORIDA LAKES Introduction Sperm count and sperm viability are import ant measures of male fertility. In several human populations, sperm counts have decreased over the past 50 years, an observation that is related to increased s ubfertility and infertility among men (Carlson et al., 1992; Swan et al., 2000; Jensen et al ., 2002). The underlying causes of reduced human sperm counts remain controversial. However, a variety of studies in non-human animals, particularly fishes, suggest a link with environmental contaminants that have been shown to disrupt endocrine function an d/or reproductive deve lopment (Gray, 1998; Toft and Guillette, 2005; Jobling et al., 2002). For example, in sexually developing and adult guppies, decreases in spermatogenesi s and stripped sperm counts have been observed after exposure to vinclozolin (fungicide) or p,p’-DDE (DDT metabolite) (Baatrup and Junge, 2001; Bayley et al., 2002) Similar observations have been reported in swordtails exposed to nonylphenol (plast icizer) (Kwak et al., 2001); goldfish or zebrafish treated with estradiol or ethynylest radiol (associated with sewage effluent), respectively (Schoenfuss et al., 2002; Van de n Belt et al., 2002); and in adult Japanese medaka exposed to 4-tert-octylphenol (e strogen mimic) (Gronen et al., 1999). In addition to these experimental studies, field studies have shown similar effects among wild English flounder and roach captu red from waterways contaminated with treated sewage effluent (Lye et al., 1998; Jobling et al., 2002b). Toft et al. (2003)

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49 previously reported reduced sperm counts am ong male mosquitofish collected from Lake Apopka in Florida (USA). Over the three months during which sampling occurred, males from Lake Apopka exhibited an average of 47% fewer stripped sperm cells per mg testis compared with cohorts captured from Lake Woodruff (reference lake). Unlike Lake Woodruff, Lake Apopka has a history of contamination by p,p’-DDE and other endocrine-disrupting contaminants with estroge nic or antiandrogenic activity (Guillette et al., 1999). In a follow-up study, Toft and Guillette (2005) observed reduced sperm counts among reference mosquitofish exposed for 1 month to water from Lake Apopka. The annual spermatogenic cycle of mosquito fish has previously been described for populations sampled in Len Province, Spain, and central Japan (Fraile et al., 1992; Koya and Iwase, 2004). Based on these reports, the cycle involves a continuous period of spermatogenesis (spring through fall), followed by a shorter period of winter quiescence. In mosquitofish, the testes are fused into a single, round, white-colored organ that is located centrally in the abdomen, dorsal to the origin of the gonopodium (Fraile et al., 1992). The grooved gonopodium is used by the male to transfer spermatozeugmata to the genital opening of the female. This structure forms during puberty by fusion and modification of the anal fin rays under stimulation by endogenous androgen (Angus et al., 2001; Ogino et al., 2004). A single vas deferens connects the gonopodium to the efferent ducts that coalesce from within the central lumen of each testis (Fraile et al., 1992). The outer wall of the testis is lined with spermatogonia (Fraile et al., 1992). In spring through fall, spermatogonia proliferat e in successive waves of mitosis, forming nests (cysts) of primary spermatocytes bounded by Sertoli cells (Fraile et al., 1992). In a process that takes approximately 30 days, sp ermatocytes within a single cyst undergo

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50 synchronized meiosis and differentiation to produce spermatids and ultimately tailed spermatozoa (Fraile et al., 1992; Koya and Iwase, 2004). As the cysts mature, they move from the periphery of the testis to the cente r, where they are released to the efferent sperm ducts as spherical aggregates of sperm (spermatozeugmata), with tails in the center and heads on the periphery (Fraile et al., 1992 ). At this point, Sertoli cells no longer surround the spermatozeugmata, but rather, th ey hypertrophy and become part of the efferent duct tubule (Fraile et al., 1992). The tubules secr ete a gelatinous matrix that holds the spherical structure of the spermatozeugma together until it reaches the oviduct of a female (reviewed by Constantz, 1989). As winter approaches, production of new spermatocytes ceases. Through the winter, st ored cysts of mature spermatozoa occupy most of the testicular volume, and will be used during early spring copulation, which occurs before the first wave of spring sp ermatogenesis is comple te (Koya and Iwase, 2004). Like most teleosts, mosquitofish exhibit seasonal variation in sperm production that is regulated by changes in temperature and photoperiod (Fraile et al., 1994). However, factors that initiate mosquitofish spermato genesis are not yet fully understood. Males that have recently entered testicular qui escence cannot be stimulated to produce new sperm by increasing temperature or photoperi od (Fraile et al., 1993). However, fish captured at the end of their quiescent period will exhibit proliferation of spermatogonia, even if temperatures remain low and days are short (Fraile et al., 1994). Increasing ambient temperatures are required for differe ntiation of spermatogonia to spermatocytes, and photoperiod must lengthen in order for sp ermatocytes to enter meiosis (Fraile et al., 1994; De Miguel et al., 1994).

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51 We conducted our study from May 2001 to September 2002 to extend our understanding of seasonal sperm production in mosquitofish populations from Lakes Apopka and Woodruff in central Florida. In addition, we tested our hypothesis that low sperm counts per mg testis, previously reporte d for fish from Lake Apopka, are related to reduced sperm counts per spermatozeugma. The resulting data will help identify possible mechanisms by which sperm count can be affected among males from Lake Apopka. In addition to sperm counts, we measured seas onal changes in sperm viability among fish from both lakes. Methods Field Collections Between May 2001 and September 2002, 15 monthly collections of adult male Gambusia holbrooki were made from Lake Apopka (nor th shore, near Beauclair Canal) and Lake Woodruff Wildlife Refuge (northwest shore, Spring Garden Lake) in central Florida, USA. Mature fish (identified by their hooked gonopodium) were captured using a 3-mm mesh dip net. Average monthly sa mple size for the sperm study was 15 fish per lake, with a range of 5 to 2 1. Concurrently, we captured additional fish for testicular histology (n = 3 to10 per mont h, per lake). Water temperature, pH, and conductivity data were obtained at the time and location where fish were sampled, using a handheld Ultrameter (Model 6P, Myron L Company, Carlsbad, CA ). Fish were held live in aerated coolers filled with lake water, fed flake fish food ad libitum, and processed 1 to 2 days after capture. Fish were over-anesthetized im mediately before sperm collection in roomtemperature 0.1% MS222 (3-aminobenzoic acid et hyl ester, methanesulfonate salt, Sigma #A5040). Standard length (SL) was measured to the nearest 0.01 cm from the snout tip to the caudal peduncle using calipers. G onopodium length was measured using an ocular

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52 micrometer mounted on a dissecting microsc ope. Fish were blotted dry and weighed with an electronic balance to the nearest milligram (range was 56 to 463 mg). Testicular Histology Fish captured for testicular histology we re anaesthetized in 0.1% MS222. Testes were removed and fixed in Smith’s fixative (aqueous solution with 0.2% potassium dichromate, 12% acetic acid, and 47% neutral buffered formalin) for 24 h, followed by storage in 75% ethanol. Testes were dehydr ated overnight, by transferring tissues to progressively drier ethanol solutions, infu sed with and embedded in paraffin, and sectioned at 10 m. Mounted sections were staine d using a trichrome procedure with Harris' hematoxylin, fast green, and Biebrich scarlet orange. Testes were considered reproductively active when spermatocyte s and/or spermatids (in addition to spermatogonia and spermatozoa), were present, and quiescent when only spermatozoa and spermatogonia were present (Fraile et al ., 1992; Koya and Iwase, 2004) (Fig. 3-1). The stages of spermatogenesis are shown in Figure 3-1. Sperm Collection Each fish was rinsed in distilled water a nd placed on its side, in a small petri dish containing enough 150 mM KCl to barely cover the fish. With the gonopodium abducted, spermatozeugmata (szm) were stripped from the fish by gently pressing down on the abdomen anterior to the gonopodi um and sweeping cauda lly, using the smooth, rounded end of large forceps with the two tip s taped together (Fi g. 3-2A). Typically, hundreds of variably sized szm are ejected fr om the fish after 1-2 sweeps (Figs. 3-2B, C, D). The fish was removed from the dish, a nd duplicate or triplicate sub-samples of a known number of szm (average = 23) were randomly drawn up in 300 l KCl using a micropipette. This collection step must be done rapidly because the KCl activates the

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53 spermatozoa, causing them to disperse from the szm in a matter of minutes (Figs. 3-2E, F). We collected and counted only intact sz m. The KCl containing the sperm was placed in a 12 x 75mm polystyrene culture tube and kept on ice until the samples were counted (up to 3.5 h). Based on validation tests, sper m maintained on ice remain viable for at least 4 h (0 to 5% change in the number of live sperm in 9 replicate samples measured repeatedly over 5 h). Sperm Staining All sperm samples were stained at the same time using the Live/Dead Sperm Viability Kit available from Molecular Probes Inc. (Cat. #L-7011). Samples containing 300 l sperm were first stained with 15 l florescent SYBR Green, diluted 500 fold from kit stock. Samples were vortexed and inc ubated on ice for 10 minutes, after which 1.5 l propidium iodide (PI) stock were added. Samples were again vortexed and incubated on ice for at least 10 minutes more. SYBR Gr een and PI are nucleic acid stains that differentially stain live sperm green and dead sperm orange, respectively. Sperm are characterized as dead if their cell membrane is compromised, allowing the rapid entry of propidium iodide. To protect florescent stai ns from light, tubes and stains were handled under low light conditions and tubes were incuba ted in a covered cooler containing ice. Sperm Counts and Viability To obtain absolute sperm number s (expressed as sperm number per spermatozeugma (szm)), we needed to quantif y the volume of sample processed by the flow cytometer. This volume was measured indirectly by adding a known number of florescent particles (average of 23,000 particles per 300 l sperm sample) to each tube and then counting the number of particles pr ocessed with the sperm sample. We used AccuCount 5.2 micron florescent particles (S pherotech Inc., Cat. #ACFP-50-5) because

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54 they were similar in size to the sperm, and their red fluorescence spectra allowed them to be distinguished from SYBR Green and PI. Particles were suspended in a small buffer volume (average = 21 l) that was added to individual samples. Sperm samples were vortexed immediately before counting to ensure homogeneous suspension of the particles. Flow cytometric analysis was perfor med on a FACSort flow cytometer (BD Biosciences, San Jose, CA). This instrument uses an argon-ion laser emitting 15 mW of 488 nm light to illuminate the cells. Data for 10,000 to 20,000 particles were collected and analyzed using CellQuest 3.3 software (B D Biosciences). Forward and side light scatter measurements and green (530 +/15nm ), orange (585 +/21 nm), and red (> 650 nm) fluorescence measurements were collected for each sample. The instrument threshold was set on forward light scatter. Sper m cells were identified using a gate on the forward vs. side light scatter dot plot (Fig. 3-3) The contents of this gate were displayed in a second plot that gated green (SYBR Gr een) and orange (PI) fluorescing particles separately (Fig. 3-3). Cells emitting only green florescence were counted as live, and cells emitting any orange florescence (indicating a breach in the cell membrane) were counted as dead cells. Calibration particle counts were quantified from their peak on a separate red-fluorescence histogram (Fig. 3-3) Our methods are similar to those used for Nile tilapia (Segovia et al., 2000) Calculations Absolute sperm count per spermatozeugma was calculated using the formula: (L+D)(B)/(b)(S), where L = nu mber of live sperm counted; D = number of dead sperm counted; B = number of calibra tion particles added per sample; b = number of calibration particles counted; and S = number of sperma tozeugmata originally added to the tube.

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55 Sperm viability was measured as the percentage of live sperm: L/(L+D). Live sperm count per szm was calculated based on this percentage. Statistics Data points lying more than three standa rd deviations from the mean value for sperm count or percentage of live sperm were excluded from the data set (applied to 6 fish or 1.5% of the total sample). Combined data were screened for correlations among SL, body weight, gonopodium length (adjusted fo r SL), sperm count per szm, percentage of live sperm, and live sperm count per sz m using a correlation matrix. Gonopodium length was adjusted for SL (using ANCOV A) because the two were significantly correlated (r2 = 0.70; p < 0.0001). Relationships between mean sperm parameters and water temperature, pH, and conductivity meas ured each month were assessed using linear regression. Differences in sperm data betw een lakes in any given month were tested using ANOVA. Monthly variation in sperm da ta within each lake was tested using Fisher’s PLSD post-hoc test for ANOVA, with cap ture date as the independent variable. Results Sperm count per spermatozeugma (szm), percent live sperm, and live sperm count per szm did not correlate with SL, body weight, or adjusted gonopodium length (r2 < 0.01). There also was no correlation between sperm count per szm and percent live sperm (r2 < 0.0001). In addition, sperm count and viability were not significantly related to monthly water temperature, pH, or conductivity (r2 < 0.14; p > 0.06), although the lowest p-value obtained (p = 0.06) suggests a seasonal effect of water temperature on percent live sperm (Figs. 3-4 and 3-5). Fi gure 3-4 illustrates seasonal change in water temperature and photoperiod during the stud y. In addition, 2002 was characterized by significant drought, particularly through the summer, and the water level of Lake Apopka

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56 was more affected than that of Lake Woodruff in the areas where Gambusia were captured. Sperm Viability In 2001, percent live sperm remained stable at about 85% (with a brief drop to 75% noted in July) from May through the beginning of December among males from Lake Woodruff (reference lake) (Fig. 3-5). Fr om December through mid-March 2002, mean viability dropped to 45%, followed by steady recovery to previous levels through mid-May. Among Woodruff fis h, viability was maintained from May through the last collection in early September (Fig. 3-5). Ba sed on testicular histology obtained from cohorts captured at the same time, male Gambusia from Lake Woodruff reduced production of new spermatocytes in early Oc tober, with reinitiation observed in early January. Through the winter, the fish stored spermatozoa, which progressively lost viability until gonadal recrudescence in the spring (Fig. 3-5). Overall, sperm viability (percent live) among fish from Lake Apopka followed a similar trend to those from Lake Woodruff, with spring recrudescence occurring at a similar time (Fig. 3-5). However, the winter decline in viability st arted 1 month earlier (November) among males from Lake Apopka (Fi g. 3-5). In addition, from June through September 2002, there was a sign ificant decline in sperm viab ility (percent viability = 50%) among fish from Lake Apopka relativ e to cohorts from Lake Woodruff. This decline was not expected based on summer data from 2001 (Fig. 3-5). Testicular histology revealed a decrease in spermatogene sis in September, but not in June. Note that no collections were made be tween June and September.

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57 Sperm Counts During the collection period, mean sperm counts varied from 3400 – 7200 sperm per spermatozeugma (szm) among male mosquito fish collected from the two lakes (Fig. 3-6). In late August 2001, males from La ke Woodruff exhibited significantly higher sperm counts per spermatozeugma (szm) relative to males from Lake Apopka, although the opposite trend was observed 1 year la ter, in early September 2002 (Fig. 3-6). Woodruff males also had higher sperm counts in late January 2002 due to a third seasonal peak in sperm count that was not observed among fish from Lake Apopka (Fig. 3-6). The other two seasonal peaks observed among male s from both lakes occurred in July and October-November 2001 (Fig. 3-6). Note that this second seasonal peak in sperm counts occurred 1 month earlier among Apopka male s, compared to Woodruff males (late October versus late November 2001) (Fig. 3-6). Although cyclic variation in sperm count s was observed during 2001, sperm counts apparently remained constant from mid March through September 2002 (Fig. 3-6). However, no samples were collected between mid June and September 2002, when the first seasonal peak should occur, as predicte d by data from 2001. Therefore the apparent lack of periodicity observed in 2002 may be an artifact of sampling date. In addition, drought conditions in 2002 may account fo r additional variation between the two sampling years. As a measure of fertility, live sperm count per szm is the most relevant of the sperm measures presented here (Fig. 3-7). For bo th lakes, live sperm count (2000 to 5800 live sperm per szm) followed the same seasonal pattern described above for total sperm counts (Fig. 3-7). However, in late Augus t 2001 and throughout the winter, males from Lake Woodruff exhibited higher live sperm counts relative to cohorts from Lake Apopka

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58 (Fig. 3-7). We observed the same effect in September 2002. At no time did live sperm counts from Apopka males exceed those obse rved among males from Lake Woodruff. Discussion Male mosquitofish from both Lake W oodruff and Lake Apopka exhibited cyclic spermatogenesis, with the number of sper m per spermatozeugma rising and falling two to three times per year. In addition, sperm vi ability followed a distinct seasonal pattern, particularly among fish from Lake Woodruff, with viability maintained at 75 to 95% during the breeding season, when sperm are continuously released and replenished, but dropping to as low as 45% during winter qu iescence, when stored sperm presumably degrade over time. We observed that spri ng testicular recrudescence was related to increasing temperature followed by increasing photoperiod. This observation is similar to previous reports of mosquitofish describe d by Fraile et al. (1994) and De Miguel et al. (1994). Seasonal Variation in Sperm Counts In our previous study (Toft et al., 2003), we observed that mosquitofish from Lake Apopka exhibited an average of 47% fewer stripped sperm cells per mg testis compared with cohorts captured from Lake Woodruff. In our study, we report sperm counts as sperm number per spermatozeugma. Based on our study, the lake-associated difference in total ejaculated sperm numb er observed by Toft et al. (2003) is probably not due to reduced sperm counts per spermatozeugma, at least not during most of the year (late January 2002 was an exception). However, th e fact that we observe d temporal variation in the number of sperm per spermatozeugma is an interesting finding in itself, because it suggests that the rates of spermatogonial mito sis or apoptosis vary on a seasonal basis. Our finding adds to previous mosquitofish st udies, which describe monthly variation in

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59 the number of cysts, or volume of tes tis, devoted to a given stage of sperm cell development (Fraile et al., 1992; Koya and Iwas e, 2004). These authors attributed most of the variation to seasonal changes in copu latory behavior, and di fferences in the time required for each stage of sperm cell devel opment. Copulatory behavior could also regulate the number of sperm per sper matozeugma. For example, since most mosquitofish broods exhibit multiple paternity (Zane et al., 1999), suggesting that sperm competition affects individual mosquitofish fitnes s (Evans et al., 2003), it is possible that males increase the number of sperm per sp ermatozeugma when competition is high. To our knowledge, this hypothesis has not been tested in Gambusia. Evans et al. (2003) found that male Gambusia housed with 3 females and another male for eight days (high risk of sperm competition) mated more ofte n and used more sperm when placed with a novel female, relative to males housed with females alone for eight days (low risk of sperm competition). In this system, the housi ng regime (eight days with or without other males) did not affect the number of stripped sperm retrieved from treated males that were not mated to novel females. However, the authors did not test the effects of mating frequency on sperm production rate. In additi on, eight days is too short a time frame to test effects on spermatogenesis, wh ich typically requires 30 days in Gambusia for a single sperm cycle (Koya and Iwase, 2004). As mentioned above, temporal variation in sperm count per spermatozeugma could be due to changing rates of spermatogonial mitosis. Spermatogonia undergo mitosis for two reasons. The first is to maintain a popu lation of spermatogonial stem cells; the second is to produce nests (called cysts in Gambusia) of primary spermatocytes that undergo meiosis (reviewed by Miura and Miura, 2003). Some of the factors that regulate

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60 spermatogonial mitosis have been determined for Japanese eels and were recently reviewed by Miura and Miura (2003). Br iefly, spermatogonial stem cell production is regulated by estradiol, which, in eels, stimul ates the expression of a protein called “eel spermatogenesis related substance” (eSRS 34). Interestingly, estradiol and eSRS34 stimulation result only in renewal of germ cells; they do not promote spermatocyte proliferation and meiosis. For these events to occur, the testis must synthesize 11ketotestosterone (11-KT), which it does in response to gonadotropins released from the pituitary. 11-KT works in conjunction with IG F-1 and activin B, both secreted by Sertoli cells, to promote spermatogonial proliferati on and meiosis. Additi onal regulation of any step in these pathways would affect sp ermatogonial mitosis and possibly explain temporal changes in sperm counts per spermatozeugma. For example, environmental cues, such as temperature and female reproductive pheromones, have been shown to stimulate gonadotropin release in ma le goldfish (Kobayashi et al., 2002). Lake-Associated Variation in Sperm Counts and Quality In the months when lake related varia tion was observed (August to September 2001, November 2001 to February 2002, June to September 2002), male mosquitofish from Lake Woodruff exhibited significantly higher live and/or total sperm counts per spermatozeugma and/or greater viability relative to males from Lake Apopka. An exception occurred in early September 2002, when males from Lake Apopka exhibited higher total sperm counts, but their live sperm counts were still significantly lower than males from Lake Woodruff. The differences in sperm quality between the two lake populations are due to three main observations. First, Woodruff males ex hibited three seasonal peaks in sperm count, in July, late November, and late January, relative to two seasonal peaks in July and

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61 October among fish from Lake Apopka. S econd, while fish from both lakes exhibited decreased sperm viability during the winter, th e period of low viability began 1 month earlier (and was therefore 1 month longer) among fish from Lake Apopka. Third, in June through September 2002, sperm viability among fish from Lake Apopka was significantly reduced relative to cohorts from Lake Woodruff, and to patterns established by both populations in 2001. The unpredicted drop may be related to the drought conditions, which severely affected La ke Apopka’s water levels in summer 2002, particularly in the shallow periphery wher e mosquitofish are found. Low water levels, especially in combination with warm temper atures and the eutrophic conditions of Lake Apopka are likely to be associated with reduced dissolved oxygen concentrations (unfortunately, daily data on oxygen concen trations are not available for the study period). In carp, chronic hypox ia has been shown to reduce serum concentrations of testosterone and estradiol, impede gonadal development, and reduce spawning success, sperm motility, and fertilization rate (Wu et al., 2003). Interestingly, the January peak in sp erm count observed among Woodruff males coincides with the time of declining winter sperm viability. This January peak may be adaptive in that it dilutes the impact of lo wer viability, allowing males to maximize their live sperm counts well into January or even February, when some females might be entering spring ovarian recrudescence (Chapter 2) If this is the case, then the lack of a January peak in sperm counts among males fr om Lake Apopka could be deleterious. Alternatively, the January st rategy among males from Lake Woodruff may be a plastic and possibly costly response that is cued to female reproductive activity. Females from Lake Woodruff, collected at the same time as males in our study, produced vitellogenic

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62 oocytes 6 to 7 weeks later in the sprin g, compared to females from Lake Apopka. Therefore the third peak in sperm count observed among Woodruff males may be a response to delayed recruitment among female cohorts. Koya and Iwase (2004) observed similar synchrony between the sexes. In addition to these social factors, the hi gher concentrations of estrogenic or antiandrogenic contaminants in Lake Apopka (G uillette et al., 1999) could explain the reduced sperm quality observed among Apopka mosquitofish in some months. This hypothesis is supported by the fact that elevated concentrations of p,p’-DDE (antiandrogenic) and toxaphene (estrogenic) ha ve been measured in the body tissues of mosquitofish collected from Lake Apopka (US Fish and Wildlife Service, unpubl. data). Estrogenic and antiandrogenic molecules, including 4-tert-pentylphenol, 4-tertoctylphenol, nonylphenol, p,p’-DDE, bisphenol A, and estradiol have been shown to cause reduction of primordial germ cell num bers or spermatogenic cysts, progressive disappearance of spermatozoa and spermatoge nic cysts, degeneration of sperm cells, or inhibition of spermatogenesis in male carp, gup pies, and platyfish (Gimeno et al., 1998a, b; Kinnberg et al., 2000; Kinnberg and Toft, 2003). Additionally, vinclozolin, a fungicide with anti-androgenic activity has been shown to reduce testis cord number, increase germ cell apoptosis, and reduce sperm motility among rats exposed during embryonic development (Uzumcu et al., 2004). One additional hypothesis is that mosquitofish are more sensitive to endocrine disruption in some months, particularly if the amplitude of endogenous signals is already low. For example, alligators exhibit seasonal variation in testicular response to gonadotropin (Edwards et al., 2004), suggestin g that dosage of components in a signaling

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63 pathway is important and seasonally variab le. Presence of endocrine disruptors may attenuate the efficacy of a signaling system, and this may be especially disruptive at either the end or beginning of the breeding season, when signal concentrations may not be optimized. This hypothesis could explain th e lack of a third January peak in sperm count per spermatozeugma and the early drop in winter sperm viability observed among fish from Lake Apopka. Conclusions Overall, we observed tempor al variation in both sperm counts per spermatozeugma and sperm viability. Mosquitofish from La ke Apopka exhibited reduced total sperm counts, live sperm counts, and sperm viability at several points du ring the 15 months of collections. Taken together with previous studies from our laboratory, in which we reported reduced total sperm counts per mg testis among mosquitofish captured from Lake Apopka and among reference mosquitofish exposed to water from Lake Apopka for 1 month (Toft et al., 2003; Toft and Guillette, 2005), we conclude that chemical components in Lake Apopka are a likely, but not sole, cause of reduced sperm quality among resident mosquitofish. The underlying causes of reduced sperm quality could include increased apoptosis or degeneration of germ cells and sperm cells, reduced mitosis of germ cells leading to germ cell renewal (estradiol mediated), and reduced efficacy of signaling pathways associated with spermatogenesis, particularly during periods of transition between br eeding and testicular quiescence.

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Figure 3-1. Testic ular histology of Gambusia holbrooki, showing breeding (main picture) and quiescent (inset, lower left ) states. SC = spermatocytes, SG = spermatogonia, ST = spermatids, SZ = spermatozoa, SZM = spermatozeugmata. Inset on lower right shows Sertoli cells (small arrows) surrounding spermatozeugmata and lining e fferent ducts. Sertoli cells enclose all spermatogenic cysts, but are most visible around spermatozoa because they hypertrophy immediately before releas ing spermatozeugma into an efferent duct. Large arrow indicates the line along which the testes fused during development.

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65

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66 Figure 3-2. Sperm methods for Gambusia holbrooki. Spermatozeugmata (szm) are stripped from an anaesthetized male mosquitofish by gently pressing down on the abdomen anterior to the gonopodi um and sweeping caudally, using the smooth, rounded end of large forceps with the two tips taped together. A) The fish is held in place by a second pair of forceps. B) Hundreds to thousands (number is highly variable) of szm are retrieved from a single fish. C) Szm vary in shape and size. D) Each sz m contains a few thousand spermatozoa, arranged with heads toward the outside of the szm, and tails in the center. E) Once szm are removed from the male, they disperse in a few minutes from the gelatinous matrix that holds them together. F) Gambusia sperm exhibit an elongated head and single flagellum. A E C B F D

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67 Two views of total cell data (scatter and topographic). Population of sperm cells is gated by oval Two views of gated sperm cells (scatter and topographic). Dead cells (stained with propidium iodide (PI)) Live cells (stained with SYBR green) Non-sperm particles Nile red bead count, gated as “M1” Two views of total cell data (scatter and topographic). Population of sperm cells is gated by oval Two views of gated sperm cells (scatter and topographic). Dead cells (stained with propidium iodide (PI)) Live cells (stained with SYBR green) Non-sperm particles Nile red bead count, gated as “M1” Figure 3-3. Gambusia sperm counts flow cytometry printout. The top panel shows two views of the total sample, gated with in the oval. The middle panel shows two views of three partic le populations (dead sperm cells, live sperm cells, and non-sperm particles) that are distinguished by their respective wavelength of florescence. The last panel indicates the gated bead count, used to calibrate the volume of sample taken up by the flow cytometer.

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68 10 15 20 25 30 35 40M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateWater Temperature (C ) 10:00 10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00Daylength (hours:mins) Apopka Woodruff Daylength Figure 3-4. Water temperature and daylength data for the collection period. Daylength data source: Astronomical Applicat ions Dept., U. S. Naval Observatory, Washington, D.C. 20392-5420. This gr aph duplicates Figure 2-2, but is presented again here because it is highly relevant to the interpretation of data in this chapter.

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69 30 40 50 60 70 80 90 100M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMean % Live Sperm Apopka Woodruff * * Figure 3-5. Mean percent live sperm (1 SE ) observed among adult male Gambusia holbrooki collected from two lakes in central Florida. Collections were made between May 2001 and September 2002. Significant variation over time within a lake is indicated by bolded trend lines (ANOVA, p 0.05). *Indicates a significant lake effect within a given month (ANOVA, p 0.05).

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70 1000 2000 3000 4000 5000 6000 7000 8000M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMean Sperm Count per Szm Apopka Woodruff * Figure 3-6. Mean sperm count per spermatozeugma (szm) (1 SE) observed among adult male Gambusia holbrooki collected from two lakes in central Florida. Collections were made between May 2001 and September 2002. Significant variation over time within a lake is indicated by bolded trend lines (ANOVA, p 0.05). *Indicates a significant la ke effect within a given month (ANOVA, p 0.05). Note that no collections were made between June and September 2002, which may explain the lack of periodicity otherwise predicted by data from 2001.

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71 1000 2000 3000 4000 5000 6000 7000 8000M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMean Live Sperm Count per Szm Apopka Woodruff * * Figure 3-7. Mean live sperm count per spermatozeugmatum (szm) (1 SE ) observed among adult male Gambusia holbrooki collected from two lakes in central Florida. Collections were made be tween May 2001 and September 2002. Significant variation over time within a lake is indicated by bolded trend lines (ANOVA, p 0.05). *Indicates a significant lake effect within a given month (ANOVA, p 0.05). Note that no collections were made between June and September 2002, which may explain the lack of periodicity otherwise predicted by data from 2001.

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72 CHAPTER 4 SEASONAL VARIATION IN BODY SIZE, MUSCLE ANDROGEN CONCENTRATIONS, AND TESTICULAR AND HEPATIC WEIGHTS AMONG MALE MOSQUITOFISH FROM TWO LAKES IN CENTRAL FLORIDA Introduction Fish reproduction is regulated by a wide va riety of abiotic and biotic environmental factors. These include temperature and phot operiod (Fraile et al., 1994), nutrition (Cech et al., 1992), and behavioral interactions between the sexe s and among individuals of the same sex (Bisazza et al., 2001; McPeek, 1992). In addition to these natural factors, most aquatic systems are now impacted by anthropog enic contaminants, which also have the potential to disrupt reproductive function of fishes (Baatrup and Junge, 2001; Jobling et al., 2002a). Many of the chemicals that now pollute aq uatic systems have been shown to affect male fish reproduction by changing steroid hormone action or metabolism. For example, estradiol and estrogenic chem icals like nonylphenol, octylphenol, pentylphenol, and the chemical mixture found in treated sewage effl uent, are associated with delayed puberty, persistently immature testes, or ovotestes, a condition in which males develop ovarian tissue within their testes (Barnhoorn et al., 20 04; Dreze et al., 2000; Gi meno et al., 1998a; Toft and Baatrup, 2001). Ovotestes are also associated with abnormal presence of an oviduct and reduced semen quality in te rms of volume, morphology, and fertilization success (Jobling et al., 2002b). In a previously published study, conducte d in conjunction with the first three months of this project, we reported redu ced sperm counts, slightly shorter gonopodia,

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73 higher whole-body testosterone concentratio ns, and increased test icular and hepatic weights among adult male mosquitofish from Lake Apopka in central Florida (Toft et al., 2003). The Lake Apopka population was compared to those from a nearby reference lake, Lake Woodruff. Unlike Lake Woodruff Lake Apopka has a history of chemical and nutrient contamination from agricultural, industrial, and municipa l sources (Guillette et al., 2000). Previously, the contaminati on in Lake Apopka has been characterized as estrogenic and anti-androgenic, based on the chemicals measured in the serum and eggs of resident alligators and body tissues of resident mosquitofish (Guillette et al., 1999; Guillette et al., 2000; Heinz et al., 1991; U.S. Fish and Wildlife Service, unpubl. data). Measured contaminants include several pesticides, like dieldrin, endrin, mirex, oxychlordane, trans-nonachlor, DDT, p,p’-DDE and DDD (DDT metabolites), and toxaphene (Guillette et al., 1999; Heinz et al., 1991; U.S. Fish and Wildlife Service, unpubl. data). These chemicals have been experimentally shown to alter steroid and thyroid hormone synthesis and degradation, cause male to female sex reversal of alligators and red-eared slider turtles, and agonize or antagonize steroid receptor binding and activity (for reviews, see Akingbemi and Hardy, 2001; Guillette et al., 2000; Guillette and Gunderson, 2001; Willingham and Crews, 2000). In a follow-up to the mosquitofish study cited above, Toft and Guillette (2005) observed reduced sperm counts and altered se xual behavior among mosquitofish after a one-month exposure to water from Lake Apopka versus water from two reference sites. In that study, fish originated from one of the reference sites. In another paper, Gallagher et al. (2001) reported elevated levels of plasma estrogens among male bullhead catfish from Lake Apopka as compared with male bullheads from Lake Woodruff. These studies

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74 expanded upon the more established Apopka-Woodruff alligator literature. That is, relative to alligators from Lake Woodruff, alligators from Lake Apopka exhibit poor hatching success (Woodward et al., 1993), changes in gonadal morphology (Guillette et al., 1994), altered hepatic enzyme expressi on and activity (Gunderson et al., 2001), and reduced plasma testosterone and phallus size in males (Guillette et al., 1999). The gonadal and steroid abnormalities are observa ble at hatching suggesting that changes occur during development (Guillette et al., 1995). Given the observed relationship between environmental contaminants and altered reproductive variables among fishes and alligators, we designed our study to further examine possible impacts of contaminants on mosquitofish reproduction. Here, we focus on male mosquitofish (Gambusia holbrooki), captured monthly for 17 mont hs from the Apopka-Woodruff model system. The seasonal component of ou r study is intended to place any observed, lake-associated alterations in reproduction in context of the seasonal cycle and “normal” environmental variation like temperature or photoperiod. Methods Field Collections Between March 2001 and September 2002, 17 monthly collections of adult male Gambusia holbrooki were made from Lake Apopka (nor th shore, near Beauclair Canal) and Lake Woodruff Wildlife Refuge (northwest shore, Spring Garden Lake) in central Florida, USA. Fish were captured using a 3-mm mesh dip net and those with a welldeveloped and hooked gonopodium were considered mature (Angus et al., 2001). Each lake collection took 1 day, plus additional days to process fish. Thus monthly collections from the two lakes were made on different days (average of 4 days apart). For 12 of the 17 months, Lake Woodruff was sampled first.

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75 Water temperature, pH, and conductivity data were obtained at the time and location where fish were sampled, using a handheld Ultrameter (Model 6P, Myron L Company, Carlsbad, CA). Each monthly sample, ranging from 37 to 67 fish per lake, was divided into three subsets. The first subset was used to obtain testicular and hepatic weight. These fish (n = 6 to 21 per month, pe r lake; mean = 18) were held live in aerated coolers filled with lake water, fed flake fish food ad libitum, and processed within 1 to 2 days of capture. Before necropsy, fish were over-anesthetized in r oom-temperature 0.1% MS222 (3-aminobenzoic acid ethyl ester, me thanesulfonate salt, Sigma #A5040). The testis (0.4 to 9.9 mg) and liver (0.1 to 8.0 mg) were removed and weighed to the nearest 0.1 mg. The second subset was used for the meas urement of androgen (testosterone and 11ketotestosterone) concentratio ns in the caudal peduncle ti ssues. Since the peduncle is primarily muscle, we will refer to it as muscle for the remainder of the chapter. Androgen concentrations were measured in muscle rather than plasma because Gambusia are too small to yield an ade quate amount of plasma. These fish (n = 6 to 12 per month, per lake; mean = 10) were frozen immediatel y at capture and held on ice while in the field. Upon return to the laboratory, the ca udal fin was removed, and all peduncle tissue posterior to the gonad was cut from the fish, weighed (average = 60 mg), and frozen at 80C. Androgens were measured on lipid extr acts of these peduncle tissues (referred to as “muscle androgens”), as described below. The third subset was used for sperm analysis as reported in Chapter 3. For all fish, standard length (SL) was measured to the ne arest 0.01 cm from the snout tip to the caudal peduncle using calipers. Fish were blotted dry and weighed with an electronic balance to

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76 the nearest milligram. Gonopodium length wa s measured to the nearest 0.05 mm, using an ocular micrometer mounted on a disse cting microscope. In mosquitofish, the gonopodium is a grooved structure, formed by the fusion of several anal fin rays, and used to transfer sperm to the female, as ferti lization is internal in this viviparous species (Batty and Lim, 1999). We have included measurement of the gonopodium because fin ray fusion and elongation are androgen dependen t, and the development of this secondary sex character can be perturbed by exposure to antiandrogens such as p,p’-DDE (present in Lake Apopka) and flutamide (Bayle y et al., 2002; Ogino et al., 2004). Muscle Androgen Measurements For measurement of muscle concentrations of testosterone and 11-ketotestosterone, frozen muscle (peduncle) tissues were thawed in glass tubes, on i ce, and homogenized in 750 l 65 mM borate buffer (pH 8.0). Homogenate was extracted twice with 5 ml diethyl ether. For each extraction, ether and homogenate were mixed together for two minutes using a multi-tube vortex mixer. For the first extraction, tubes were allowed to settle for three minutes to separate phases. For the second extraction, phases were separated by centrifugation for two minutes. The aqueous phase was frozen in a methanol bath chilled to -25C with dry ice. The ether from bot h extractions was combined in a second glass tube and evaporated under dry forced air. Hormone concentrations were determined using validated enzyme immunoassay (EIA) kits (Cat No. 582701 (T); 582751 (1 1-KT) purchased from Cayman Chemical Company (Ann Arbor, Michigan). Dry extract was reconstituted in 600-800 L EIA buffer so that samples would fall within the ra nge of the standard curve. EIAs were run as recommended by Cayman with an 18 h refr igerated incubation to increase sensitivity.

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77 Data were quantified against a standard curv e linearized using a l ogit transformation of B/Bo (bound sample/maximum bound). Duplic ate interassay variance (IAV) samples at two dilutions were included with each plat e. The coefficient of variance among all plates, averaged for the two dilutions was 18.5% for T, and 11.7% for 11-KT. To normalize sample androgen concentrations across assays, we multiplied by a correction factor derived from the relationship between individual plate IAV values and the mean IAV values for all plates. Statistics Androgen data points lying more than three standard deviations from the mean (n = 3 for T assays (1%); 7 for 11-KT assays (2%)) were excluded from the data set. Data were screened for correlations between st andard length (SL) or body weight, and gonopodium length, testicular and hepatic wei ght, and androgen concentrations using a correlation matrix. Apparent correlations were visualized using linear regression. For variables that scaled signif icantly with body size, body size was entered as a covariate when ANCOVAs were performed. Monthly means for each lake were co mpared using ANOVA or ANCOVA. To improve homogeneity of variance, all vari ables (with the exception of SL and body weight, when analyzed alone) were log10 transformed. Androgen concentrations were log10 (y+1) transformed to avoid nega tive log values (Zar, 1999). A priori pairwise LSD comparisons were made between lakes for each month and between consecutive months within each lake. This latter analysis was used to describe seasonal variation within a lake. Correlation, regression, and ANOVA an alyses were completed using Statview 5.0. ANCOVA analyses and calculati ons of adjusted means were completed using SPSS 13.0.

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78 Results Abiotic Factors Seasonal variability in water temperature (12C in January to about 35C during May to August) was similar between the two la kes, with the exception that Lake Apopka exhibited an early warming period ( about 7C warmer) between February and March of both years (Fig. 4-1). Photoperiod varied seas onally from 10.5 to 14 h (Fig. 4-1). Water pH varied from 6.5 to 8.5, with a mean of 7.2 in Lake Woodruff, and from 6.8 to 8.7, with a mean of 7.5 in Lake Apopka. Conductivity was generally higher in Lake Woodruff, ranging from 700 to 1807 S/cm, with a mean of 1196 S/cm (peaks occurred in March/April both years). Conductivity in Lake Apopka ranged from 433 to 765 S/cm, with a mean of 583 S/cm. Body Size Body weight and standard length (S L) were significantly related (r2 = 0.74; p < 0.0001). In 2001, males from Lake Apopka were significantly larger in six out of nine months, in terms of SL (Fig. 4-2) and body weight (Fig. 4-3), than males from Lake Woodruff. In the first half of 2002, fish fr om both lakes were similar in SL and weight (Figs. 4-2 and 4-3), but in late summer, fish from Lake Woodruff were longer and heavier (Figs. 4-2 and 4-3). As a general pattern average male body size appears to decline during the breeding season, beginning some time in March to May, and reaching a seasonal low in September to October, quickly rising again by October or November. In 2001, the spring decline among males from Lake Apopka was delayed by 2 months compared to males from Lake Woodruff (Fig s. 4-2 and 4-3). However, in 2002, mean body size of males from both lakes bega n to decline at about the same time.

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79 Gonopodium Length Gonopodium length was positively related to SL (r2 = 0.67, p < 0.0001). In some months, in both 2001 and 2002, we observed a lake-associated difference in adjusted gonopodium length (Fig. 4-4). However, there was no pattern with regards to which lake had fish with longer or shorter gonopodia. In addition, the maximum lake-associated difference in adjusted gonopodium length wa s about 0.25 mm (Fig. 4-4). Seasonally, adjusted gonopodium length appeared to fluctu ate greatly, particularly during the winter months (Fig. 4-4). This is likely due to variation in the body size of sampled males. Androgens Testosterone concentrations were negatively correlated with SL (r2 = 0.12; p < 0.0001). Seasonally, there was substant ial lake-associated variation in androgen concentrations (Fig. 4-5). For 11-KT, peak s easonal concentrations represent an increase of 2 to10 fold (Woodruff), or 2 to 5 fold (A popka) relative to seasonal lows (Fig. 4-5A). For adjusted testosterone, peak seasonal con centrations represent an increase of 2 to 5 fold (Woodruff), or 4 to 6 fold (Apopka) rela tive to seasonal lows (Fig. 4-5B). Males from Lake Woodruff exhibited two annual p eaks of 11-KT, one in March-April-May and one in September-October (Fig. 4-5A). This second peak occurred in 2001, but was not apparent by September 2002 when the last samples were collected. The annual low in 11-KT concentrations occurred in January among fish from Lake Woodruff. Like males from Lake Woodruff, a spring peak in 11 -KT among males from Lake Apopka occurred between March and May (Fig. 4-5A). Howeve r, males from Lake Apopka did not exhibit a significant rise in 11-KT in September, al though they did exhibit a small peak in June 2002, a peak that was not observed in 2001 (Fig. 4-5A). Finally, males from Lake

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80 Apopka exhibited an additional peak in 11-KT in January, the time that coincided with the lowest annual 11-KT concentrations am ong males from Lake Woodruff (Fig. 4-5A). Like muscle 11-KT concentrations, muscle testosterone (T) concentrations varied both seasonally and between lakes. In 2002, the March-April peak in T coincided with the spring peak in 11-KT among fish from both lakes (Fig. 4-5B). However, this pattern was not observed in 2001. Likewise, fish from both lakes exhibited high T concentrations in September-O ctober 2001 (Fig. 4-5B). As with 11-KT concentrations, this pattern had not yet developed by Se ptember 2002. Fish from Lake Woodruff exhibited a third peak in T in December just before the annual low in January-February (Fig. 4-5B). This peak was not observed am ong fish from Lake Apopka. However, fish from Lake Apopka exhibited a third rise in muscle T concentrations in June (2002 only), which coincided with the June 2002 rise in muscle 11-KT concentrations (Fig. 4-5B). This June 2002 peak in T was not observed among fish from Lake Woodruff. Testicular Weight Testicular and hepatic weights were positively related to body weight (r2 = 0.35, p < 0.0001; r2 = 0.46, p < 0.0001, respectively). Except during November 2001 and March 2002, adjusted testicular weights were si gnificantly higher among males from Lake Apopka compared to males from Lake Woodr uff (Fig. 4-6). The difference in actual adjusted testicular weight in most months is substantial. For example, the greatest lakeassociated difference was observed at the e nd of April 2001, when adjusted testicular weight among fish from Lake Apopka was 145 % greater than that for fish from Lake Woodruff (Fig. 4-6). Among fish from Lake W oodruff, testicular weight peaked in late March, early August (2001), and late Octobe r (2001) (Fig. 4-6). The March peak was consistent between 2001 and 2002 and coinci ded with the beginning of the spring period

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81 of elevated androgen (both T and 11-KT) con centrations (Figs. 4-5 and 4-6). Likewise, the early August peak precedes the elevated androgen concentrations observed in late August-September, and the late October peak in testicular size precedes elevated testosterone concentrations observed in late November. No testicular measurements were made in August or October 2002, so a year-toyear comparison of this variable is not available. As observed among fish from Lake Wood ruff, males from Lake Apopka exhibited similar seasonality in testicular size; w ith increased size often preceding observed elevations in androgen concen trations (Figs. 4-5 and 4-6) In 2001 and 2002, testicular size peaked in April, coinciding with elevat ed 11-KT concentrations during both years. Similarly, testicular weights were increased in early August, preceding high testosterone concentrations in late Septembe r. It should be noted that testicular size data are missing for fish from Lake Apopka in late August. The testosterone data s uggest that testicular weight remained high throughout August and early September among these fish. Finally, testicular size peaked in late January, po ssibly explaining the high 11-KT concentrations also observed at that time. This late Janu ary peak in testicular weight also precedes elevated concentrations of both 11-KT and T in March. Hepatic Weight The most notable lake associat ed differences in adjusted hepatic weight occurred in May – June 2001 and 2002, when mean hepati c weight was significantly greater among fish from Lake Apopka (Fig. 4-7). Tempor al fluctuation occurred throughout the study period among fish from both lakes, although the patterns on each la ke are dissimilar and frequently oppose each other (Fig. 4-7). Fi sh from Lake Woodruff exhibited temporal peaks in early August 2001, November 2001, an d March/late April 2002. Each peak in

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82 hepatic weight was followed by a seasonal low, suggesting a pulsatile pattern. Fish from Lake Apopka exhibited seasonal peaks in June 2001 and 2002, and in late January 2002. Except in March 2002, hepatic weights among fish from Lake Apopka remain elevated year-round with respect to seasonal lows obse rved among fish from Lake Woodruff (Fig. 4-7). Discussion Body Size Size at maturity among male mosquitofish is highly variable, being affected by photoperiod, mating strategy, female preferen ce, and sex ratio (Bisazza et al., 1996; Zulian et al., 1993). Although females app ear to prefer larger males, small males generally have a mating advantage, possibly because they can approach females without detection (Bisazza et al., 2001). Small size reduces mating success only when the sex ratio is male-biased, as is often the case at the end of the re productive season (Bisazza and Marin, 1995; Zulian et al., 1995). Male body size is not a strongl y heritable trait. Zulian et al. (1993) report that, regardless of paternal size, leng th at maturity is greater for males raised in groups, and this larger body size sometimes occurs in conjunction with delayed maturation. In the same study, they report that mosquitofish reared under short photoperiods (9 h of light) matured earlier an d at a smaller size. Taken together, these studies predict that male body size should be small at the beginning of the breeding season, and larger at the end. Our data did not follow this pattern. Instead, we observed a general decline in body size among samp led fish during the breeding season, until October, when females become reproductively quiescent (Chapter 2). Although we did not collect sex ratio data, our body size data suggest that, in Lake Apopka and Lake Woodruff, the sex ratio does not become male biased as the breeding season progresses.

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83 Declining body size during the breeding season could reflect increasing recruitment of small males that were born in the same year that they were sampled. The increase in body size during reproductive quiescence proba bly reflects continued winter growth by the established male population. Although the sampled males from both la kes exhibited declining body size during the breeding season in 2001, we noted that the spring decline among males from Lake Apopka was delayed by 2 months compar ed to males from Lake Woodruff. Interestingly, females from Lake Apopka become reproductively active in late January, about 1.5 months earlier than those from La ke Woodruff (Chapter 2). If the Apopka breeding season begins early, and males mature rapidly at a small size because photoperiod is short and the sex ratio is female biased (as suggested by the studies described above), then we would expect mean male body size to d ecline earlier in the breeding season rather than later. The obser ved delay in body size decline suggests that maturation was delayed among males from Lake Apopka, relative to those from Lake Woodruff, at least in 2001. Possible causes for delayed maturation include an overall higher density of males year-round (Zulian et al., 1993) or exposure to an antiandrogen such as p,p’-DDE (Bayley et al., 2002). DDE makes up most of the contaminant load measured in the serum of alligators from Lake Apopka (Guillette et al., 1999). Furthermore, elevated concentrations of p,p ’-DDE have been measur ed in mosquitofish from Lake Apopka (5300 mg/kg) (US Fish an d Wildlife Service, unpubl. data). Although delayed maturation among males from La ke Apopka is a likely explanation for differences in body size between the two lake populations in 2001, the effect did not persist in 2002.

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84 Gonopodium Length Lake associated differences in adjusted gonopodial length were maximized at about 0.25 mm or 4%. However, fish with shor ter (or longer) gonopodia did not always come from the same lake. We reported simila rly low variation in gonopodial length in a previous paper (Toft et al., 2003). To our kn owledge, no data are available to indicate if this degree of variation is large enough to confer a ny functional changes. Androgens Both 11-ketotestosterone (11-KT) and testosterone (T) exhibited temporal variation. We previously reported wholebody testosterone con centrations of 1000 – 1600 pg/g for male mosquitofish capture d from Lake Apopka and Lake Woodruff in March – May 2001 (Toft et al., 2003). Muscle testosterone concentrations reported here for the same period are about 150 pg/g for fish from both lakes. These data suggest that testosterone concentrations in the testis and possibly the brain, which are included in the whole-body measurement, are higher than ci rculating testosterone concentrations, as measured in the muscle. Our measured concen trations of muscle 11-KT were similar to muscle 11-KT concentrations measured in male gag grouper (Heppell and Sullivan, 2000). In African catfish and Japane se eels, 11-KT, rather than T, is implicated in the stimulation of spermatogenesis (Cavaco et al ., 1998; Miura and Miura, 2001). Based on sperm count and viability data (Chapter 3), male mosquitofish exhibit active spermatogenesis by May, after a period of wi nter quiescence. Our data suggest that 11KT, which rises in March-April, is associat ed with spermatogenic activation among fish from both lakes. A small peak in 11-KT in September among fish from Lake Woodruff, but not Lake Apopka may explain why male s from Lake Woodruff have more viable

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85 sperm later in the year (Chapter 3). Intere stingly, the January peak in 11-KT among fish from Lake Apopka was not associated with any observable effects on spermatogenesis, even up to three months later (Chapter 3). It is likely that during winter quiescence, testes are not responsiv e to androgen stimulation in terms of spermatogenesis, thus the reason for the January 11-KT peak among fish from Lake Apopka is not clear. In 2002, males from Lake Apopka exhibi ted a large rise in muscle 11-KT concentrations in April, preceding an expected rise in sperm viability in May (Chapter 3). However, viability dropped substantially and unexpectedly in June, in association with a large rise in muscle T concentrations. In Af rican catfish, the stimulatory effect of 11-KT on spermatogenesis can be blocked by co -treatment with T (Cavaco et al., 2001). Although we cannot explain the June rise in muscle T concentrations, which did not occur in 2001, nor among males from Lake Woodruff, the June rise in muscle T concentrations may explain the concomitant decrease in sperm viability observed among fish from Lake Apopka in 2002 (Chapter 3). Testicular Weight Among males from both lakes, temporal variation in testicular size was often positively related to changes in muscle androgen concentrations. The period of increased testicular size, implying increased testicul ar activity, ranged from March to November, followed by a period of winter quiescence. This observation is supported by our data on sperm quantity and viability (Chapter 3). It is also similar to previously published observations examining Gambusia reproduction in Japan, although the reproductive season for the Japanese populations was sh orter (Koya and Iwase, 2004). Although the pattern among fish from the two lakes was sim ilar, testicular weight s were significantly higher among males from Lake Apopka compar ed to males from Lake Woodruff. On the

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86 other hand, this did not translate into subs tantially higher androgen concentrations or sperm counts (Chapter 3) among Apopka fish. This observation suggests that testes of males from Lake Apopka are hypertrophied, a pat hological state that may be necessary to maintain androgen homeostasis. That is, pe r unit mass, androgen synthesis by testicular tissues is reduced among males from Lake Apopka, but androgen concentrations are maintained at normal levels by the compensa tory growth of Leydig cells. Histology data associated with this project will be evaluated in a future study. Leydig cell hyperplasia a nd hypertrophy can be induced by exposure to both androgen receptor antagonists (i.e., flutamide, p,p’-DDE, or the fungicide Vinclozolin) and estrogen agonists (diethylstilbestrol (DES ) or 17-estradiol) (Du Mond et al., 2001; Mylchreest et al., 2002; O’Connor et al ., 2002; Rai and Haider, 1991). Both DDE (antiandrogenic) and toxaphene (estrogenic) oc cur in elevated concentrations in tissue samples from mosquitofish collected from La ke Apopka (US Fish and Wildlife Service, unpubl. data). In our study, the estrogenic nature of Apopka cont aminants may be more relevant in terms of testicular enlargement si nce a study of the eff ects of antiandrogens on the gonadosomatic index of guppies yielded no effect (Bayley et al., 2002). Some risk assessment studies suggest that hormonally induced Leydig cell proliferation is related to development of Leydig cell adenoma and po ssibly carcinoma (Clegg et al., 1997). Hepatic Weight As with testicular weight, adjusted hepatic weight was generally higher among mosquitofish from Lake Apopka compared to Lake Woodruff, particularly between May and September. Increased hepatic weight ca n be associated with long or short-term exposure to toxicants, as is the case for ma le rodents exposed to di-n-butylphthalate (Wine et al., 1997), p,p’-DDE (Kang et al., 2004), or toxaphene (Hedli et al., 1998); or

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87 fed contaminated salmon from the Great La kes (Arnold et al., 1998). Increased hepatic weight can also be due to induction of vitell ogenesis, which can occur in juvenile or male fishes, including mosquitofish, exposed to estrogenic substances (Tolar et al., 2001; Verslycke et al., 2002). Summary The study presented here illustrates tem poral variation in body size, androgen concentrations, and testicular and hepatic weights in two populations of male mosquitofish inhabiting subtropical lakes in central Florida, USA. Males are reproductively active from March (temperature 20C; daylength 11.5 h) through November (temperature = 20-25C; daylength 11 h). Induction of reproductive activity appears to be controlled by a combination of daylength and temperature since, in central Japan (Koya and Iwase, 2004), male mos quitofish reproduction begins when the temperature is approximately 14C and daylength is 13 h. Our data suggest that 11-KT, rather than testosterone, is responsible for induction of spermatogenesis, as reported in other fish species (Cavaco et al., 1998; Miura and Miura, 2001). Given previous studies of alligators by our laboratory, we hypothesized that mosquitofish captured from Lake Apopka w ould exhibit altered reproductive characters in comparison to a reference population. We observed delayed maturation among males from Lake Apopka and enlarged testicular a nd hepatic size during most of the year. As discussed above, these altered characters could be indicative of exposure to antiandrogenic or estrogenic contaminants such as those found in Lake Apopka.

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88 10 15 20 25 30 35 40M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateWater Temperature (C ) 10:00 10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00Daylength (hours :mins) Apopka Woodruff Daylength Figure 4-1. Water temperature for Lake Apopka and Lake Woodruff, shown with ambient photoperiod for each collection date. This graph duplicates Figures 2-2 and 3-4, but is presented again here because it is highly relevant to the interpretation of data in this chapter.

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89 1.7 1.8 1.9 2 2.1 2.2 2.3M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateSL (cm) Apopka Woodruff * * ** * Figure 4-2. Mean standard length (SL) (1 SE) of adult male mosquitofish from Lake Apopka and Lake Woodruff. *Months in which the mean SL of fish from the two lakes are signifi cantly different (ANOVA, p 0.05). A heavier line between data points indicates significan t month-to-month variation within a lake (ANOVA, p 0.05). Slashed bars (//) indicate that time between consecutive collections exceeds 1 mont h and that the indicated trend is extrapolated.

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90 90 110 130 150 170 190M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateBody Weight (mg) Apopka Woodruff * * * *Figure 4-3. Body weight of adult male mosquitofish from Lake Apopka and Lake Woodruff. Graph shows means 1 SE. *Months in which the mean body weights of fish from the two lakes are significantly different (ANOVA, p 0.05). A heavier line between data po ints indicates significant month-tomonth variation within a lake (ANOVA, p 0.05). Slashed bars (//) indicate that time between consec utive collections exceed s 1 month and that the indicated trend is extrapolated.

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91 5.8 5.9 6 6.1 6.2 6.3 6.4M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Gonopodium Length (mm) Apopka Woodruff * * * 5.8 5.9 6 6.1 6.2 6.3 6.4M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Gonopodium Length (mm) Apopka Woodruff * * * Figure 4-4. Mean gonopodium length (1 SE), adjusted for standard length, of adult male mosquitofish from Lake Apopka and Lake Woodruff. *Months in which the mean adjusted gonopodium lengt hs of fish from the two lakes are significantly different (ANCOVA, p 0.05). A heavier line between data points indicates significant monthto-month variation within a lake (ANCOVA, p 0.05). Slashed bars (//) indicate that time between consecutive collections exceeds 1 mont h and that the indicated trend is extrapolated.

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92 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMuscle 11-KT (pg/mg) Apopka Woodruff * 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Muscle Testosterone (pg/mg) Apopka Woodruff * * A B 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMuscle 11-KT (pg/mg) Apopka Woodruff * 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Muscle Testosterone (pg/mg) Apopka Woodruff * * 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMuscle 11-KT (pg/mg) Apopka Woodruff * 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateMuscle 11-KT (pg/mg) Apopka Woodruff * 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Muscle Testosterone (pg/mg) Apopka Woodruff * * 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Muscle Testosterone (pg/mg) Apopka Woodruff * * A B Figure 4-5. Mean muscle androgen concentrations (1 SE) for adult male mosquitofish from Lake Apopka and Lake Woodruff. A) 11-Ketotestosterone (11-KT). B) Testosterone (T). T concentrations are adjusted for male standard length. *Months in which the mean androgen c oncentrations of fish from the two lakes are significantl y different (AN(C)OVA, p 0.05). A heavier line between data points indicates significan t month-to-month variation within a lake (AN(C)OVA, p 0.05). Slashed bars (//) show that time between collections exceeded 1 month and that the indicated trend is extrapolated.

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93 1 2 3 4 5M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Testicular Weight (mg) Apopka Woodruff * * * * * * 1 2 3 4 5M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Testicular Weight (mg) Apopka Woodruff * * * * * * Figure 4-6. Testicular weight, adjusted for body weight, of adult male mosquitofish from Lake Apopka and Lake Woodruff. Graph shows means 1 SE. *Months in which the mean adjusted testicular weights of fish from the two lakes are significantly different (ANCOVA, p 0.05). A heavier line between data points indicates significant monthto-month variation within a lake (ANCOVA, p 0.05). Slashed bars (//) indicate that time between consecutive collections exceeds 1 mont h and that the indicated trend is extrapolated.

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94 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Hepatic Weight (mg) Apopka Woodruff * * * * 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4M-01 A-01 M-01 J-01 J-01 A-01 S-01 O-01 N-01 D-01 J-02 F-02 M-02 A-02 M-02 J-02 J-02 A-02 S-02Collection DateAdj. Hepatic Weight (mg) Apopka Woodruff * * * * Figure 4-7. Hepatic weights, adjusted for bod y weight, of adult male mosquitofish from Lake Apopka and Lake Woodruff. Graph shows means 1 SE. *Months in which the mean adjusted hepatic weights of fish from the two lakes are significantly different (ANCOVA, p 0.05). A heavier line between data points indicates significant monthto-month variation within a lake (ANCOVA, p 0.05). Slashed bars (//) indicate that time between consecutive collections exceeds 1 mont h and that the indicated trend is extrapolated.

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95 CHAPTER 5 WATER QUALITY INFLUENCES REPRODU CTION IN FEMALE MOSQUITOFISH (Gambusia holbrooki) FROM EIGHT FLORIDA SPRINGS1 Introduction Freshwater nitrate contamination is a gr owing international concern. While the drinking water standard is 10 mg/L NO3-N in the United States and 11.3 mg/L NO3-N in Europe (European Council 1998; US EPA 1996 ), natural water bodies can exceed 100 mg/L nitrate (reviewed by Rouse et al. 1999) In Iowa, a statewide well-water survey reported that 18% of rural drinking water wells were contaminated with nitrate concentrations that exceeded 10 mg/L NO3-N (Kross et al. 1993). Nitrate usually enters surface and ground water in runoff from point and non-point sources, including fields, golf courses, private gardens, livestock feedlots, and sewage treatment facilities (Berndt et al. 1998; Katz et al. 1999). Under norma l circumstances, nitrogen is naturally cycled by bacterial and plant communities in aquatic ecosystems. However, if these organisms are limited (e.g., low light, low phosphorus ) and unable to remediate excess nitrate concentrations, nitrate can accumulate. El evated aquatic nitrat e potentially affects reproduction and survival of exposed animal s by directly influencing their physiology (Guillette and Edwards, 2005). Aquatic animals are exposed to nitrate primarily through ingestion or epithelial absorption across gills or skin (Onken et al. 2003 ). In crabs, nitrate can cross the gills, 1Disclaimer : The views expressed herein are those of the au thor and do not necessarily reflect the views of the Florida Department of Environmental Protection, which provided funding for this project.

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96 sometimes against a concentration gradient, by substituting for chloride in the chloridebicarbonate exchange mechanism that norma lly regulates the osmo tic and respiratory functions of the gill (Lee and Pritchard 1985; Onken et al. 2003). The ability of the gill epithelium of freshwater fishes to accumulate Clsuggests that nitrate can also accumulate, as shown in shrimp (Cheng an d Chen 2002). Thus, like chloride, the circulating nitrate concentration could ex ceed that of the surrounding water. Evidence suggests that sensitivity to nitrat e is species specific. Kincheloe et al. (1979) reported larval mortality of Chinook sa lmon, rainbow trout, and cutthroat trout at concentrations as low as 2.3 to 7.6 mg/L NO3-N. The 96 h median lethal concentration (LC50) for fathead minnow larvae is 1,341 mg/L NO3-N (Scott and Crunkilton 2000), and the lethal dose for adult and juvenile medaka is 100 mg/L NO3-N (Shimura et al. 2002). A range of sublethal effects of n itrate has also been reported. For example, Greenlee et al. (2004) observed increased ap optosis and reduced cell number in cultured pre-implantation mouse embryos exposed to 1 mg/L ammonium nitrate. In an accumulated nitrate test, in which nitrate built up over the course the experiment, Shimura et al. (2002) observed delayed hatc hing time and reduced fertilization and hatching rates of eggs produced by adult medaka exposed for two months to a maximum of 75 mg/L NO3-N. In that test, the offspring also exhibited reduced juvenile growth rates. At 50 mg/L NO3-N, Shimura et al. (2002) observed reduced spawning and fecundity (measured as egg number) among adult medaka exposed to nitrate as juveniles. Several authors have suggested that nitrat e can impact reproduction in vertebrates through effects on steroid hormone balan ce or impacts on nitric oxide regulation (DelPunta 1996; Panesar and Chan 2000; Vanvoorhis et al. 1994).

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97 Nitrate metabolism has not been studied in detail in fish. However, in mammals, nitrate can be converted in vivo by reversible reactions to nitrite and then nitric oxide (NO) by a number of suggested mechanisms (Kozlov et al. 1999; Lepore 2000; Panesar and Chan 2000; Samouilov et al. 1998; Weitz berg and Lundberg 1998). Nitric oxide is a gas that diffuses through tissues, playing diverse roles in vas odilation, cell-to-cell signaling, neurotransmission, and immun ity. The mammalian ovarian cycle and ovulation are regulated, in part, by inte ractions among gonadotropins, progesterone, estradiol, and NO (Al-Hijji et al. 2001; R upnow et al. 2001; Vanvoorhis et al. 1994; Yamagata et al. 2002). In a broad sense, NO appears to inhibit steroid hormone synthesis by inhibiting several steroidogenic enzymes or other major factors in this pathway. These include steroidogenic acute regulator y protein (StAR), and the enzymes P450sidechain cleavage (P450SCC), 3-hydroxysteroid dehydrogenase (3HSD), and aromatase (DelPunta et al. 1996; Panesar an d Chan 2000; Stocco and Guillette, unpubl. data; Vanvoorhis et al. 1994; Weitzberg and Lundberg 1998; Yamagata et al. 2002). Given the observed and hypothesized eff ects of nitrate on vertebrate reproduction and growth, we investigated the relationshi ps between nitrate and several reproductive variables in wild female mosquitofish captu red from eight Florida springs with varying concentrations of nitrate. We also consid ered the potential infl uence of four other environmental parameters, including temp erature, pH, conductivity, and dissolved oxygen. The Florida springs present an excellent sy stem for our study because they vary in nitrate concentration; yet appear similar in many other respects, such as relatively constant year-round temperature, pH, conductiv ity, and clarity. In addition, unlike most

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98 surface water sites, spring water arises from ground water sources. This suggests that water quality of spring water is more stable over time, compared to other surface waters. Specifically, water quality and chemistry of spring water primarily reflects the composition of the underground aquifer rock with which it comes in contact during its time underground (residence time) (Scott et al., 2004). Residence times range from several days to thousands of years, dependin g on the geology and flow rate of the spring (reviewed in Scott et al., 2004). Our study de pends on water data taken only at the time of our fish collections. Thus we cannot desc ribe temporal variati on in water quality. However, given the underground source of spring water, it is likely that our measured values are representative of spring conditio ns over the short term (weeks to months) preceding our study. This statement is suppor ted by other data we collected during 2003 (unpublished) and the emerging database on spring water quality initiated by Florida’s Suwannee River Water Management District (available online at http://www.srwmd.state.fl.us/water+data/su rfacewater+quality/sear ch+surfacewater+qual ity+data.asp?county_code=F001&Submit=GO ). Methods Field Collections and Water Quality Between May 18 and June 7, 2003, adult female Gambusia holbrooki (eastern mosquitofish) were collected using 3-mm mesh dip nets or seines from eight Florida springs with varying degrees of nitrate cont amination. The sampled springs are located along the Santa Fe and Suwannee Rivers in nort h-central Florida. Fish were selected if they were mature. This was judged by si ze in the field and confirmed during necropsy based on presence of differentiated follicles Mature fish from the sampled springs exhibited a standard length 2 cm.

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99 As fish were captured, they were randomly parsed into one of two groups. Fish placed in the group for estradiol analysis (n = 13 to 17 per spring) were immediately chilled on ice. Fish used for necropsy (n = 30 per spring) were taken live to the laboratory, using aerated coolers of water taken from the capture site. Fish in the necropsy group were dissected within 1 day of capture to examine ovarian and hepatic weight, embryo number, and em bryo dry and wet weight. Water quality data were obtained at the time and location where fish were captured. Water temperature, pH, and conductivity were measured using a handheld Ultrameter (Model 6P, Myron L Company, Carlsbad, CA). Dissolved oxygen was measured using an YSI oxygen probe (Model 550A). In addi tion, water samples were filtered through a 1-micron glass fiber filter (Millipore Cat. No. AP4004700), chilled on ice, and stored at 20C until they were analyzed for nitrate using an auto-analyzer (Bran+Luebbe Technicon II with colorimeter). This method uses a copper-cadmium column to reduce nitrate to nitrite, which then reacts to form a colored solution that can be assessed colorimetrically. Therefore, nitrate concen trations are reported as ppm (mg/L) nitrogen in the forms of nitrate and nitrite combined (NO3-N). Body Size and Dissections Adult standard length (SL) was measured from the snout tip to the caudal peduncle using calipers. Fish were blotted dry and weighed with an elect ronic balance to the nearest milligram. Ovaries and livers were removed and weighed to the nearest 0.1 mg. Ovarian wet weight ranged from 1.6 to 874.2 mg ovarian dry weight ranged from 0.3 to 200.3 mg, and hepatic weight ranged from 1.6 to 94.8 mg. Mature females were considered reproductive if their ovaries cont ained at least 1 vitellogenic (yellow rather than white) oocyte. To assess fecundity, we determined the developmental stage of the

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100 embryos (based on Haynes 1995), and counted embryos that were stage 3 or older. Counted embryos were dried in an oven for 24 h at 40 C. In Gambusia, embryos develop within the ovary in synchronized waves and account for most of the ovarian weight. Therefore, mean embryo weight, both wet and dry, for each female was calculated by dividing the total wet and dry weight of a brood by the embryo number (Meffe and Snelson, 1993). For stage 11 em bryos (just before bi rth), wet weights are slightly exaggerated by the presence of yo lked ovarian follicles under development as part of the subsequent brood. Estradiol Concentration Estradiol-17 concentrations were measur ed on extracts of mosquitofish tissue using enzyme immuno-assay (EIA) kits (C at No. 582251) purchased from Cayman Chemical Company (Ann Arbor, Michigan), and validated in our lab for this purpose. All body tissue posterior to the gonad and an al fin was collected from each fish, and the fresh wet weight obtained after the caudal fin was removed. This tissue is primarily muscle, and will be referred to as muscle fo r the remainder of the chapter. Tissue was stored at –80C until it was thawed on ice, homogenized in 1 ml 65mM borate buffer (pH 8.0), and extracted twice with 5 ml diethyl ether. For each extraction, the ether and homogenate were mixed for two minutes using a multi-tube vortex mixer. For the first extraction, tubes were allowed to settle for three minutes to separate phases. For the second extraction, phases were separated by centr ifugation for two minutes. After phase separation, the aqueous portion was frozen in a methanol bath chilled to -25C with dry ice. The lipophilic layers from both extrac tions were combined in a new tube, and the ether was evaporated under dry forced air. Dry extract was reconstituted in up to 4 ml

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101 EIA buffer and diluted as necessary (up to 1:100) so that samples would fall within the range of the standard curve. EIAs were run as recommended by Cayman with an 18 h refrigerated incubation to increase sensitivity. Data were quantified against a standard curve that was linearized using a logit tran sformation of B/Bo (bound sample/maximum bound). Statistics At the beginning of our analysis, we inte nded to evaluate relationships between water quality factors, such as nitrate, and various measur ed reproductive variables. However, as we progressed through the anal ysis, it became clear that several response variables were interrelated and that these rela tionships needed to be described before we could examine the influence of water quality on reproduction. Relationships among reproductive variables To discover how different reproductive va riables related to each other, we combined the study populations and constructed a correlation matrix based on data from individual fish. Estradiol concentrations we re not included in the matrix because they were measured on a separate subset of fish (separate subsets were used to avoid altered sex steroid concentrations due to capture stre ss). To improve linea rity, all data (except embryo stage) were log10-transformed. After the correla tion analysis, colinear pair-wise combinations of reproductive variables were vi sualized using simple regression. Ovarian weight and embryo number were strongly related to more than one other response variable (Table 5-1). Therefore, for thes e two variables, we used forward stepwise regression to rank the relative im portance of each regressor.

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102 Relationships among water quality parameters and reproductive variables To determine which environmental parameters were important predictors of the measured reproductive variables, we used forward stepwise regres sion. The five water parameters (NO3-N, temperature, conductivity, dissolved oxygen, pH), expressed as a mean for each spring, were entered as inde pendent variables, and their collective statistical influence was evaluated for each de pendent variable, also expressed as a mean or adjusted mean. Adjusted means were calculated using ANCOVA following log-log transformation. Dependent variables incl uded body size (SL, weight, and condition (expressed as mean (log10 weight) adjusted for (log10 SL)), estradiol concentration (log10 (E2+1)), embryo weight (wet and dry), numb er of non-reproductive, mature females captured (out of 30 total from each spring), hepatic weight adjusted for body weight, and embryo number adjusted for standard length2. Possible colinearities between pairs of independent variables were assessed usi ng a correlation matrix. No significant colinearities were detected (r2 < 0.41, p > 0.09 for all pairwise correlations). When more than one independent variable entered into the stepwise model, we calculated partial correlation coefficients using a partial correl ation matrix of the dependent and relevant independent variables. At the conclusion of the stepwise analys is, we visualized the effects of single independent variables (water parameters) on individual response variables using simple linear regression. We observed that temper ature was an important predictor for several 2 Results from the correlation/simple regression an alyses indicated that em bryo number correlated positively with both standard length ( r2 = 0.64; p < 0.0001) and maternal body weight ( r2 = 0.74; p < 0.0001). Compared to body weight, standard length is a more appropriate covariate because it is independent of the response variable (embryo number).

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103 reproductive variables. However, for all these variables, part icularly condition, the significant influence of temperature was driven by a lower temperature at Ruth Spring. The temperature of Ruth Spring was 0.9 to 1.8C less than the other seven sites. Given that this is a seemingly small difference, we repeated the stepwise analysis after excluding temperature as an independent variable. In addition to the above stepwise analysis, log10 (E2+1)-transformed estradiol concentrations were also compared among fish from the different springs using ANOVA. Adjusted means were calculated and compared using an ANCOVA model in SPSS, version 12.0. All other analyses were perf ormed using Statview 5.0, and results were considered significant at = 0.05. Outliers During the analysis, we omitted three measured estradiol values (2.5%) that were more than three standard deviations from the mean for all fish in the study. One female from Ruth Spring was omitted because she exhibited unusually high fecundity compared to the mean for all females in the stu dy (245 versus an average of 27 embryos in ovario). Results Relationships among Reproductive Variables Standard length and female body mass (log10-log10 transformed) were highly correlated (r2 = 0.95) (Table 5-1). In addition, hepatic weight correlated positively with maternal body weight (r2 = 0.62) and embryo number corre lated positively with standard length (r2 = 0.64) (Table 5-1). Adjusted hepatic weight was influenced by stage of embryonic development, being highest duri ng the period of yolk deposition to the embryos (stages 0.5 – 2.5) and then dropping fo r the remainder of gestation (Fig. 5-1A). Likewise, ovarian weight and embryo number were also influenced by other factors

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104 (Table 5-1). Gambusia embryos develop inside the mate rnal ovary and, according to our data, consistently gain wet weight during the c ourse of gestation (as stage increases) (Fig. 5-1B). Embryo dry weight also increases at the beginning of gest ation, but stabilizes between stages 4.5 – 8, and then decreases as offspring approach parturition (Fig. 5-1C). There appears to be a tradeoff between embr yo number and embryo dry weight (but not wet weight) such that a female may have many smaller embryos or fewer large ones (Table 5-1). The outcome of this tradeoff is influenced by maternal body weight, as larger females generally produce more offspr ing, and those offspring exhibit increased wet weights in a manner that may be stagedependent (Table 5-1). Therefore, the relationship between ovarian weight and body mass, traditionally expressed as the gonadosomatic index (GSI), is complicated by gestational wet weight gain and embryo number, which in turn is influenced by maternal body mass and embryo dry weight (Table 5-1). Since females in any given popul ation are not synchroniz ed with regard to gestational stage, it could be misleading to compare populations using GSI as a singular measure of reproductive health or fecund ity (as is a common practice in the piscine literature) without knowing the gestational stage or degree of tradeoff between embryo size and number. Water Quality Table 5-2 shows the collection sites and pr ovides abiotic water data. Ranges across the eight springs for each wa ter parameter were as follows: temperature: 21.4 to 23.2 C; pH: 7.02 to 7.35; conductivity: 347 to 479 S; dissolved oxygen: 0.39 to 5.22 mg/L; and NO3-N: 0.22 to 5.06 mg/L.

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105 Relationships between Water Quality and Reproduction Detailed results of the stepwise regression analyses are shown in Table 5-3. The direction of individual interactions is indi cated by the partial correlation coefficients. With all five water parameters included, ni trate significantly predicted the number of non-reproductive females sampled from the springs (Fig. 5-2). Temperature exhibited a negative relationship with condition and adju sted embryo number (Fig. 5-3), and a positive relationship with embryo dry wei ght; embryo dry weight was also related negatively to nitrate (Table 5-3). However, for these three variables, the influence of temperature appears to be driven by the Ruth Spring data point, which is cooler than the other sites by less than 2 C. If Ruth Spring is excluded from the model, the negative relationship between temperature and adju sted embryo number becomes marginal (r2 = 0.53; p = 0.06), and the relationshi ps between temperature and condition or embryo dry weight are lost (p > 0.3). With temperature excluded from the list of potential independent variables in the stepwise model, we found that nitrate still pl ayed a significant role in predicting embryo dry weight (negative relationship, Fig. 5-4). We checked our data to be sure that this negative association between nitrate and embr yo dry weight could not be explained by differences in stage among embryos from different springs (Fig. 5-1C). Dissolved oxygen was the only variable to enter into the stepwise model for predicting adjusted hepatic weight (Fig. 55). Again, this association between oxygen concentration and hepatic weight could not be explained by differences in developmental stage of the embryos (Fig. 5-1A). Based on our data, embryo wet weight and maternal estradiol concentrations were not influenced by the water quality parameters we

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106 measured (Table 5-3 and Fig. 5-6). In addition, we did not observe significant differences in muscle estradiol concentrations among springs (p = 0.15). Discussion At the outset of our study, we hypothe sized that low concentrations of environmental nitrate (1 – 5 mg/L NO3-N) would be related to changes in reproduction and growth of mosquitofish captured from Florida springs. Our data indicate a significant association between increasing ni trate and reduced embryo dry weight. We also observed a strong relationship between increased nitrate and reduced reproductive activity among mature females. In addition to these findings regarding nitrate, we observed that many of the measured reproducti ve variables were inte rrelated. In addition, variation in Gambusia body size and embryo number and dry weight were related to temperature, and hepatic wei ght was related to dissolved oxygen concentration. We hypothesize that the observed negativ e relationship between nitrate and embryo dry weight is due to nitrat e-induced alterations in thyroid function. Environmentally relevant concentrations of n itrate have been shown to reduce thyroid function, feeding behavior, and growth rate in a variety of vertebrates such as sharks, amphibians, and mammals (Allen et al. 1996; Crow et al 1998; Jahreis et al. 1991; Schuytema and Nebeker 1999; Zaki et al. 2004; Zraly et al. 1997). Nitrate exposure has been associated with goiter and reductions in plasma thyroxine (T4), plasma tri-iodothyronine (T3), iodine availability, iodine uptake, hypothalamic con centrations of growth hormone releasing factor, and plasma concentrations of soma tomedin-C and IGF-1, which are part of the growth hormone axis (Crow et al. 1998; Jahr eis et al. 1991; Kursa et al. 2000; Simon et al. 2000; Zraly et al. 1997). The importanc e of thyroid function during development and

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107 growth suggests that embryos, fetuses, and juveniles could be more susceptible than adults to the disruptive eff ects of nitrate exposure. In addition to the observed relationship be tween low embryonic growth and nitrate, we noted that the number of reproductive females captured during sampling was negatively related to nitrate concentration. That is, as nitrate levels went up, fewer reproductive females (less than 54% in Fanni ng Spring) were captured relative to the total number of sexually mature fema les caught. In our other studies of Gambusia collected from Florida lakes, we typically observe that 95% of mature females are reproductively active (Edwards et al. unpubl. data). Since Gambusia incorporate yolk into oocytes before fertilization (Koya et al. 2000), our observation does not imply disrupted fertilization. Rather, it suggests th at nitrate, or its metabolites (nitrite, nitric oxide) may influence some aspect of vite llogenesis or vitellogenin sequestering during oogenesis. Vitellogenesis occurs in the liver, and is stimulated by es trogens (Tolar et al. 2001). If estrogens are decreased by nitrate or its metabolites, then vitellogenesis could be similarly decreased. Yamagata et al. (2002) demonstrated that in vitro steroidogenesis by rat granulosa cells could be decreased by ex posure to a nitric oxide donor. We did not find a relationship between estradiol con centration in the body tissue and nitrate concentrations in the springs. Nor did we observe a relationship between estradiol and frequency of reproductive females. However, it is possible that nitrate may alter the action of estradiol in the liver. Alternativ ely, the reduced frequency of reproductively active females could be due to a delayed onset of seasonal reproductive activity among some females in the population. Both hypotheses require further testing.

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108 Hypoxia in the springs was related to increased adjusted hepatic weight, and explained 84% of the variance in this vari able. In cultured mammalian hepatocytes and whole animals, hypoxia and related acidosis stimul ate sodium accumulation in liver cells, which causes cell swelling. If this swellin g is not excessive, the cell membrane will remain intact and the cell will av oid necrosis (Carini et al. 19 99). In addition, necrosis is also avoided if hepatocytes are preconditioned by early but intermittent exposure to hypoxia (Carini et al. 2001), as may be the case for wild Gambusia. We informally screened hepatic histology for several male fish captured with the females in our study and did not observe necrotic cells in fish from low or high oxygen sites. Matrotrophy We have noted in the literature that Gambusia are classified as lecithotrophs (Constantz, 1989). However, our data suggest that Gambusia are matrotrophic, at least during the first two thirds of developm ent (through stage 8), when both embryo dry weight and wet weight are stab le or rising (Figs. 5-1B and C). Meffe and Snelson (1993) observed a similar increase in Gambusia embryo dry weight during gestation. In our study, hepatic weight (adjusted for body we ight) was highest duri ng the period of yolk deposition to the embryos (stages 0.5 – 2.5) (Fig. 5-1A) and then dropped for the remainder of gestation. This suggests that vitellogenesis is greates t at the beginning of gestation (Koya et al. 2000). Although v itellogenesis apparently drops by stage 3, embryos gain or maintain dry weight through to stage 8. Between stages 8 and 11, the yolk sac diminishes rapidly and some dry wei ght is lost (Fig. 5-1C). Based on these observations, we suggest that Gambusia exhibit some direct, matrotrophic support of embryo growth during the first two thirds of development, and rely on egg yolk reserves for the completion of gestation. Th is observation of matrotrophy in Gambusia is

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109 supported by other recent evid ence of maternal nutrient tran sfer (Marsh-Matthews et al. 2001). The gain in wet weight before bi rth (a 4-fold increase on average) could be adaptive in that larger larvae often exhib it better survivorship (Hare and Cowen 1997). Conclusion Our data suggest that growth and reproductive parameters in Gambusia are highly interrelated and subject to influence from a variety of environmental factors, including nitrate, temperature, and dissolved oxygen. In particular, nitrate exposure is related to reduced dry weight of developing Gambusia embryos during gestation and reduced rate of reproductive activity among mature females. These findings, along with those of other studies cited here, suggest that nitrate ma y act as an endocrine disruptor, possibly affecting signaling patterns associated with the thyroid, liver, and gonad. The mechanisms associated with these altera tions require extensive study, as nitrate contamination of aquatic ecosy stems is a global concern.

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110 Table 5-1. Relationships of reproductive response variables measured in adult female Gambusia holbrooki collected from eight Florida springs*. Response Variable Correlated with r (indicates direction of relationship) r2 p -value Body weight Standard Length 0.98 0.95 < 0.0001 Hepatic weight Body weight 0.79 0.62 < 0.0001 Embryo dry weight Embryo number -0.32 0.10 < 0.0001 Embryo wet weight Stage** 0.81 0.66 < 0.0001 Embryo wet weight Maternal body weight** 0.46 0.21 < 0.0001 Response Variable Step r2 p -value Other Reproductive Variables Partial r Ovary weight 1 0.86 < 0.0001 Body weight 0.93 2 0.91 < 0.0001 Body weight 0.92 Stage 0.57 3 0.97 < 0.0001 Body weight 0.69 Stage 0.82 Embryo number 0.82 Embryo number 1 0.64 < 0.0001 Standard Length 0.80 2 0.77 < 0.0001 Standard Length 0.86 Embryo dry weight -0.61 *Analyses involving embryo number or data deri ved from embryo number (embryo weight) do not include females with embryos younger than stage 3 (Haynes, 1995) because such embryos are variable in size and difficult to count. ** Both stage and maternal body weight are signi ficantly correlated with embryo wet weight. However, if both are included in a stepwise regressi on model for embryo wet weight, only stage enters the model. Table 5-2: Florida collection sites for female Gambusia holbrooki. Parameter values ( 1 SE) were obtained at the time and location(s) of the fish collection. Spring Location (GPS) Water Temperature (C) pH Conductivity (uS) Dissolved O2 (mg/L) NO3-N (mg/L) Blue N 2949’49.2”; W 08240’56.6” 23.2 7.27 346.5 5.22 1.51 Fanning N 2935’15.0”; W 08256’08.0” 22.60 0.06 7.09 0.01 470.9 3.6 1.89 0.20 4.03 0.41 Hart N 2940’30.4”; W 08257’05.0” 22.35 0.25 7.10 0.01 402.1 0.9 0.39 0.07 0.81 0.04 Lily N 2949’48.6”; W 08239’37.7” 22.3 7.19 425.1 0.84 0.32 Manatee N 2929’20.6”; W 08258’40.0” 22.85 0.25 7.16 479.1 0.6 1.94 0.14 1.26 0.16 Peacock N 3007’18.0”; W 08307’57.0” 22.50 0.44 7.35 0.07 362.2 1.6 2.02 0.27 1.69 0.15 Poe N 2949’33.0”; W 08238’58.0” 22.40 0.06 7.19 0.01 415.1 0.2 0.59 0.28 0.22 0.01 Ruth N 2959’44.0”; W 08258’38.0” 21.37 0.50 7.02 0.07 404.2 4.3 1.17 0.44 5.06 0.61

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111 Table 5-3. Relationships of water quality parame ters and response variables measured in adult female Gambusia holbrooki collected from eight Florida springs. Response Variable Step r2 p -value Water Parameter Partial r Standard length (SL) 0 Body weight 0 Condition 1 0.56 0.03 Temperature -0.75 Adjusted hepatic weight 1 0.85 0.001 Dissolved O2 -0.92 Adjusted embryo number 1 0.76 0.005 Temperature -0.87 1 0.68 0.012 Temperature 0.82 Mean embryo dry weight 2 0.83 0.01 Temperature NO3-N 0.78 -0.69 Mean embryo wet weight 0 Estradiol 0 Number of non-reproductive females 1 0.57 0.03 NO3-N 0.75 Temperature removed from the analysis* Condition 0 Adjusted hepatic weight 1 0.85 0.001 Dissolved O2 -0.92 Adjusted embryo number 0 Mean embryo dry weight 1 0.56 0.03 NO3-N -0.75 Number of non-reproductive females 1 0.57 0.03 NO3-N 0.75 *With the exception of the robust relationship between temperature and embryo number, the significant influence of temperature is largel y driven by a lower temperature at Ruth Spring, which is 0.9-1.8C less than the other seven sites. Given that this is a seemingly small difference, we repeated the stepwise analysis after excluding temperature as an independent variable.

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112 1.4 1.6 1.8 2 2.2 2.4 2.6 2.80 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5StageEmbryo Dry Weight (mg) 4 5 6 7 8 9 10 11 120 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5StageEmbryo Wet Weight (mg) 0 5 10 15 20 250 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5StageAdj. Hepatic Weight (mg) B C A 1.4 1.6 1.8 2 2.2 2.4 2.6 2.80 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5StageEmbryo Dry Weight (mg) 4 5 6 7 8 9 10 11 120 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5StageEmbryo Wet Weight (mg) 0 5 10 15 20 250 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5StageAdj. Hepatic Weight (mg) B C A Figure 5-1. A) Mean maternal hepatic weig ht, adjusted for body weight; B) embryo wet weight; and C) embryo dry weight plo tted by embryonic stage (stages based on Haynes, 1995). Data at stage 3.5 were limited to a single female. Embryo weights represent the sum of the embr yo and yolk sac. We did not obtain oocyte/embryo weight data at stages younger than 3 because those oocytes are small and highly variable in size. *The collective mean dry weight of embryos between stages 4.5 – 8 was signif icantly greater than the collective mean dry weights of embryos either younger or older (ANOVA, p = 0.0004).

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113 r2 = 0.57; p = 0.03 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 0123456 NO3-N (mg/L)Non-pregnant Females Figure 5-2. Percentage of non-reproductiv e, mature females sampled from Florida springs with varying nitrate concentr ations. Total samplings from each spring consisted of 30 mature females.

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114 r2 = 0.76; p = 0.005 12 15 18 21 24 27 30 33 36 39 42 2121.52222.52323.5 Temperature (C)Adj. Embryo Number Figure 5-3. Mean embryo number, adjust ed for maternal body weight for females captured in Florida springs with varying mean temperatures. Graph shows means 1 SE.

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115 r2 = 0.56; p = 0.03 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 0123456 NO3-N (mg/L)Embryo Dry Weight (mg) Figure 5-4. Embryo dry weight (mg) for em bryos taken from females captured in Florida springs with varying concentrations of nitrate. Graph shows means 1 SE.

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116 r2 = 0.85; p = 0.001 6 8 10 12 14 16 18 0123456 Dissolved Oxygen (mg/L)Adj. Mean Hepatic Weight (mg) Figure 5-5. Mean hepatic weight, adjusted for body weight, for females captured in Florida springs with varying dissolved oxygen concentrations. Graph shows adjusted means 1 SE.

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117 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6B l u e Fa n ning H a r t Lil y Manatee Pe acoc k Poe R uthEstradiol (pg/mg muscle ) 1317131417141516 Figure 5-6. Muscle estradiol concentrations for females from each spring. Graph shows means 1 SE. Means are not statistically different (ANOVA, p = 0.15). Numbers at base of data columns indicate sample size.

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118 CHAPTER 6 WATER QUALITY INFLUENCES REPR ODUCTION IN MALE MOSQUITOFISH (Gambusia holbrooki) FROM EIGHT FLORIDA SPRINGS3 Introduction In the past 30 years, nitrate concentra tions in many Florida springs and coastal waters have increased signi ficantly, due largely to anthropogenic sources such as fertilizer application and disc harge of treated sewage (Bar ile, 2004; Katz, 2004; Katz et al., 2004). Nitrate concentra tions in Florida springs vary from a presumed background concentration of 1 mg/L NO3-N to 38 mg/L NO3-N (Katz et al., 1999). This latter value is almost 4-fold higher than the current U.S. drinking water standard for nitrate (10 mg/L NO3-N) (US EPA, 1996). Florida springs are not the only sites of groundwater nitrate contamination. According to the Minnesot a Pollution Control Agency (2000), 13.7% of sampled wells exceed 10 mg/L NO3-N. In Pennsylvania’s Susquehanna River Basin, 45% of sampled wells in agri cultural areas exceed 10 mg/L NO3-N (Lindsey et al., 1997), and in Iowa, a statewide well-water survey reported that 18% of rural drinking water wells were contaminated with nitrate co ncentrations that exceeded 10 mg/L NO3-N (Kross et al. 1993). Clearly, drinking water is at risk for nitrate contamination, and consequently, health risks associated with nitrate exposure must be determined. A number of recent vertebrate studies report that nitrate exposure reduces reproductive hormone concentrations, fertiliz ation rates, and seme n quality, suggesting a direct, negative effect on reproductive func tion (DelPunta et al., 1996; Kostic et al., 1998; 3Disclaimer : The views expressed herein are those of the au thor and do not necessarily reflect the views of the Florida Department of Environmental Protection, which provided funding for this project.

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119 Panesar and Chan, 2000; Rosselli et al., 1995; Shimura et al ., 2002; Zraly et al., 1997; for review, see Guillette and Edwards, 2005) Nitrate can elicit its effects though in vivo conversion to nitrite and then nitric oxid e (NO), a potent signaling molecule that can reduce androgen concentrations and sper m motility, maturation, and viability through a variety of mechanisms (DelPunta et al., 1996; Kostic et al., 1998; Panesar and Chan, 2000; Ratnasooriya and Dharmasiri, 2001; Rosselli et al., 1995). Nitric oxide has been shown to inhibit several steroidogenic enzyme s, including steroidogenic acute regulatory protein (StAR) and cytochrome P450 enzyme s such as P450-sidechain cleavage (SCC) and 3-hydroxysteroid dehydrogenase (3HSD) (DelPunta et al., 1996; Panesar and Chan, 2000). The effects of nitrate on sperma togenesis could either be indirect through alterations of steroidogenic enzyme action or expression, or direct, via the influence of nitric oxide on germ cell or sperm cell apopto sis. Nitric oxide is implicated in both normal and abnormal regulation of apoptosis of spermatogonia, spermatocytes, and spermatids (Lue et al., 2003; Di Meglio et al ., 2004; El-Gohary et al., 1999; Zini et al., 1996). Given these observed effects of nitrate or its metabolite, NO, on reproduction, we hypothesized that adult male mosquitofish (Gambusia holbrooki), captured from springs contaminated with nitrate would exhibit alte red reproduction relative to those from less contaminated springs. Our evaluation of re productive health was based on adult body size, gonopodium length, gonadoand hepato-s omatic indices, androgen concentrations (testosterone and 11-ketotestosterone), total and live sperm counts, and sperm viability. In addition to nitrate, we measured wate r temperature, pH, conductivity, and dissolved oxygen. In mosquitofish, the gonopodium is a gr ooved, bony extension of the anal fin

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120 that elongates during puberty in response to testosterone, and is used for copulation (Rosa-Molinar et al., 1994). Male mosquito fish package their sperm in spherical sperm packets called spermatozeugmata. Since the spermatozeugma, which facilitates transfer of sperm to the female, is a functional and quantifiable reproductive unit, we measured the number of sperm per spermatozeugma. Methods Field Collections and Water Quality Between May 18 and June 3, 2003, adult male Gambusia holbrooki (eastern mosquitofish) were collected from eight arte sian springs located along the Suwannee and Santa Fe Rivers in north-central Florida (Tab le 6-1). Fish were captured using a 3-mm mesh dipnet or seine. The eight springs were selected for two reasons. First, they all had mosquitofish populations large enough fo r sampling, and second, they represent a gradient of nitrate concentrations, which, at the time of sampling ranged from 0.2 to 5.1 mg/L NO3-N. Fish maturity was assessed in the field by looking for a well-developed and hooked gonopodium (Angus et al., 2001). Sampled fish from each spring were divided into three subsets. Sample sizes for each response variab le are given in Table 6-2. One subset was immediately chilled on ice for androgen (tes tosterone (T) and 11-ketotestosterone (11KT)) analysis. The other two subsets were tr ansported live to the laboratory in aerated coolers containing water from the capture site. Within 2 days of capture, these fish were anesthetized in 0.1% MS-222 (3-aminobenzoi c acid ethyl ester methanesulfonate salt, Sigma #A5040), and processed for testicular and hepatic weights from one subset, or sperm counts and sperm viability from the second subset. Testicular weight and sperm

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121 data were not taken from the same fish as the process of stripping sperm is likely to distort the gonad, and the removal of sperm affects test icular weight. On the day of the collection, between n oon and 3 pm, water quality data were obtained at the spring boil and the fish collecti on site(s) within the spring basin. If fish were captured at multiple locations within th e spring basin, the associated water quality measurements were averaged (standard errors given in Table 6-1). Water temperature, conductivity, and pH were measured using a handheld Ultrameter (Model 6P, Myron L Company, Carlsbad, CA). Dissolved oxyge n was measured using an YSI oxygen probe (Model 550A). Nitrate concentrations were measured as follows. Water samples (50 ml) were filtered through a 47-mm glass fiber filte r (0.7 m nominal pore size), chilled on ice, and stored in clean glass containers at -20C until they were analyzed in duplicate using an auto-analyzer (Bran+Luebbe Technicon II with colorimeter). This method uses a copper-cadmium column to reduce nitrate to nitrite, which then reacts to form a colored solution that can be assessed colorimetricall y. Therefore, nitrate concentrations are reported as ppm (mg/L) nitrogen in the forms of nitrate and nitrite combined (NO3-N). All instruments were calibrated using standa rd solutions before each sampling event. Ranges across the eight springs for each wate r parameter were as follows: temperature: 21.4 to 23.2 C; pH: 7.02 to 7.35; conductivity: 347 to 479 S; dissolved oxygen: 0.59 to 5.22 mg/L; and NO3-N: 0.22 to 5.06 mg/L (Table 6-1). Body Size and Dissections Standard length (SL), body weight, and gonopodium length were measured on all fish. SL was measured from the snout tip to the caudal peduncle using calipers. Fish were blotted dry and weighed with an elec tronic balance to the nearest milligram (range

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122 was 68 to 615 mg). Gonopodium length wa s measured to the nearest 0.01 cm, using an ocular micrometer mounted on a dissecting micr oscope. For GSI and HSI, testes (1.0 to 13.6 mg) and livers (0.1 to 5.6 mg) were re moved and weighed to the nearest 0.1 mg. Muscle Androgen Measurements Testosterone (T) and 11-ketotestosterone (11-KT) were measured on extracts of mosquitofish caudal peduncle tissue (referred to as “muscle androgens” for the remainder of the chapter). Upon return to the labor atory, all peduncle tissue posterior to the gonopodium and gonad was collected from each fish iced in the field. After removal of the caudal fin, the caudal peduncle was weighe d (average = 60 mg) and stored at -80C. For extraction, peduncle tissues were hom ogenized (Polytron homogenizer, Kinematica AG, Littau, Switzerland) in 750 l of 65 mM borate buffer (pH 8.0), and extracted twice with 5 ml diethyl ether, mixed with the homogenate for 2 minutes using a multi-tube vortex mixer. For the first extraction, tubes were allowed to settle for three minutes to separate phases. For the second extraction, ph ases were separated by centrifugation for 2 minutes. The aqueous phase was frozen in a me thanol bath cooled to -25C with dry ice. The ether from both extractions was combined in a second tube and evaporated under dry forced air. Hormone concentrations were determined using validated enzyme immunoassay (EIA) kits (Cat No. 582701 (T); 582751 (11-KT), Cayman Chemical, Ann Arbor, Michigan). Dry extract was reconstituted in 1000 to 1500l EIA buffer so that samples would fall within the range of the standard curve. EIAs were run as recommended by Cayman with an 18 h refrigerated incubation to increase sensitivity. Data were quantified against a standard curve linearized usi ng a logit transformation of B/Bo (bound

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123 sample/maximum bound). Duplicate interassay variance (IAV) samples at 2 dilutions were included with each plate. The coeffici ent of variance among all plates, averaged for the 2 dilutions was 10.0% for T, and 12.3% for 11-KT. To normalize sample androgen concentrations across assays, we multiplied by a correction factor derived from the relationship between individual pl ate IAV values and the mean IAV values for all plates. Sperm Counts and Sperm Viability Males were over-anesthetized immediately before sperm collection in room temperature MS-222. Each fish was rinsed in distilled water and placed on its side, with gonopodium abducted, in a petri dish containi ng enough 150 mM KCl to cover the fish. Spermatozeugmata were stripped from the fish by gently pressing down on the abdomen, anterior to the gonopodium, and sweeping cau dally using the smooth, rounded end of a pair of large forceps with the two tips ta ped together. Triplicat e samples of a known number of spermatozeugmata (20 to 40) were drawn up in 300l KCl using a micropipette, and placed in tubes on ice until the sample was counted using a flow cytometer. The method described belo w was previously characterized for Gambusia sperm (Chapter 3). Based on validation te sts, sperm are stable for 3 to 4 h. Just before counting, the sperm samples were stained with 15 l florescent SYBR green dye, incubated on ice for 20 to 30 mi nutes, and then counter-stained with 1.5 l florescent propidium iodide (PI) for 10 minutes. These stains are available in a Live/Dead Sperm Viability Kit (#L-7011, Mol ecular Probes, Inc.). Before staining, the purchased SYBR green was diluted 500 fold in DMSO. SYBR Green and PI are nucleic acid stains that differentially stain live sperm green and dead sperm orange, respectively.

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124 Sperm are characterized as dead if their cell membrane is compromised, allowing the rapid entry of PI. To obtain absolute sperm number s (expressed as sperm number per spermatozeugma), the flow rate of the flow cytometer was calibrated by adding a known number of nile-red florescent beads to each sperm sample (20,000 beads suspended in 20 l buffer per 300 l sperm sample). Pre-counte d fluorescent beads (SPHERO AccuCount fluorescen t particles, 5.2 m, Cat. No. ACFP-50-5) were purchased from Spherotech (Libertyville, Illi nois). Beads were added to sperm and vortexed immediately before counting. Flow cytometric analysis for 20,000 part icles was performed on a FACSort flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using CellQuest 3.3 software (BD Biosciences). Forward and side light scatter measur ements and green (530 +/15 nm), orange (585 +/21 nm), and red (> 650 nm) fluorescence measurements were collected for each sample. The instrument th reshold was set on forward light scatter. Sperm cells were identified using a gate on the forward vs. side light scatter dot plot. The contents of this gate were displayed in a second plot that gates green (SYBR Green) and orange (PI) fluorescing particles separately. Cells emitting only green florescence were counted as live, and cells emitting any orange florescence (indicating a breach in the cell membrane) were counted as dead cells. Nile-r ed bead counts were quantified from their peak on a red-fluorescence histogram. Absolute sperm count per spermatozeugmatum (szm) was calculated as follows: sperm/szm = [(L+D)(B)]/[(b)(S)] where L = number of live sperm counted; D = number of dead sperm counted; B = number of b eads added per sample; b = number of beads

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125 counted; and S = number of spermatozeugmat a originally added to the tube. Sperm viability, measured as the percentage of live sperm, was calculated as % live = [L/(L+D)]*100. Statistical Analysis Relationships among reproductive and morphometric variables To examine if different reproductive and morphometric response variables were related, we combined the spring populations and constructed a correlation matrix based on data from all individual fish. The thr ee subsets of response va riables (GSI and HSI; androgens; and sperm data) were evaluated as separate studies since different fish were used in each subset. To improve lin earity and homogeneity of variance, log10-transformation was used for body, hepatic, and testicular weights, and androgen data (log10 (y+1)) (Zar, 1999). Percentage of live sperm was arcsine-square-root transformed (Zar, 1999). During the androgen analyses, we omitted four fish (3%) with measured androgen values that were more th an three standard deviations from the mean. After the correlation analysis, colinear pair-w ise combinations of re sponse variables were visualized using simple regression. Correlation and regression results indicated that standard length and body mass were significantly related (r2 = 0.92). Similarly, testicular (r2 = 0.68) and hepatic weights (r2 = 0.55) scaled positively with body weig ht, and gonopodium length scaled with SL (r2 = 0.75). Therefore, in subsequent step-wise regression analyses (described below), mean values for testicular and hepatic weights were adjusted for body weight, gonopodium length was adjusted for SL, and condition was calculated as mean body weight adjusted for SL, after log10 transformation of both variable and covariate.

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126 Relationships among water quality parameters and reproductive variables Measured variables among fish from th e eight springs were compared using ANOVA or ANCOVA (when a cova riate was appropriate). We observed spring related differences in all response variables (p < 0.02) except muscle testosterone concentration (p = 0.07) and percentage of live sperm (p = 0.32). For response variables that differed, we identified which of the water quality parameters were important predictors of the variables using forward stepwise regre ssion. The five water parameters (NO3-N, temperature, conductivity, dissolved oxygen, pH), expressed as a mean for the fish collection sites at each spring, were entered as independent variables, and their collective statistical influence was evaluated for each de pendent variable, also expressed as a mean or adjusted mean. Dependent variables included SL weight, condition, adjusted gonopodium length, GSI (adjusted mean testicul ar weight), HSI (adjusted mean hepatic weight), muscle 11-KT concentrations and total and live sperm counts per spermatozeugma. When more than one independent variable entered into the stepwise model, we calculated partial correlation coefficients using a partial correla tion matrix of the dependent and relevant independent variables. Possible colinearities between pairs of independent variables were assessed usi ng a correlation matrix. No significant colinearities were detected (r2 < 0.41, p > 0.09 for all pairwise correlations). We did, however, note that the two springs with the highest nitrate concentrations had the lowest pH readings, an observation that we address in our interpretation of the results. At the conclusion of the stepwise analysis, we visual ized the effects of single water parameters on response variables (means or adjusted means for each spring) using simple linear regression.

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127 Temperature appeared to be an impo rtant predictor for several reproductive variables. However, this effect was driven by the lower temperature at Ruth Spring. The measured temperature of Ruth Spring at th e time of the fish collection was 0.9 to 1.8C less than the other seven sites. Given that this small difference may be within the error of natural variation, we repeated the stepwise an alysis without including temperature as an independent variable. Likewise, we observed that water pH was predictive of muscle 11KT concentration and HSI. However, because the sites with lowest pH had the highest nitrate, and because we have hypothesized that nitrate can impact the variables we have measured, we also compared muscle 11-KT concentrations and HSI between fish from the high nitrate sites (Fanning and Ruth) and fish from all other “low” nitrate sites using ANOVA. This component of our investigation is of interest because water pH can affect gill permeability and nitrate uptake (Jensen, 1995). Adjusted means were calculated using an ANCOVA model in SPSS, version 13.0. All other analyses were performed using Statview 5.0, and results were considered significant at = 0.05. Results Water Quality There were no significant colinearities am ong pairs of water quality variables (r2 < 0.41, p > 0.09). However, the springs with the highest nitrate concentrations also exhibited the lowest pH values. The rela tionship between these two variables for the eight springs is better descri bed by a curve than a line, wh ich is why colinearity was not detected (Fig. 6-1).

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128 Relationships among Reproductive and Morphometric Variables Standard length and male body mass were highly correlated (r2 = 0.88; p < 0.0001). Likewise, hepatic and testicular weights were positively correlated with body weight (r2 = 0.57, p < 0.0001 and r2 = 0.68, p < 0.0001, respectively). Gonopodium length was significantly related to SL (r2 = 0.75; p < 0.0001). We did not observe a significant correlation between muscle testosterone an d 11-ketotestosterone concentrations. The ratios of T to 11-KT in individual fish were both above and below 1.0, suggesting that the two androgens are regulated independently. Finally, neither androgens, nor the T to 11KT ratio, nor sperm parameters were strongl y related to body size. Total sperm counts and percentage of live sperm we re unrelated. Sample sizes for all variables are given in Table 6-2. Relationships among Water Quality Parameters and Reproductive Variables We observed significant spring-related differences in all response variables (p < 0.02, based on ANOVA/ANCOVA) except musc le testosterone concentration (p = 0.07) and percent live sperm (p = 0.32), which averaged 86.5%. The low p-value associated with variation in muscle testosterone varia tion among fish from the different springs is driven by higher T concentrations in fish from Peacock spring (Fig. 6-2A). Mean testosterone concentrations were generally hi gher than 11-KT concentrations (Fig. 6-2A, B) Results of the stepwise regression analys es are shown in Table 6-3. The direction of individual interactions is indicated by th e partial correlation coefficients. Although SL, body weight, and condition differed signif icantly among fish from the different springs, the differences were not explaine d by any of the measured water parameters, based on regression analyses. Nonetheless, fi sh pooled from the two high nitrate sites (4-

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129 5.1 mg/L NO3-N) were significantly smaller (in terms of SL and weight) than fish pooled from the six lower nitrate sites (0.2 – 1.7 mg/L NO3-N) (p <0.0001). With all five water parameters included, adjusted mean gonopodium length was positively related to nitrate concentration (r2 = 0.67; p = 0.01) (Figure 6-3A). GSI and HSI were negatively related to temperature (r2 = 0.73, p = 0.007 and r2 = 0.75, p = 0.005, respectively), whereas total and live sperm counts per spermatozeugma were positively related to temperature (r2 = 0.61, p = 0.02 and 0.53, p = 0.04, respectively) (Fig. 6-4A, B, C, D). However, if Ruth spring, which had the lowest temperature, is removed from the analysis, the regression relationships betw een these four variables and temperature become nonsignificant. Muscle 11-KT concentrations were positively related to pH (r2 = 0.56; p = 0.03) (Fig. 6-5). After controlling fo r the effects of pH in the stepwise regression, 11-KT was also negatively related to disso lved oxygen concentrations (r2 = 0.79; p = 0.003). As with testosterone, fish from Peacock spring exhibited the highest muscle concentrations of 11-KT (Fig. 6-2B). However, unlike the anal ysis of testosterone, if fish from Peacock are removed from the ANOVA, significant spring related variation in muscle 11-KT is still detected (p = 0.03). Furthermore, muscle 11-KT concentrations were significantly lower among males from high nitrate springs co mpared to males from low nitrate springs (p < 0.001, with or without Peacock fish included in the analysis) (Fig. 6-6). With temperature excluded from the list of potential independent variables in the stepwise model, we found that nitrate played a more significant role in predicting GSI (positive relationship, r2 = 0.63; p = 0.02) (Fig. 6-3B) and total sperm count per spermatozeugma (negative relationship, r2 = 0.50; p = 0.05) (Fig. 6-3C) Despite the

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130 total sperm count results, we noted that ni trate did not predict the number of live sperm per spermatozeugma when temperature was removed from the model. This is because the percentage of live sperm among fish from the two highes t nitrate springs (Ruth and Fanning) averaged 88%, compared to 85% amon g fish from the six low nitrate springs. The change was not significantly different between the two groups (p = 0.06), but accounts for some discrepancy between total versus live sperm counts. Functionally, live sperm counts are more relevant. Discussion Relationships between Nitrate and Reproduction As predicted by our hypothesis, nitrate c oncentration in the springs was predictive of reproductive variation among resident ma le mosquitofish. Fish from high nitrate springs exhibited a suite of symptoms that included tes ticular hypertrophy, decreased muscle concentrations of 11-KT, and lower total sperm counts per spermatozeugma. Muscle 11-KT concentrations were also posi tively related to water pH. Because springs with low pH had the highest nitrate, we c onclude that the inter action between pH and nitrate is important, although little data are available to explain the interaction. One possible mechanism, based on data from crabs, is as follows: nitrate and nitrite enter the body of a freshwater fish by crossing the gill epithelia and accumulating in extracellular fluid; these ions are transported against a concentration gradient by substituting for chloride in the bicarbonate-chloride exchange mechanism that normally participates in the osmoregulatory and respiratory functions of the gill (Dobland er and Lackner, 1996; Jensen, 1996; Lee and Pritchard, 1985; Panesar, 1999); because chloride cells in the gill also participate in acid-base regulation (Hir ose et al., 2003), their f unction is likely to be affected by pH. In crayfish, nitrate uptake is pH dependent, with uptake increasing as

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131 water pH declines (Jensen, 1995). Thus, as suggested by our data, the physiological influence of nitrate may be magnified by lower water pH. Gonopodium Length In addition to the above variables, fish from high nitrate springs possessed longer gonopodia (adjusted for standard length). It is not clear whether this significant association results from direct or indirect eff ects of nitrate on mosqu itofish physiology or if the association is accidental. In Gambusia, gonopodium length appears to be highly plastic and affected by a number of selective pressures. For example, Langerhans et al. (2005) observed that female G. affinis and G. hubbsi preferred males with longer gonopodia, whereas males with shorter gon opodia survive better when predators are present. This is because a longer gonopodium hinders the burst-sw imming behavior used to evade predators (Langerhans et al., 2005). However, Jennions and Kelly (2002) observed that male Brachyrhaphis episcopi (Poeciliidae) exhibited longer gonopodia in the presence of predators. The authors of bot h these studies suggested that differences in food availability (potentially nitrate depend ent) or flow rates (which vary among the springs) might also explain va riation in gonopodial length. Testicular Hypertrophy and Mu scle 11-KT Concentrations Testicular hypertrophy could be a compensatory response to reduced steroidogenesis (11-KT in our study) (Mylchrees t et al., 2002). As explained previously, nitrate can be converted in vivo to nitrite and then nitric oxide, especially under changing pH conditions, and nitric oxide has been shown to reduce testicular steroidogenesis (Panesar and Chan, 2000). When target tissues detect that circulating androgens are low, either because they are reduced, or because the androgen receptors are blocked or downregulated, gonadotropins are released from th e pituitary. In male rats treated with

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132 antiandrogens, the gonadotropin release is followed by comp ensatory hypertrophy and/or hyperplasia of Leydig cells (O'Connor et al ., 2002). This Leydig cell increase could account for the increased testicular size associ ated with nitrate exposure. The observed decreases in 11-KT could also, in part, expl ain the lower total sperm counts exhibited by males from high nitrate springs, since 11-KT is required for successful spermatogenesis in teleosts (Miura and Miura, 2003). In Af rican catfish and Japane se eels, 11-KT is the primary androgen involved in spermatogenesis, but its concentration in relation to that of testosterone is also relevant, as both androgens provide feedback to the pituitary (Cavaco et al., 2001; Schulz and Miura, 2002). Spermatogenesis In addition to possible indirect negati ve effects on spermatogenesis through reductions in synthesis of 11-KT, the influen ce of nitrate on spermatogenesis may also be direct. In Gambusia, multiple spermatogonia are grouped in cysts, surrounded by Sertoli cells (Fraile et al., 1992). At the onset of spermatogenesis, these germ cells undergo mitosis, forming hundreds of spermatocytes, which then complete meiosis to form a few thousand spermatids (Koya and Iwase, 2004). After spermatozoa are formed, the cyst is released to the sperm duct as a spermato zeugma (Koya and Iwase, 2004). Therefore, sperm count per spermatozeugma, which we measured in our study, depends on the rates of cell division and the incidence of apoptosis. If nitrate is converted in vivo to nitrite and then nitric oxide (Modin et al., 2001; Pa nesar and Chan, 2000; Weitzberg and Lundberg, 1998), it has the potential to reduce sperm numbers per spermatozeugma by increasing apoptosis of sperm cells at various stages of development (germ cells, spermatocytes, and spermatids), as has been observed in mammals (Di Meglio et al., 2004; El-Gohary et al., 1999; Lue et al., 2003; Zini et al., 1996). This would explain the significant, negative

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133 relationship we observed betw een nitrate and total sperm counts per spermatozeugma. Although we observed a negative relationship between nitrate and total sperm count, we did not observe spring-related differences in the percentage of live sperm, suggesting that, if increased apoptosis is responsible for lower tota l sperm counts among fish from high nitrate springs, then it occurs mostly at the level of the germ cell, or degenerate sperm cells must be cleared from the sperma tozeugmata before they are released to the sperm duct. Conclusions Though correlative, our findings indicate th at environmental exposure to nitrate is related to changes in several reproductive variables among adult male mosquitofish. Future experimental studies are needed to establish cause and effect. These studies should focus on the processes of steroidoge nesis and spermatogenesis in relation to nitrate concentrations as low as 4 mg/L NO3-N. In addition, studies are needed to investigate possible interactions between nitr ate uptake and pH, given the widespread and well-documented occurrences of fresh water acidification (Dunson et al., 1992).

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134 Table 6-1: Florida collection sites for male Gambusia holbrooki. Parameter values ( 1 SE) were obtained at the time and location(s) of the fish collection. Spring Location (GPS) Water Temperature (C) pH Conductivity (uS) Dissolved O2 (mg/L) NO3-N (mg/L) Blue N 2949’49.2”; W 08240’56.6” 23.2 7.27 346.5 5.22 1.51 Fanning N 2935’15.0”; W 08256’08.0” 22.60 0.06 7.09 0.01 470.9 3.6 1.89 0.20 4.03 0.41 Hart N 2940’30.4”; W 08257’05.0” 22.35 0.25 7.10 0.01 402.1 0.9 0.39 0.07 0.81 0.04 Lily N 2949’48.6”; W 08239’37.7” 22.3 7.19 425.1 0.84 0.32 Manatee N 2929’20.6”; W 08258’40.0” 22.85 0.25 7.16 479.1 0.6 1.94 0.14 1.26 0.16 Peacock N 3007’18.0”; W 08307’57.0” 22.50 0.44 7.35 0.07 362.2 1.6 2.02 0.27 1.69 0.15 Poe N 2949’33.0”; W 08238’58.0” 22.40 0.06 7.19 0.01 415.1 0.2 0.59 0.28 0.22 0.01 Ruth N 2959’44.0”; W 08258’38.0” 21.37 0.50 7.02 0.07 404.2 4.3 1.17 0.44 5.06 0.61 Table 6-2. Male Mosquitofish Sample Sizes Spring SL, body weight Gonopodium length GSI HSI Muscle 11-KT Muscle T Total sperm per szm Viable Sperm (% live) Live sperm count Blue 35 34 19 20 19 19 15 14 14 Fanning 50 49 20 20 15 15 15 15 15 Hart 39 38 20 20 19 19 16 16 16 Lily 34 34 20 19 20 20 14 13 13 Manatee 41 41 20 20 18 18 15 15 15 Peacock 51 51 18 18 13 13 15 12 12 Poe 55 55 20 20 14 15 18 19 18 Ruth 51 51 20 20 14 14 14 15 14 SL = Standard Length GSI = Gonadosomatic Index HSI = Hepatosomatic Index 11-KT = 11-ketotestosterone T = testosterone Szm = spermatozeugma

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135 Table 6-3. Significant relationships among water quality parameters from fish-collection sites and response variables measured in adult male Gambusia holbrooki collected from eight Florida springs. Response Variable Step r2 p -value Water Parameter Partial r Standard length 0 Body weight 0 Condition 0 GSI (log10 testicular weight adjusted for log10 body weight) 1 0.73 0.007 Temperature -0.85 2 0.91 0.003 Temperature -0.87 NO3-N 0.81 HSI (log10 hepatic weight adjusted for log10 body weight) 1 0.75 0.005 Temperature -0.87 Gonopodium length (adjusted) for SL) 1 0.67 0.01 NO3-N 0.82 Sperm count per szm 1 0.61 0.02 Temperature 0.78 Live sperm count per szm 1 0.53 0.04 Temperature 0.73 1 0.56 0.03 pH 0.75 Muscle 11-ketotestosterone 2 0.91 0.003 pH 0.95 Dissolved oxygen -0.89 Temperature removed from the analysis* GSI (log10 testicular weight adjusted for log10 body weight) 1 0.63 0.02 NO3-N 0.79 Sperm count per szm 1 0.50 0.05 NO3-N -0.71 HSI (log10 hepatic weight adjusted for log10 body weight) 0 Live sperm count per szm 0 *The significant influence of temperature on GSI, HSI, and sperm count data are largely driven by a lower temperature at Ruth Spring, just 0.9-1.8C less than the other seven sites. Given that this small difference may be within the error of natural variation, we repeated the stepwise analysis without temperature as an independent variable.

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136 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.007.107.207.307.40 pHNO3-N (mg/L) Figure 6-1. Relationship (second order polynomial), between water nitrate concentrations and water pH among eight Florida springs

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137 0 6 12 18 24B l ue F anning Hart L i ly Ma nat e e P e acock Po e RuthMuscle T (pg/mg) 0.0 0.4 0.8 1.2 1.6 2.0Bl ue Fanning Hart L il y Manatee Peacock P oe RuthMuscle 11-KT (pg/mg) A B 0 6 12 18 24B l ue F anning Hart L i ly Ma nat e e P e acock Po e RuthMuscle T (pg/mg) 0.0 0.4 0.8 1.2 1.6 2.0Bl ue Fanning Hart L il y Manatee Peacock P oe RuthMuscle 11-KT (pg/mg) A B Figure 6-2. Muscle androgen concentrations for adult male mosquitofish collected from eight Florida springs. A) Testostero ne. B) 11-KT. Graphs show means 1 SE. Note that the y-axis scales are not the same for the two androgens. Muscle 11-KT concentrations were si gnificantly related to spring pH, oxygen concentration, and nitrate load (Table 3 and Fig. 6-6).

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138 r2 = 0.67 640 650 660 670 680 690 700 710 0123456Adj. Gonop. Length (mm) r2 = 0.62 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 0123456Adj. Testicular Weight (mg) r2 = 0.50 2500 3000 3500 4000 4500 5000 0123456Sperm Count per Szm NO3-N (mg/L)A B C r2 = 0.67 640 650 660 670 680 690 700 710 0123456Adj. Gonop. Length (mm) r2 = 0.62 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 0123456Adj. Testicular Weight (mg) r2 = 0.50 2500 3000 3500 4000 4500 5000 0123456Sperm Count per Szm NO3-N (mg/L)A B C Figure 6-3. Linear relationships between water nitrate concentr ations and A) adjusted mean gonopodium length; B) adjusted mean testic ular weight; and C) mean sperm count per spermatozeugma of adult male mosquitofi sh captured from eight Florida springs. Graphs show adjusted means 1 SE. Regressions are significant at p 0.05.

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139 Water Temperature ( C) r2 = 0.61 2500 3000 3500 4000 4500 5000 21.021.522.022.523.023.5Total Sperm Count per Szm r2 = 0.73 4 4.5 5 5.5 6 6.5 7 7.5 21.021.522.022.523.023.5Adj. Testicular Weight (mg) Water Temperature ( C) r2 = 0.75 1.4 1.6 1.8 2 2.2 2.4 2.6 21.021.522.022.523.023.5Adj. Hepatic Weight (mg) r2 = 0.53 2000 2500 3000 3500 4000 4500 21.021.522.022.523.023.5Live Sperm Count per Szm B C A DWater Temperature ( C)Water Temperature ( C) Water Temperature ( C) r2 = 0.61 2500 3000 3500 4000 4500 5000 21.021.522.022.523.023.5Total Sperm Count per Szm r2 = 0.73 4 4.5 5 5.5 6 6.5 7 7.5 21.021.522.022.523.023.5Adj. Testicular Weight (mg) Water Temperature ( C) r2 = 0.75 1.4 1.6 1.8 2 2.2 2.4 2.6 21.021.522.022.523.023.5Adj. Hepatic Weight (mg) r2 = 0.53 2000 2500 3000 3500 4000 4500 21.021.522.022.523.023.5Live Sperm Count per Szm B C A DWater Temperature ( C)Water Temperature ( C) Figure 6-4. Linear relationships between water temperature (C) and A) adjusted mean testicular weight; B) adjusted mean hepatic weight; C) mean sperm count per spermatozeugma; and D) mean live sperm count per spermatozeugma of adult male mosquitofish captured from eight Florida springs. Graphs show adjusted means 1 SE. Regressions are significant at p 0.05.

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140 r2 = 0.57 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 7.007.107.207.307.40 pHMuscle 11-KT (pg/mg) Figure 6-5. Linear relationship between muscle 11-KT concentrations and water pH for adult male mosquitofish captured from eight Florida springs. Graph shows adjusted means 1 SE. Regression is significant at p 0.05.

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141 0 0.2 0.4 0.6 0.8 1 1.2 Low Nitrate SpringsHigh Nitrate SpringsMuscle 11-KT (pg/mg) 0 0.2 0.4 0.6 0.8 1 1.2 Low Nitrate SpringsHigh Nitrate SpringsMuscle 11-KT (pg/mg) Figure 6-6. Mean muscle 11-KT concentrations ( 1 SE) of adult male mosquitofish captured from high (4.0 – 5.1 mg/L NO3-N) or low (0.2 – 1.7 mg/L NO3-N) nitrate springs. *P = 0.0008 based on ANOVA of log10 (y+1).

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142 CHAPTER 7 SUMMARY AND FUTURE DIRECTIONS Overview This study is the first re latively long-term reproductive study of adult female and male mosquitofish to join the expanding e ndocrine disruption literature. It builds on a few previous endocrine disruption studies (su mmarized in Table 7-1) along with several available papers on Gambusia basic biology (i.e., Bisazza an d Marin, 1995; Fraile et al., 1994; Koya et al., 2000). Th e result greatly expands and integrates our knowledge of Gambusia reproduction in terms of seasonal va riation, life history, and potential influences of environmental contaminants. In addition, it forms the needed foundation on which to build a more detailed understa nding of endocrine disruption, using Gambusia as a model species. This second idea is desc ribed in greater detail below. Summary Our results show that both females a nd males exhibit altered reproductive parameters in association with exposure to several endocrine-disrupting contaminants (EDCs), including organochlorines and nitrate (s ummarized in Table 7-1). In Chapters 2 through 4, we investigated seasonal reproduction of adult Gambusia from Lake Apopka (organochlorine pesticide contamination) and Lake Woodruff (reference). As predicted by the authors of previous studies (Table 7-1), both males and females from Lake Apopka exhibited increased hepatosomatic index relative to the population from Lake Woodruff. Apopka females also exhibited te mporally altered estradiol patterns and a substantial increase in fecundity. Relative to males for Lake Woodruff, males from Lake

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143 Apopka exhibited increased testicular size, al though with no apparent benefit in terms of improved sperm production. In fact, their sperm counts and viability were significantly reduced in some months, particularly at the end of the reproductive season. In Chapters 5 and 6, we assessed repro duction at one point during the breeding season among male and female mosquitofish captured from eight Florida springs with varying concentrations of nitrate. These la st two chapters represent the first detailed reproductive studies of wild fish from aquatic systems with elevated nitrate concentrations. Our results are summarized in Table 7-1. In males exposed to high nitrate, we observed increased gonadosom atic index, reduced 11-ketotestosterone concentrations, and reduced total sperm counts (per spermatozeugma). Females exposed to high nitrate exhibited reduced embryo dry weights and a decreased rate of reproductive activity, based on the presence or ab sence of vitellogenic oocytes. New Hypotheses and Development of Gambusia as a Model Species As with most scientific studies, our study has generated new data, has both corroborated and contradicted previous fi ndings (Table 7-1), and has suggested new hypotheses that logically arise from the resu lts presented in each chapter (summarized in Table 7-2). These outcomes, coupled with the ubiquitous global distribution of Gambusia and their ability to tolerate polluted water (Courtenay and Meffe, 1989), suggest that Gambusia are well suited to be designated a sentinel species. In addition, several aspects of their biology are conduc ive to novel avenues of inquiry that are relevant to the fields of endocrine disruption and aquatic toxicology. For example, unlike other established fish models used to study endocrine disruption, such as English roach (Jobling et al., 2002a), Japanese medaka (Nakayama et al., 2005), fathead minnow (Leino et al., 2005) or common carp (Gimeno et al., 1998b),

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144 Gambusia are viviparous, and, based on our data, matrotrophic, at leas t through the first two thirds of gestation (Chapters 2 and 5). In Chapter 5, we observed a decreased rate of pregnancy and reduced embryo dry weights among female mosquitofish from high nitrate springs. In a viviparous species such as Gambusia, these observations suggest that nitrate exposure impairs maternal provisioning. A viviparous model makes it possible to differentiate between potential effects of nitr ate on vitellogenesis, which occurs largely before fertilization, and effects on direct ma ternal provisioning during gestation. Also, the mosquitofish model promotes investiga tion of offspring health after maternal exposure that occurred before versus during gestation. In addition to their importance to wildlife conservation, these types of studi es are compelling from the perspective of human pregnancy. They inform fetal risk asse ssment by comparing the effects of female body burdens obtained before pregnancy versus exposure during pregnancy. In Chapters 3 and 6, we documented re duced sperm counts among males collected from Lake Apopka and high nitrate sp rings. As detailed in Chapter 3, Gambusia males produce and package their sperm in spherical packets (spermatozeugmata). Spermatogenesis begins with mitotic prolifer ation of spermatogonia w ithin cysts that are bounded by Sertoli cells (Fraile et al., 1992). The resulting primary spermatocytes undergo meiosis and differentiation to produce spermatids and ultimately tailed spermatozoa (Fraile et al., 1992; Koya and Iwase, 2004). Because we measured sperm count per spermatozeugma, our observation of lowered sperm counts suggests specific follow-up hypotheses (Table 7-2) th at are ideally addressed using the Gambusia model. Sperm count per spermatozeugma is most likel y to be affected by altered spermatogonial mitosis or meiosis, germ cell or sperm cell a poptosis, or reduced functionality of Sertoli

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145 cells. The clustering of all the products of cell division from a single spermatogonium into one spermatozeugma makes it possible to quantify the spermatogenic capacity of a male. In addition, cells resulting from meio sis versus mitosis are distinguishable based on size and morphology (Fraile et al., 1992) making the two proce sses separable in the search for mechanisms of endocrine disruption. Fraile et al. (1992) suggest that a single Sertoli cell surrounds each sperm packet. If this is the case, then the number of spermatozeugmata in the testis reflects the numbe r of available Sertoli cells. In addition, reduced sperm number may reflect decreased capacity on the part of the Sertoli cell to support sperm cells. Investigation of thes e hypotheses could identify new potential mechanisms by which endocrine disruption can occur. In Chapter 4, we observed temporal varia tion in the patterns of mean male body size that we interpreted as re flective of changes in adult male recruitment patterns. In Chapter 4, we speculated that males from Lake Apopka exhibited delayed puberty, an outcome that could be either beneficial or de leterious to fitness. Combined with data from Gambusia studies by Bisazza and Marin (1995), in which they report female preference for large males contrasted with increased copulatory success by small males (sneakers), it is clear that Gambusia are an interesting model with regard to the interface of endocrine disruption, sexual selection, and male reproductive strategy. For example, in Chapter 4, males from Lake Apopka were larger than males from Lake Woodruff in most months. This larger size could be due to nutrition, or it could be a response to the observed increase in female size (Chapter 2) coupled with sexual selection for larger size in males. The timing of puberty is marked by development of the gonopodium and is thus easily assessed in male Gambusia. If puberty were delayed in males from Lake

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146 Apopka, it would be necessary to differentiate between the effects of organochlorines on puberty versus the effects of an energetic trade-off between growth and sexual maturation that was under selective pressure. Another consideration that could influence Gambusia populations living in close contact with environmental contaminants is that they potentially adapt to these environments. Again, as a model species, Gambusia offers an interesting opportunity to investigate adaptation to chemical perturbati on. In Chapter 2, we observed that female mosquitofish exhibited substantially greater f ecundity (adjusted for body size) relative to females from Lake Woodruff. One easily tested possibility is that increased fecundity is a response to higher larval mortality. Altern atively, females from Lake Apopka may in fact be better adapted to their environm ent that females from Lake Woodruff. Anecdotally, adult fish from Lake Apopka ge nerally had higher short-term survivorship in the laboratory relative to t hose from Lake Woodruff. In other studies, the ability of mosquitofish to excel in polluted environments has been marked by increased heterozygosity and overall genetic diversit y among exposed individuals (Downhower et al., 2000; Stockwell and Vinyard, 2000; Theo dorakis and Shugart, 1997). In addition, genetic diversity is supported by Gambusia’s mating system, in which females often mate with more than one male, resulting in broods characterized by multiple paternity (Zane et al., 1999). Thus, mosquitofish potentially pr ovide an opportunity to study interactions among genetic diversity, adaptation, and environmental contamination. Not only are mosquitofish a model for the potentially ne gative effects of endocrine disruption, but also, they may provide adaptive answers that wi ll allow long-term survival of this species in environments contaminated with anth ropogenic, biologically active agents.

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147 Table 7-1. Results, integrated with other studies of endocrine disruption in fishes Class of Endocrine Disruptors† Sample Compounds Observed Alterations in Reproduction Caused By or Associated With Endocrine Disruptor Exposure Edwards, 2005 Estrogen Estradiolc ghrstx 4-Tertpentylphenolfgh 4-Tertoctylphenolsx Octylphenolt P-nonylphenola 4-Nonylphenold e Nonylphenolw Endosulfanw Keponeb DDDbyz Bisphenol Ai PCBsjyz Treated sewage effluentklmn o p Chlordaneqyz Chronic hypoxia (1 0.2 mg/L)v Toxapheney z DDEyz Organochlorine mixtureyz* Plasma vitellogeninghlmnps Hepatosomatic indexlt Ovotestes/intersexaghkmnqs Oviduct formationgfkm Delayed puberty/persistent immature testesd Gonadosomatic indextux Sertoli cell structuret Gonadal developmentv Diameter of seminiferous tubulesh Atrophy of germinal epitheliumh Primordial germ cell numberfg PCG distribution in developing gonadw Malformed germ cellsn (intersex fish) Delayed spermatogenesisghn Loss of spermatogenic cystsh Sperm Countsikn z Milt volumeknt (intersex fishes) Sperm motilitykv (intersex fishes) Occluded reproductive ductsnt (intersex fishes) Delayed gonopodial developmentcdo Genital papilla lengthr Adult colorationx Courtship behaviorc Fertilization successkv (intersex fishes) Embryonic/larval survivorshipejuv Oocyte maturationb Oocyte atresian Embryo growths Ca++, amino acid availability to fetuses during gestations Delayed hatchingjv E2 binding in livers Serum E2, Tv Plasma T, 11-KTy Plasma E2,Tn (intersex fish) Serum E2 vy Serum T, 11-KTpuv 17, 20-DHPu Body size HSI GSI (without increased androgen or sperm production) Premature loss of sperm viability at end of reproductive season Live/total sperm counts per spermatozeugma Sperm viability (based on membrane integrity) No strong effect on gonopodium length Fecundity with stronger relationship between fecundity and body size No apparent effect on embryo growth Temporal E2 pattern, with much higher E2 in spring and lower E2 in fall

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148 Table 7-1 Continued Class of Endocrine Disruptors† Sample Compounds Observed Alterations in Reproduction Caused By or Associated With Endocrine Disruptor Exposure Edwards, 2005 Anti-Androgen Vinclozolinabc DDEabd e Flutamideab Sperm countabc e Fertilization successc Adult colorationabc GSIa Courtship behaviorabc Delayed maturationb Gonopodial developmentb Serum E2 d Plasma T, 11-KTd Premature loss of sperm viability at end of reproductive season Live/total sperm counts per spermatozeugma Sperm viability (based on membrane integrity) No strong effect on gonopodium length GSI (without increased androgen or sperm production) Androgen 11ketotestosteroneb Paper mill effluentacd ef Methyltestosteronefg Gonopodial developmentabcd f Male biased sex ratioe Male colorationf Number of reproductive femalesf Intersexg Aromatase** Inhibition Tributyltinabcd Male-biased sex ratiod Sperm countsa Sperm lacking flagellad ATP content of spermb Lactate dehydrogenase activity in spermb Sperm motilitybd Fertilization successc Hatchabilityc Embryo survivorshipc Other Landfill leachateab Male-biased sex ratioab GSIab Brain aromatase activitya Plasma T, E2 a Delayed vitellogenesisb

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149 Table 7-1 Continued Class of Endocrine Disruptors† Sample Compounds Observed Alterations in Reproduction Caused By or Associated With Endocrine Disruptor Exposure Edwards, 2005 Other Nitratec Spawningc Egg numberc Fertilization ratec Delayed hatching timec Hatching rate of the eggsc GSI 11-KT Total sperm count per spermatozeugma Embryo dry weight Rate of pregnancy †Endocrine disruptor classification is general and non -exclusive. Several ch emicals operate through a number of mechanisms that vary with context. Fo r example, DDE has been called an estrogen, an antiestrogen, and an anti-androgen. Classifications presented here depend on the papers cited. *Organochlorine mixture – refers to the chemical mixtur e detected in the plasma of juvenile alligators from Lake Apopka. The mix includes PCBs, DDE, DDD, mirex, endrin, dieldrin, trans-nonachlor, and oxychlordane. These chemicals cause male to female sex reversal of reptile embryos (reviewed by Guillette et al., 2000) **Aromatase catalyzes the conversion of testosterone to estradiol, and androstene dione to estrone (Johnson and Everitt, 1995). Italicized descriptors refer to papers on Gambusia species. Superscripts match sample compounds and observed effects. Citations are given in Table 1-1. Highlighted descriptors are related to hypotheses tested in our study. = female. = male. (Intersex fish(es)) = the sex of the fish(es) in the cited study was an abnormal mix of female and male. = increase. = decrease or inhibition. = altered. 11-KT = 11ketotestosterone. ATP = adenosin e triphosphate. DDD, DDE = metabolites of the insecticide DDT. GSI = gonadosomatic index. E2 = estradiol. HSI = hepatosomatic index. PCBs = polychlorinated biphenyls. PGC = primordial germ cell. T = testosterone. T3 = tri-iodothyronine. 17, 20-DHP = 17 -20 -dihydroxyprogesterone.

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150 Table 7-2. Summary of New Hypotheses Suggested by Dissertation Results Predicted reproductive variation among female Gambusia holbrooki living in Lake Apopka relative to Lake Woodruff Chapter 2 Vitellogenesis Genetic diversity of females and offspring Larval survivorship Predicted changes in sperm quality and seasonal sperm availability among male Gambusia holbrooki living in Lake Apopka relative to Lake Woodruff Chapter 3 Apoptosis or degeneration of germ and sperm cells. Mitosis/meiosis of germ cells. Sertoli cell number/function Efficacy or proper timing of 11-KT or other cellular signals associated with spermatogenesi s, especially during transition between breeding and winter quiescence Predicted reproductive variation (except sperm quality) among male Gambusia holbrooki living in Lake Apopka relative to Lake Woodruff Chapter 4 Delayed puberty among males based on temporal variation in mean body size Vitellogenesis Compensatory hypertrophy or hyperplasia (most likely) of Leydig cells to ensure adequate androgen production Predicted reproductive variation among female Gambusia holbrooki living in high nitrate springs Chapter 5 Thyroid function Delayed puberty or onset of spring ovarian recrudescence Vitellogenesis or vitellogenin sequestering by ovary Maternal nutrient availability to embryos during gestation Larval survivorship Predicted reproductive variation among male Gambusia holbrooki living in high nitrate springs Chapter 6 Sperm cell apoptosis Spermatogonial mitosis/meiosis Sertoli cell number/function Activity of steroidogenic enzymes Compensatory hypertrophy or hyperp lasia of Leydig cells to ensure adequate androgen production Possible deleterious effects from interaction between rising nitrate and low pH

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151 LIST OF REFERENCES Akingbemi, B. T., Hardy, M. P., 2001. Oestr ogenic and antiandrogenic chemicals in the environment: effects on male reproductive health. Ann. Med. 33, 391-403. Al-Hijji, J., Larsson, I., Batra, S., 2001. Effect of ovarian steroids on nitric oxide synthase in the rat uterus, cervix, and vagina. Life Sci. 69, 1133-1142. Allen, A. L., Townsend, H. G. G., Doige, C. E., Fretz, P. B., 1996. A case-control study of the congenital hypothyroidism and dysmaturity syndrome of foals. Can. Vet. J. Rev. Vet. Can. 37, 349. Angus, R. A., 1989. Inheritance of melanistic pigmentation in the eastern mosquitofish. J. Hered. 80, 387-392. Angus, R. A., McNatt, H. B., Howell, W. M., Peoples, S. D., 2001. Gonopodium development in normal male and 11-ketote stosterone-treated female mosquitofish (Gambusia affinis): A quantitative study using computer image analysis. Gen. Comp. Endocrinol. 123, 222-234. Angus, R. A., Weaver, S. A., Grizzle, J. M., Watson, R. D., 2002. Reproductive characteristics of male mosquitofish (Gambusia affinis) inhabiting a small southeastern us river receiv ing treated domestic sewage effluent. Environ. Toxicol. Chem. 21, 1404-1409. Arnold, D. L., Bryce, F., Miller, D., St apley, R., Malcolm, S., Hayward, S., 1998. The toxicological effects foll owing the ingestion of Chinook salmon from the great lakes by sprague-dawley rats during a two-generati on feeding-reproduction study. Regulatory Toxicology and Pharmacology. 27, S18-S27. Arthington, A. H., Lloyd, L. N., 1989. In troduced poeciliids in Australia and New Zealand. In Ecology and Evolution of Liveb earing Fish (Poeciliidae), (ed. G. K. Meffe and F. F. Snelson), pp. 333-348. Englewood Cliffs, NJ: Prentice Hall. Attayde, J. L., Hansson, L. A., 1999. Effect s of nutrient recycling by zooplankton and fish on phytoplankton commun ities. Oecologia. 121, 47-54. Baatrup, E., Junge, M., 2001. Antiandrogenic pe sticides disrupt sexua l characteristics in the adult male guppy (Poecilia reticulata). Environ. Health Perspect. 109, 10631070.

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152 Barbeau, T. 2004. Influence of insulin-like growth factor-1, steroids, and nitrate on reproduction in amphibians. PhD Dissertati on, University of Florida, Gainesville, FL. Barile, P. J., 2004. Evidence of anthropogenic nitrogen enrichment of the littoral waters of east central Florida. J. Coast. Res. 20, 1237-1245. Barnhoorn, I. E. J., Bornman, M. S., Pieterse, G. M., van Vuren, J. H. J., 2004. Histological evidence of intersex in feral sharptooth catfish (Clarias gariepinus) from an estrogen-polluted water source in Gauteng, South Africa. Environ. Toxicol. 19, 603-608. Batty, J., Lim, R., 1999. Morphological and reproductive characteristics of male mosquitofish (Gambusia affinis holbrooki) inhabiting sewage-contaminated waters in New South Wales, Australia. Arch. Environ. Contam. Toxicol. 36, 301-307. Bayley, M., Junge, M., Baatrup, E., 2002. Exposure of juvenile guppies to three antiandrogens causes demasculinization and a reduced sperm count in adult males. Aquat. Toxicol. 56, 227-239. Berndt, M. P., Hatzell, H. H., Crandall, C. A., Turtora, M., Pittman, J. R. and Oaksford, E. T. (1998). Water quality in the Georgi a-Florida coastal plain, U.S. Geological Survey, Tallahassee, FL. Bisazza, A., Marin, G., 1995. Sexual selection an d sexual size dimorphism in the eastern mosquitofish Gambusia holbrooki (Pisces Poeciliidae). Ethol. Ecol. Evol. 7, 169183. Bisazza, A., Pilastro, A., Pal azzi, R., Marin, G., 1996. Sexual behavior of immature male eastern mosquitofish: A way to measure intensity of intra-sexual selection? J. Fish Biol. 48, 726-737. Bisazza, A., Vaccari, G., Pila stro, A., 2001. Female mate choice in a mating system dominated by male sexual co ercion. Behav. Ecol. 12, 59-64. Black, D. A., Howell, W. M., 1979. North-American mosquitofish, Gambusia affinis – a unique case in sex-chromosome evolution. Copeia. 509-513. Black, D. E., Gutjahr-Gobell, R., Pruell, R. J., Bergen, B., Mills, L., McElroy, A. E., 1998. Reproduction and polychlorinated biphenyls in Fundulus heteroclitus (Linnaeus) from New Bedford Harbor, Ma ssachusetts, USA. Environ. Toxicol. Chem. 17, 1405-1414. Bortone, S. A., Cody, R. P., 1999. Morphologi cal masculinization in poeciliid females from a paper-mill-effluent-receiving tributary of the St. Johns River, Florida, USA. Bull. Environ. Contam. Toxicol. 63, 150-156.

PAGE 168

153 Cadenas, E., Poderoso, J. J., Antunes, F., Bo veris, A., 2000. Analysis of the pathways of nitric oxide utilizati on in mitochondria. Free Radic. Res. 33, 747-756. Carini, R., Autelli, R., Bellomo, G., Al bano, E., 1999. Alterations of cell volume regulation in the developmen t of hepatocyte necrosis. Exp. Cell Res. 248, 280-293. Capriulo, G. M., Smith, G., Troy, R., Wikfor s, G. H., Pellet, J., Yarish, C., 2002. The planktonic food web structure of a temperat e zone estuary, and its alteration due to eutrophication. Hydrobiologia. 475, 263-333. Carini, R., De Cesaris, M. G., Splendore, R., Albano, E., 2001. Stimulation of p38 map kinase reduces acidosis and Na+ overload in preconditioned hepatocytes. FEBS Lett. 491, 180-183. Carlsen, E., Giwercman, A., Keiding, N ., Skakkebaek, N. E., 1992. Evidence for decreasing quality of semen during pa st 50 years. Br. Med. J. 305, 609-613. Cavaco, J. E. B., Bogerd, J., Goos, H., Sc hulz, R. W., 2001. Testosterone inhibits 11ketotestosterone-induced spermat ogenesis in African catfish (Clarias gariepinus). Biol. Reprod. 65, 1807-1812. Cavaco, J. E. B., Vilrokx, C., Trudeau, V. L., Schulz, R. W., Goos, H. J. T., 1998. Sex steroids and the initiation of pube rty in male African catfish (Clarias gariepinus). Am. J. Physiol.-Regul. Integr Comp. Physiol. 44, R1793-R1802. Cech, J. J., Schwab, R. G., Coles, W. C., Br idges, B. B., 1992. Mosquitofish reproduction — effects of photoperiod and nutrition. Aquaculture 101, 361-369. Cheng, S.Y., Chen, J.C., 2002. Accumulations of nitrite and nitrat e in the tissues of Penaeus monodon exposed to a combined enviro nment of elevated nitrite and nitrate. Archives of Environmental Contamination and Toxicology 43, 64-74. Clegg, E. D., Cook, J. C., Chapin, R. E., Fost er, P. M. D., Foster, G. P., 1997. Leydig cell hyperplasia and adenoma formation: m echanisms and relevance to humans. Reprod. Toxicol. 11, 107-121. Cochran, R. C., 1992. In vivo and in vitro evidence for the role of hormones in fish spermatogenesis. J. Exp. Zool. 261, 143-150. Constantz, G. D., 1989. Reproductive biology of poeciliid fish. In Ecology and Evolution of Livebearing Fish (Poeciliidae), (ed. G. K. Meffe and F. F. Snelson), pp. 33-50. Englewood Cliffs, NJ: Prentice Hall. Courtenay, W. R. Jr., Meffe, G. K. 1989. Sma ll fish in strange places: a review of introduced poeciliids. In Ecology and Evolution of Liveb earing Fish (Poeciliidae), (ed. G. K. Meffe and F. F. Snelson), pp. 319-331. Englewood Cliffs, NJ: Prentice Hall.

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154 Crain, D. A., 1997. Effects of endocrinedisrupting contaminants on reproduction in the American alligator, Alligator mississippiensis. PhD Dissertation, University of Florida, Gainesville, FL. Crow, G. L., Atkinson, M. J., Ron, B., Atkins on, S., Skillman, A. D. K., Wong, G. T. F., 1998. Relationship of water chemistry to se rum thyroid hormones in captive sharks with goitres. Aquat. Geochem. 4, 469-480. Danielopol, D. L., Griebler, C., Gunatilaka A., Notenboom, J., 2003. Present state and future prospects for groundwater eco systems. Environ. Conserv. 30, 104-130. Daniels, G. L., Felley, J. D., 1992. Life history and foods of Gambusia affinis in 2 waterways of southwestern Louisi ana. Southw. Natural. 37, 157-165. Danielson, T. L., 1968. Differential predation on Culex pipiens and Anopheles albimanus mosquito larvae by two species of fish (Gambusia affinis and Cyprinodon nevadensis) and the effects of simulated re eds on predation. PhD Dissertation, University of Califor nia, Riverside, CA. Das, S., Thomas, P., 1999. Pesticides interfere with the nongenomic action of a progestogen on meiotic maturation by binding to its plasma membrane receptor on fish oocytes. Endocrinology 140, 1953-1956. DelPunta, K., Charreau, E. H., Pignataro, O. P., 1996. Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 137, 5337-5343. De Miguel, M. P., Fraile, B., Saez, F. J., Vicentini, C. A., Paniagua, R., 1994. Influence of ocular and extraocular phot oreception on spermatogenesis in Gambusia affinis holbrooki (Teleostei, Poeciliidae). J. Exp. Zool. 269, 367-372. Di Meglio, S., Tramontano, F., Cimmino, G., Jo nes, R., Quesada, P., 2004. Dual role for poly (ADP-ribose) polymerase-1 and-2 an d poly (ADP-ribose) glycohydrolase as DNA-repair and pro-apoptotic f actors in rat germinal cells exposed to nitric oxide donors. Biochim. Biophys. Ac ta-Mol. Cell Res. 1692, 35-44. Doblander, C., Lackner, R., 1996. Metabolis m and detoxification of nitrite by trout hepatocytes. Biochim. Biophys. Acta-Gen. Subj. 1289, 270-274. Downhower, J. F., Brown, L. P., Matsui, M. L., 2000. Life history variation in female Gambusia hubbsi. Environ. Biol. Fish 59, 415-428. Doyle, C. J., Lim, R. P., 2002. The effect of 17-beta-estradiol on the gonopodial development and sexual activity of Gambusia holbrooki. Environ. Toxicol. Chem. 21, 2719-2724. Dreze, V., Monod, G., Cravedi, J. P., Biagianti-Risbourg, S., Le Gac, F., 2000. Effects of 4-nonylphenol on sex differentiatio n and puberty in mosquitofish (Gambusia holbrooki). Ecotoxicology 9, 93-103.

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155 Du Mond, J. W., Singh, K. P., Roy, D., 2001. Th e biphasic stimulation of proliferation of Leydig cells by estrogen exposure. Int. J. Oncol. 18, 623-628. Dunson, W. A., Wyman, R. L ., Corbett, E. S., 1992. A symposium on amphibian declines and habitat acidification. J. Herpetol 26, 349-352. Durhan, E. J., Lambright, C., Wilson, V., Bu tterworth, B. C., Kuehl, D. W., Orlando, E. E., Guillette, L. J., Gray, L. E., Ankley, G. T., 2002. Evaluation of androstenedione as an androgenic component of river water downstream of a pulp and paper mill effluent. Environ. Toxicol. Chem. 21, 1973-1976. Edwards, T. M., Gunderson, M. P., Milnes, M. R., Guillette, L. J., 2004. Gonadotropininduced testosterone response in pe ripubertal male alligators. Gen. Comp. Endocrinol. 135, 372-380. El-Gohary, M., Awara, W. M., Nassar, S ., Hawas, S., 1999. Deltamethrin-induced testicular apoptosis in rats: The protective e ffect of a nitric oxide synthase inhibitor. Toxicology 132, 1-8. Environmental Protection Agency (US EPA) 1996. Drinking water regulations and health advisories. Washington, D.C. Environmental Protection Agency (US EPA) 2005. Modernized STORET database. Website published by US EPA. Accessed at http://www.epa.gov/ storet/dbtop.html on 7/6/05. European Council. 1998. Council Directive 98/ 83/EC of 3 November 1998 on the quality of water intended for human consumption. Off. J. Eur. Commun. L 330, 32-54. Evans, J. P., Pierotti, M., Pilastro, A ., 2003. Male mating behavior and ejaculate expenditure under sperm competition risk in the eastern mosquitofish. Behav. Ecol. 14, 268-273. Fairchild, W. L., Swansburg, E. O., Arsena ult, J. T., Brown, S. B., 1999. Does an association between pesticide use and subs equent declines in catch of atlantic salmon (Salmo salar) represent a case of endocrine disruption? Environ. Health Perspect. 107, 349-357. Folmar, L. C., Denslow, N. D., Kroll, K., Orlando, E. F., Enblom, J., Marcino, J., Metcalfe, C., Guillette, L. J., 2001. Altered serum sex steroids and vitellogenin induction in walleye (Stizostedion vitreum) collected near a metropolitan sewage treatment plant. Arch. Environ. Contam. Toxicol. 40, 392-398. Folmar, L. C., Denslow, N. D., Rao, V., C how, M., Crain, D. A., Enblom, J., Marcino, J., Guillette, L. J., 1996. Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant. Environ. Health Perspect. 104, 1096-1101.

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156 Fraile, B., Saez, F. J., Vicentini, C. A., De Miguel, M. P., Paniagua, R., 1992. The testicular cycle of Gambusia affinis holbrooki (Teleostei, Poeciliidae). J. Zool. 228, 115-126. Fraile, B., Saez, F. J., Vicentini, C. A., De Miguel, M. P., Paniagua, R., 1993. Effects of photoperiod on spermatogenesis in Gambusia affinis holbrooki (Teleostei, Poeciliidae) during the period of testic ular quiescence. J. Zool. 230, 651-658. Fraile, B., Saez, F. J., Vicentini, C. A., G onzalez, A., De Miguel, M. P., Paniagua, R., 1994. Effects of temperature and photoperiod on the Gambusia affinis holbrooki testis during spermatogenesis period. Copeia. 216-221. Gallagher, E. P., Gross, T. S., Sheehy, K. M., 2001. Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameriurus nebulosus). Aquat. Toxicol. 55, 223-237. Gatseva, P., Lazarova, A., Donchev, N ., 1999. Pathomorphological study of rats submitted to a drinking water regime with high nitrate content. Fresenius Environ. Bull. 8, 45-52. Gatseva, P., Lazarova, A., Maximova, S., Pavlova, K., 1996. Experimental data on the effect of nitrates entering the organism w ith the drinking water. Folia Medica. 37, 75-83. Gimeno, S., Komen, H., Gerritsen, A. G. M., Bowmer, T., 1998a. Feminisation of young males of the common carp, Cyprinus carpio, exposed to 4-tert-pentylphenol during sexual differentiation. Aquat. Toxicol. 43, 77-92. Gimeno, S., Komen, H., Jobling, S., Sumpter, J., Bowmer, T., 1998. Demasculinisation of sexually mature male common carp, Cyprinus carpio, exposed to 4-tertpentylphenol during spermatogenesis. Aquat. Toxicol. 43, 93-109. Gimeno, S., Komen, H., Venderbosch, P. W. M., Bowmer, T., 1997. Disruption of sexual differentiation in genetic male common carp (Cyprinus carpio) exposed to an alkylphenol during different life stages. Environ. Sci. Technol. 31, 2884-2890. Gray, L. E., 1998. Xenoendocrine disrupters: Laboratory studies on male reproductive effects. Toxicol. Lett. 103, 331-335. Greene, J. M., Brown, K. L., 1991. Demogra phic and genetic-characteristics of multiply inseminated female mosquitofish (Gambusia affinis). Copeia. 2, 434-444. Greenlee, A. R., Ellis, T. M., Berg, R. L., 2004. Low-dose agrochemicals and lawn-care pesticides induce developmental toxicity in murine pre-implantation embryos. Environ. Health Perspect. 112, 703-709.

PAGE 172

157 Grether, G. F., Millie, D. F., Bryant, M. J ., Reznick, D. N., Mayea, W., 2001. Rain forest canopy cover, resource availability, and life history evolution in guppies. Ecology. 82, 1546-1559. Gronen, S., Denslow, N., Manning, S., Barn es, S., Barnes, D., Brouwer, M., 1999. Serum vitellogenin levels and reproductive imp airment of male Japanese medaka (Oryzias latipes) exposed to 4-tert-octylphenol. Environ. Health Perspect. 107, 385-390. Guillette, L. J., Brock, J. W., Rooney, A. A., Woodward, A. R., 1999. Serum concentrations of various environmental co ntaminants and their relationship to sex steroid concentrations and phallus size in juvenile American alligators. Arch. Environ. Contam. Toxicol. 36, 447-455. Guillette, L. J., Crain, D. A., Gunderson, M. P ., Kools, S. A. E., Milnes, M. R., Orlando, E. F., Rooney, A. A., Woodward, A. R., 2000. Alligators and endocrine-disrupting contaminants: A current perspective. Am. Zool. 40, 438-452. Guillette, L. J., Crain, D. A., Rooney, A. A ., Pickford, D. B., 1995. Organization versus activation — the role of endocrine-dis rupting contaminants (EDCs) during embryonic-development in wildlife. Environ. Health Perspect. 103, 157-164. Guillette, L.J., Edwards, T.M., 2005. Is ni trate an ecologically relevant endocrine disruptor in vertebrates? Integr. Comp. Biol. 45, 19-27. Guillette, L. J., Gross, T. S., Masson, G. R., Matter, J. M., Percival, H. F., Woodward, A. R., 1994. Developmental abnormalities of the gonad and abnormal sex-hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ. Health Perspect. 102, 680-688. Guillette, L. J., Gunderson, M. P., 2001. Altera tions in development of reproductive and endocrine systems of wildlife populations expose d to endocrine-disrupting contaminants. Reproduction 122, 857-864. Gunderson, M. P., LeBlanc, G. A., Guille tte, L. J., 2001. Alterations in sexually dimorphic biotransformation of testosterone in juvenile American alligators (Alligator mississippiensis) from contaminated lakes. Environ. Health Perspect. 109, 1257-1264. Hahlbeck, E., Griffiths, R., Bengtsson, B. E ., 2004. The juvenile three-spined stickleback (Gasterosteus aculeatus) as a model organism for endocrine disruption — sexual differentiation. Aquat. Toxicol. 70, 287-310. Harding, G. C., LeBlanc, R. J., Vass, W. P., Addison, R. F., Hargrave, B. T., Pearre, S., Dupuis, A., Brodie, P. F., 1997. Bioaccumulation of polychlorinated biphenyls (PCBs) in the marine pelagic food web, base d on a seasonal study in the southern Gulf of St. Lawrence, 1976-1977. Mar. Chem. 56, 145-179.

PAGE 173

158 Hare, J. A., Cowen, R..K., 1997. Size, growth, development, and survival of the planktonic larvae of Pomatomus saltatrix (Pisces: Pomatomidae). Ecology 78, 2415-2431. Harshbarger, J. C., Coffey, M. J., Young, M. Y., 2000. Intersexes in Mississippi River shovelnose sturgeon sampled below Saint Louis, Missouri, USA. Mar. Environ. Res. 50, 247-250. Haubruge, E., Petit, F., Gage, M. J. G., 2000. Reduced sperm counts in guppies (Poecilia reticulata) following exposure to low levels of tributyltin and bis phenol A. Proc. R. Soc. Lond. Ser. B-Biol. Sci. 267, 2333-2337. Haynes, J. L., 1995. Standardized classificatio n of poeciliid development for life-history studies. Copeia. 1, 147-154. Haynes, J. L., Cashner, R. C., 1995. Life-h istory and population-dynamics of the western mosquitofish — a comparison of natural and introduced populations. J. Fish Biol. 46, 1026-1041. Hecnar, S. J., 1995. Acute and chronic toxic ity of ammonium nitrate fertilizer to amphibians from southern Ontario. Environ. Toxicol. Chem. 14, 2131-2137. Hedli, C. C., Snyder, R., Kinoshita, F. K., Steinberg, M., 1998. Investigation of hepatic cytochrome p-450 enzyme induction and DNA adduct formation in male CD/1 mice following oral administration of toxaphene. J. Appl. Toxicol. 18, 173-178. Heinz, G. H., Percival, H. F., Jennings, M. L., 1991. Contaminants in American alligator eggs from Lakes Apopka, Griffin, and Okeechobee, Florida. Environ Monit. Assess. 16, 277-285. Heppell, S. A., Sullivan, C. V., 2000. Identi fication of gender and reproductive maturity in the absence of gonads: muscle tissue le vels of sex steroids and vitellogenin in gag (Mycteroperca microlepis). Can. J. Fish. Aquat. Sci. 57, 148-159. Hess, R. A., Bunick, D., Lee, K. H., Bahr, J ., Taylor, J. A., Korach, K. S., Lubahn, D. B., 1997. A role for oestrogens in the male reproductive system. Nature. 390, 509-512. Hirose, S., Kaneko, T., Naito, N., Takei, Y., 2003. Molecular biology of major components of chloride cells. Comp. Bioc hem. Physiol. B-Biochem. Mol. Biol. 136, 593-620. Howell, W. M., Black, D. A., Bortone, S. A., 1980. Abnormal expression of secondary sex characters in a population of mosquitofish, Gambusia affinis holbrooki — evidence for environmentally induced masculinization. Copeia. 676-681. Hubbs, C., 1999. Effect of light inte nsity on brood production of livebearers Gambusia spp. Trans. Am. Fish. Soc. 128, 747-750.

PAGE 174

159 Irfanullah, H. M., Moss, B., 2004. Factors influe ncing the return of submerged plants to a clear-water, shallow temperate lake. Aquat. Bot. 80, 177-191. Jahreis, G., Hesse, V., Rohde, W., Prange H., Zwacka, G., 1991. Nitrate-induced hypothyroidism is associated with a re duced concentration of growth hormone releasing factor in hypothalamic tissue of rats. Exp. Clin. Endocrinol. 97, 109-112. Jenkins, R., Angus, R. A., McNatt, H., Howe ll, W. M., Kemppainen, J. A., Kirk, M., Wilson, E. M., 2001. Identific ation of androstenedione in a river containing paper mill effluent. Environ. Toxicol. Chem. 20, 1325-1331. Jennions, M. D., Kelly, C. D., 2002. Geogr aphical variation in male genitalia in Brachyphaphis episcopi (Poeciliidae): Is it sexually or naturally selected? Oikos. 97, 79-86. Jensen, F. B., 1995. Uptake and effects of nitrite and nitrate in animals. In Nitrate Metabolism and Excretion, (ed. P. J. Walsh and P. Wright), pp. 289-303. CRC Press, Inc. Boca Raton, FL. Jensen, F. B., 1996. Uptake, elimination and e ffects of nitrite and ni trate in freshwater crayfish (Astacus astacus). Aquat. Toxicol. 34, 95-104. Jensen, T. K., Carlsen, E., Jorgensen, N., Be rthelsen, J. G., Keiding, N., Christensen, K., Petersen, J. H., Knudsen, L. B., Skakkeb aek, N. E., 2002. Poor semen quality may contribute to recent decline in fert ility rates. Hum. Reprod. 17, 1437-1440. Jobling, S., Beresford, N., Nolan, M., Rodgers-G ray, T., Brighty, G. C., Sumpter, J. P., Tyler, C. R., 2002a. Altered sexual matura tion and gamete production in wild roach (Rutilus rutilus) living in rivers that receive trea ted sewage efflue nts. Biol. Reprod. 66, 272-281. Jobling, S., Coey, S., Whitmore, J. G., Kime, D. E., Van Look, K. J. W., McAllister, B. G., Beresford, N., Henshaw, A. C., Brighty, G., Tyler, C. R., Sumpter, J. P., 2002b. Wild intersex roach (Rutilus rutilus) have reduced fertility. Biol. Reprod. 67, 515524. Johnson, M. H., Everitt, B. J., 1995. Essential reproduction. 4th edition. Blackwell Science, Ltd., Cambridge, Mass. Kang, I. H., Kim, H. S., Shin, J. H., Kim, T. S., Moon, H. J., Kim, I. Y., Choi, K. S., Kil, K. S., Park, Y. I., Dong, M. S., Han, S. Y., 2004. Comparison of anti-androgenic activity of flutamide, vinclozolin, procymidone, linuron, and p,p'-DDE in rodent 10-day Harshbarger assay. Toxicology 199, 145-159. Katz, B. G., 2004. Sources of nitrate contam ination and age of water in large karstic springs of Florida. Environ. Geol. 46, 689-706.

PAGE 175

160 Katz, B. G., Chelette, A. R., Pratt, T. R., 2004. Use of chemical and isotopic tracers to assess nitrate contamination and ground-wat er age, Woodville karst plain, USA. J. Hydrol. 289, 36-61. Katz, B. G., Hornsby, D., Bohlke, J. F., Mokray, M. F., 1999. Sources and chronology of nitrate contamination in spring waters, Suwannee River basin, Florida. WaterResources Investigations Report No. 994252. US Geological Survey, Tallahassee, Florida. Kime, D. E., Nash, J. P., 1999. Gamete viability as an indicator of reproductive endocrine disruption in fish. Sci. Total Environ. 233, 123-129. Kincheloe, J. W., Wedemeyer, G. A., Ko ch, D. L., 1979. Tolerance of developing salmonid eggs and fry to nitrate exposure. Bull. Environ. Contam. Toxicol. 23, 574-578. Kinnberg, K., Korsgaard, B., Bjerregaard, P ., Jespersen, A., 2000. Effects of nonylphenol and 17-beta-estradiol on vitellogenin synt hesis and testis morphology in male platyfish Xiphophorus maculatus. J. Exp. Biol. 203, 171-181. Kinnberg, K., Toft, G., 2003. Effects of es trogenic and antiandrogenic compounds on the testis structure of the adult guppy (Poecilia reticulata). Ecotox. Environ. Safe. 54, 16-24. Kirby, M. F., Bignell, J., Brown, E., Craft, J. A., Davies, I., Dyer, R. A., Feist, S. W., Jones, G., Matthiessen, P., Megginson, C., Robertson, F. E., Robinson, C., 2003. The presence of morphologically inte rmediate papilla syndrome in United Kingdom populations of sand goby (Pomatoschistus spp.): endocrine disruption? Environ. Toxicol. Chem. 22, 239-251. Kobayashi, M., Sorensen, P. W., Stacey, N. E., 2002. Hormonal and pheromonal control of spawning behavior in the goldfis h. Fish Physiol. Biochem. 26, 71-84. Kobayashi, T., Sakai, N., Adachi, S., Asahin a, K., Iwasawa, H., Nagahama, Y., 1993. 17alpha, 20-alpha-dihydroxy-4-pregnen-3-one is the naturally occurring spermiationinducing hormone in the testis of a frog, Rana nigromaculata. Endocrinology 133, 321-327. Kostic, T., Andric, S., Kovacevic, R., Maric, D., 1998. The involvement of nitric oxide in stress-impaired testicular steroidogene sis. Eur. J. Pharmacol. 346, 267-273. Koya, Y., Fujita, A., Niki, F., Ishihara, E., Miyama, H., 2003. Sex differentiation and pubertal development of gonads in the viviparous mosquitofish, Gambusia affinis. Zool. Sci. 20, 1231-1242. Koya, Y., Inoue, M., Naruse, T., Sawaguchi, S., 2000. Dynamics of oocyte and embryonic development during ovarian cycle of the viviparous mosquitofish Gambusia affinis. Fish. Sci. 66, 63-70.

PAGE 176

161 Koya, Y., Itazu, T., Inoue, M., 1998. Annual reproductive cycle based on histological changes in the ovary of the female mosquitofish, Gambusia affinis, in central Japan. Ichthyol. Res. 45, 241-248. Koya, Y., Iwase, A., 2004. Annual reproducti ve cycle and rate of the spermatogenic process in male mosquitofish Gambusia affinis. Ichthyol. Res. 51, 131-136. Koya, Y., Kamiya, E., 2000. Environmental re gulation of annual reproductive cycle in the mosquitofish, Gambusia affinis. J. Exp. Zool. 286, 204-211. Kozlov, A. V., Staniek, K., Nohl, H., 1999. N itrite reductase activity is a novel function of mammalian mitochondria. FEBS Lett. 454, 127-130. Kross, B. C., Hallberg, G. R., Bruner, D. R., Cherryholmes, K., Johnson, J. K., 1993. The nitrate contamination of private well wate r in Iowa. Am. J. Public Health 83, 270272. Kuo, R. K., Baxter, G. T., Thompson, S. H., Stricker, S. A., Patton, C., Bonaventura, J., Epel, D., 2000. NO is necessary and suffici ent for egg activation at fertilization. Nature 406, 633-636. Kursa, J., Travnicek, J., Rambeck, W. A., Kroupova, V., Vitovec, J., 2000. Goitrogenic effects of extracted rapeseed meal and n itrates in sheep and their progeny. Vet. Med. 45, 129-140. Kwak, H. I., Bae, M. O., Lee, M. H., Lee, Y. S., Lee, B. J., Kang, K. S., Chae, C. H., Sung, H. J., Shin, J. S., Kim, J. H., Mar, W. C., Sheen, Y. Y., Cho, M. H., 2001. Effects of nonylphenol, bisp henol A, and their mixture on the viviparous swordtail fish (Xiphophorus helleri). Environ. Toxicol. Chem. 20, 787-795. Langerhans, R. B., Layman, C. A., DeWitt, T. J., 2005. Male genital size reflects a tradeoff between attracting mates and avoidi ng predators in two live-bearing fish species. Proc. Natl. Acad. Sci. U. S. A. 102, 7618-7623. Larsson, D. G. J., Forlin, L., 2002. Male-biase d sex ratios of fish embryos near a pulp mill: temporary recovery after a short-term shutdown. Environ. Health Perspect. 110, 739-742. Larsson, D. G. J., Kinnberg, K., Sturve, J ., Stephensen, E., Skon, M., Forlin, L., 2002. Studies of masculinization, detoxification, and oxidative stress responses in guppies (Poecilia reticulata) exposed to effluent from a pulp mill. Ecotox. Environ. Safe. 52, 13-20. Lee, S. H., Pritchard, J. B., 1985. Bicarbo nate-chloride excha nge in gill plasma membranes of blue crab. Am. J. Physiol. 249, R544-R550.

PAGE 177

162 Leino, R. L., Jensen, K. M., Ankley, G. T., 2005. Gonadal histol ogy and characteristic histopathology associated with endocrine disruption in the adult fathead minnow (Pimephales promelas). Environ. Toxicol. Pharmacol. 19, 85-98. Lepore, D. A., 2000. Nitric oxide synthase-i ndependent generation of nitric oxide in muscle ischemia-reperfusion injury Nitric Oxide-Biol. Chem. 4, 541-545. Lindsey, B.D., Loper, C.A., and Hainly, R. A., 1997, Nitrate in gr ound water and stream base flow in the Susquehanna River Basin, Pennsylvania and Maryland: U.S. Geological Survey Water-Resources Investigations Report 97-4146, 66 p. Lue, Y. H., Hikim, A. P. S ., Wang, C., Leung, A., Swerdlo ff, R. S., 2003. Functional role of inducible nitric oxide sy nthase in the induction of male germ cell apoptosis, regulation of sperm number, and determin ation of testes size: evidence from null mutant mice. Endocrinology. 144, 3092-3100. Lye, C. M., Frid, C. L. J., Gill, M. E., 1998. Seasonal reproductive health of flounder Platichthys flesus exposed to sewage effluent. Mar. Ecol.-Prog. Ser. 170, 249-260. Lye, C. M., Frid, C. L. J., Gill, M. E., McCormick, D., 1997. Abnormalities in the reproductive health of flounder Platichthys flesus exposed to effluent from a sewage treatment works. Mar. Pollut. Bull. 34, 34-41. Marsh-Matthews, E., Skierkowski, P., DeMarais, A., 2001. Direct evidence for motherto-embryo transfer of nutrients in the livebearing fish Gambusia geiseri. Copeia. 1, 1-6. McAllister, B. G., Kime, D. E., 2003. Early li fe exposure to environmental levels of the aromatase inhibitor tributyltin causes ma sculinisation and irreversible sperm damage in zebrafish (Danio rerio). Aquat. Toxicol. 65, 309-316. McKinsey, D. M., Chapman, L. J., 1998. Di ssolved oxygen and fish distribution in a Florida spring. Environ. Biol. Fish. 53, 211-223. McPeek, M. A., 1992. Mechanisms of se xual selection operating on body size in the mosquitofish (Gambusia holbrooki). Behav. Ecol. 3, 1-12. Meffe, G. K., Snelson, F. F., 1989. An ecol ogical overview of poeciliid fish. In Ecology and Evolution of Livebearing Fish (Poeciliidae), (ed. G. K. Meffe and F. F. Snelson), pp. 13-31. Englewood Cliffs, NJ: Prentice Hall. Meffe, G. K., Snelson, F. F., 1993. Lipid dyna mics during reproduction in 2 livebearing fish, Gambusia holbrooki and Poecilia latipinna. Can. J. Fish. Aquat. Sci. 50, 2185-2191. Meffe, G. K., Weeks, S. C., Mulvey, M., Kandl, K. L., 1995. Genetic differences in thermal tolerance of eastern mosquitofish (Gambusia holbrooki, Poeciliidae) from ambient and thermal ponds. Can. J. Fish. Aquat. Sci. 52, 2704-2711.

PAGE 178

163 Meyer, D. J., 1995. Enzymatic and non-enzyma tic formation of nitric-oxide. Nat. Med. 1, 1103-1103. Minnesota Pollution Control Agency, 2000. Status of water resource monitoring programs and emerging trends in Minnesota ground water. Supplement. 7 p. Miura, T., Miura, C. I., 2001. Japanese eel: A model for analysis of spermatogenesis. Zool. Sci. 18, 1055-1063. Miura, T., Miura, C. I., 2003. Molecular c ontrol mechanisms of fish spermatogenesis. Fish Physiol. Biochem. 28, 181-186. Miura, T., Yamauchi, K., Takahashi, H., Nagahama, Y., 1991. Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proc. Natl. Acad. Sci. U.S.A. 88, 5774-5778. Modin, A., Bjorne, H., Herulf, M., Alving, K ., Weitzberg, E., Lundberg, J. O. N., 2001. Nitrite-derived nitric oxide: a possible mediator of 'acidic-metabolic' vasodilation. Acta Physiol. Scand. 171, 9-16. Mylchreest, E., Sar, M., Wallace, D. G., Foster, P. M. D., 2002. Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di-(n-butyl)-phthalate. Reprod. Toxicol. 16, 19-28. Nakamura, M., 1978. Morphological and experi mental studies on sex differentiation of the gonad in several teleost fish. Ph D Thesis. Hokkaido University, Hokkaido, Japan. Nakayama, K., Oshima, Y., Nagafuchi, K., Hano, T., Shimasaki, Y., Nakayamaj, K., 2005. Early-life-stage toxicity in offs pring from exposed parent medaka, Oryzias latipes, to mixtures of tributyltin and polyc hlorinated biphenyls. Environ. Toxicol. Chem. 24, 591-596. Noaksson, E., Gustavsson, B., Linderoth, M ., Zebuhr, Y., Broman, D., Balk, L., 2004. Gonad development and plasma steroid profiles by HRGC/HRMS during one reproductive cycle in reference an d leachate-exposed female perch (Perca fluviatilis). Toxicol. Appl. Pharmacol. 195, 247-261. Noaksson, E., Tjarnlund, U., Bosveld, A. T. C., Balk, L., 2001. Evidence for endocrine disruption in perch (Perca fluviatilis) and roach (Rutilus rutilus) in a remote Swedish lake in the vicinity of a public refuse dump. Toxicol. Appl. Pharmacol. 174, 160-176. Nohl, H., Kozlov, A. V., Staniek, K., Gille, L., 2001. The multiple functions of coenzyme Q. Bioorganic Chem. 29, 1-13. Norris, D. O., 1997. Vertebrate Endocrinology. Academic Press, San Diego, CA.

PAGE 179

164 O'Connor, J. C., Frame, S. R., Ladics, G. S., 2002. Evaluation of a 15-day screening assay using intact male rats for identify ing antiandrogens. Toxicol. Sci. 69, 92-108. Ogino, Y., Katoh, H., Yamada, G., 2004. Androgen dependent development of a modified anal fin, gonopodium, as a model to understand the mechanism of secondary sexual character expression in vertebrates. FEBS Lett. 575, 119-126. Onken, H., Tresguerres, M., Luquet, C. M., 2003. Active NaCl absorption across posterior gills of hyper-osmoregulating Chasmagnathus granulatus. J. Exp. Biol. 206, 1017-1023. Orlando, E. F., Davis, W. P., Guillette, L. J ., 2002. Aromatase activity in the ovary and brain of the eastern mosquitofish (Gambusia holbrooki) exposed to paper mill effluent. Environ. Health Perspect. 110, 429-433. Palace, V. P., Evans, R. E., Wautier, K., Baron, C., Vandenbyllardt, L., Vandersteen, W., Kidd, K., 2002. Induction of vitellogenin a nd histological effects in wild fathead minnows from a lake experimentally treated with the synthetic estrogen, ethynylestradiol. Water Qual. Res. J. Canada 37, 637-650. Panesar, N. S., 1999. Role of chloride an d inhibitory action of inorganic nitrate on gonadotropin-stimulated steroidogenesis in mouse Leydig tumor cells. Metab. Clin. Exp. 48, 693-700. Panesar, N. S., Chan, K. W., 2000. Decreased steroid hormone synthesis from inorganic nitrite and nitrate: Studies in vitro and in vivo. Toxicol. Appl. Pharmacol. 169, 222230. Parenti, L. R. and Rauchenberger, M., 1989. Sy stematic overview of the Poeciliines. In: Ecology and Evolution of Liveb earing Fish (Poeciliidae), (ed. G. K. Meffe and F. F. Snelson), pp. 3-12. Englewood Cliffs, NJ: Prentice Hall. Parks, L. G., Lambright, C. S., Orlando, E. F., Guillette, L. J., Ankley, G. T., Gray, L. E., 2001. Masculinization of female mosquitofish in kraft mill effluent-contaminated Fenholloway River water is associated with androgen receptor agonist activity. Toxicol. Sci. 62, 257-267. Patyna, P. J., Davi, R. A., Parkerton, T. F., Brown, R. P., Cooper, K. R., 1999. A proposed multi-generation protoc ol for Japanese medaka (Oryzias latipes) to evaluate effects of endocrine disrup ters. Sci. Total Environ. 233, 211-220. Piferrer, F., 2001. Endocrine se x control strategies for the feminization of teleost fish. Aquaculture 197, 229-281. Porte, C., Barcelo, D., Albaiges, J., 1992. Monitoring of organophosphorus and organochlorinated compounds in a rice crop field (Ebro Delta, Spain) using the mosquitofish Gambusia affinis as indicator organism. Chemosphere 24, 735-743.

PAGE 180

165 Rai, U., Haider, S., 1991. Testis and ep ididymis of the Indian wall lizard (Hemidactylus flaviviridis) — effects of flutamide on FSH and testosterone influenced spermatogenesis, Leydig cells, and epididymis. J. Morphol. 209, 133-142. Rasmussen, T. H., Andreassen, T. K., Pedersen, S. N., Van der Ven, L. T. M., Bjerregaard, P., Korsgaard, B., 2002. Effects of waterborne exposure of octylphenol and oestrogen on pregnant viviparous eelpout (Zoarces viviparus) and her embryos in ovario. J. Exp. Biol. 205, 3857-3876. Rasmussen, T. H., Korsgaard, B., 2004. Estrogenic octylphenol affects seminal fluid production and its biochemical composition of eelpout (Zoarces viviparus). Comp. Biochem. Physiol. C, Toxicol. Pharmacol. 139, 1-10. Ratnasooriya, W. D., Dharmasiri, M. G., 2001. L-arginine, the substrate of nitric oxide synthase, inhibits fertility of male rats. Asian J. Androl. 3, 97-103. Revelli, A., Costamagna, C., Moffa, F., Aldieri, E., Ochetti, S., Bosia, A., Massobrio, M., Lindblom, B., Ghigo, D., 2001. Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa. Biol. Reprod. 64, 1708-1712. Rodgers-Gray, T. P., Jobling, S., Kelly, C ., Morris, S., Brighty, G., Waldock, M. J., Sumpter, J. P., Tyler, C. R., 2001. Exposure of juvenile roach (Rutilus rutilus) to treated sewage effluent induces dose-de pendent and persistent disruption in gonadal duct development. Environ. Sci. Technol. 35, 462-470. Rooney, A., 1998. Variation in the endocrine an d immune system of juvenile alligators: environmental influence on physiology. PhD Dissertation. University of Florida, Gainesville, Florida. Rosa-Molinar, E., Fritzsch, B., Hendricks S. E., 1996. Organizational-activational concept revisited: sexual differentiation in an atherinomorph teleost. Horm. Behav. 30, 563-575. Rosa-Molinar, E., Hendricks, S. E., Rodr iguezsierra, J. F., Fritzsch, B., 1994. Development of the anal fin appendicula r support in the western mosquitofish, Gambusia affinis affinis (Baird and Girard, 1854 ) — a reinvestigation and reinterpretation. Acta Anat. 151, 20-35. Rosselli, M., Dubey, R. K., Imthurn, B., Macas, E., Keller, P. J., 1995. Effects of nitric oxide on human spermatozoa — evidence that nitric-oxide decreases sperm motility and induces sperm toxic ity. Hum. Reprod. 10, 1786-1790. Rouse, J. D., Bishop, C. A., Struger, J., 1999. Nitrogen pollution: An assessment of its threat to amphibian survival. En viron. Health Perspect. 107, 799-803.

PAGE 181

166 Rupnow, H. L., Phernetton, T. M., Shaw, C. E ., Modrick, M. L., Bird, I. M., Magness, R. R., 2001. Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS Am. J. Physiol.-Heart Circul. Physiol. 280, H1699-H1705. Rurangwa, E., Biegniewska, A., Slominska, E., Skorkowski, E. F., Ollevier, F., 2002. Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechan isms of toxicant-reduced spermatozoa motility. Comp. Biochem. Physiol. C, Toxicol. Pharmacol. 131, 335-344. Saiki, M. K., Ogle, R. S., 1995. Evidence of impaired reproduction by western mosquitofish inhabiting seleniferous agricu ltural drainwater. Trans. Am. Fish. Soc. 124, 578-587. Samouilov, A., Kuppusamy, P., Zweier, J. L., 1998. Evaluation of the magnitude and rate of nitric oxide production from nitrite in biological systems. Arch. Biochem. Biophys. 357, 1-7. Schoenfuss, H. L., Levitt, J. T., Van der Kraak, G., Sorensen, P. W., 2002. Ten-week exposure to treated sewage discharge ha s relatively minor, variable effects on reproductive behavior and sperm production in goldfish. Environ. Toxicol. Chem. 21, 2185-2190. Scholz, S., Gutzeit, H. O., 2000. 17-alpha-e thinylestradiol affects reproduction, sexual differentiation and aromatase gene expression of the medaka (Oryzias latipes). Aquat. Toxicol. 50, 363-373. Schultz, I. R., Skillman, A., Nicolas, J. M., Cy r, D. G., Nagler, J. J., 2003. Short-term exposure to 17-alpha-ethynylestradiol decr eases the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem. 22, 12721280. Schulz, R. W., Miura, T., 2002. Spermatoge nesis and its endocrine regulation. Fish Physiol Biochem 26, 43-56. Schuytema, G. S., Nebeker, A. V., 1999. Comp arative toxicity of ammonium and nitrate compounds to Pacific tree frog and African clawed frog tadpoles. Environ. Toxicol. Chem. 18, 2251-2257. Scott, G., Crunkilton, R. L., 2000. Acute and chronic toxicity of nitrate to fathead minnows (Pimephales promelas), Ceriodaphnia dubia, and Daphnia magna. Environ. Toxicol. Chem. 19, 2918-2922. Scott, T. M., Means, G. H., Meegan, R. P., Means, R. C., Upchurch, S. B., Copeland, R. E., Jones, J., Roberts, T., Willet, A., 2004. Springs of Florida, Bulletin no. 66, Florida Geological Survey.

PAGE 182

167 Scribner, K. T., Avise, J. C., 1994. Populatio n cage experiments with a vertebrate — the temporal demography and cytonucl ear genetics of hybridization in Gambusia fish. Evolution. 48, 155-171. Scribner, K. T., Datta, S., Arnold, J., Avise, J. C., 1999. Empirical evaluation of cytonuclear models incorporating genetic drift and tests for neutrality of mtdna variants: Data from experimental Gambusia hybrid zones. Genetica. 105, 101-108. Segovia, M., Jenkins, J. A., Paniagua-Chav ez, C., Tiersch, T. R. 2000. Flow cytometric evaluation of antibiotic effects on vi ability and mitochondrial function of refrigerated spermatozoa of Nile tilapia. Theriogenology. 53, 1489-1499. Shimura, R., Ijiri, K., Mizuno, R. and Naga oka, S., 2002. Aquatic animal research in space station and its issues — focus on suppor t technology on nitrate toxicity. In Space Life Sciences: Biologica l Research and Space Radiation, Vol. 30, pp. 803808. Pergamon-Elsevier Science, Ltd. Oxford, England. Simon, C., Bostedt, H., Adams, W., 2000. Juvenile goiter in a herd of goats in northwest Germany. Schweiz. Arch. Tierheilkd. 142, 339-347. Skakkebaek, N. E., Rajpert-De Meyts, E., Jorgensen, N., Carlsen, E., Petersen, P. M., Giwercman, A., Andersen, A. G., Jense n, T. K., Andersson, A. M., Muller, J., 1998. Germ cell cancer and disorders of spermatogenesis: An environmental connection? Apmis. 106, 3-11. Sohoni, P., Tyler, C. R., Hurd, K., Caunter, J., Hetheridge, M., Williams, T., Woods, C., Evans, M., Toy, R., Gargas, M., Sumpter, J. P., 2001. Reproductive effects of longterm exposure to bisphenol A in the fathead minnow (Pimephales promelas). Environ. Sci. Technol. 35, 2917-2925. Sone, K., Hinago, M., Itamoto, M., Katsu, Y ., Watanabe, H., Guillette, L., Iguchi, T., 2005. Effects of an androgenic growth promoter 17-beta-trenbolone on masculinization of mosquitofish (Gambusia affinis). Gen. Comp. Endocrinol. In press. Stockwell, C. A., Vinyard, G. L., 2000. Life history variation in recently established populations of western mosquitofish (Gambusia affinis). West. North Am. Naturalist. 60, 273-280. Stuehr, D. J., Marletta, M. A., 1985. Ma mmalian nitrate biosynthesis: mouse macrophages produce nitrite a nd nitrate in response to Escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sci. U.S.A. 82, 7738. Suwannee River Water Management District 2005. Surface water quality search. Website published by SRWMD. Accessed at http://www.srwmd.state.fl.us/water+data/ surfacewater+quality /search+surfacewater +quality+data.asp?county_code=F001&Submit=GO on 7/6/05.

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168 Swan, S. H., Elkin, E. P., Fenster, L., 2000. The question of declining sperm density revisited: an analysis of 101 studi es published 1934-1996. Environ. Health Perspect. 108, 961-966. Theodorakis, C. W., Shugart, L. R., 1997. Genetic ecotoxicology 2. Population genetic structure in mosquitofish exposed in situ to radionuclides. Ecotoxicology. 6, 335354. Thibaut, R., Monod, G., Cravedi, J. P., 2002. Residues of C-14-4n-nonylphenol in mosquitofish (Gambusia holbrooki) oocytes and embryos du ring dietary exposure of mature females to this xenohormone. Mar. Environ. Res. 54, 685-689. Toft, G., Baatrup, E., 2001. Sexual characteri stics are altered by 4tert-octylphenol and 17-beta-estradiol in the adult male guppy (Poecilia reticulata). Ecotox. Environ. Safe. 48, 76-84. Toft, G., Edwards, T. M., Baatrup, E ., Guillette, L. J., 2003. Disturbed sexual characteristics in male mosquitofish (Gambusia holbrooki) from a lake contaminated with endocrine disruptors. Environ. Health Perspect. 111, 695-701. Toft, G., Guillette, L. J., 2005. Decrease d sperm count and sexual behavior in mosquitofish exposed to water from a pesticide-contaminated lake. Ecotox. Environ. Safe. 60, 15-20. Tolar, J. F., Mehollin, A. R., Watson, R. D., Angus, R. A., 2001. Mosquitofish (Gambusia affinis) vitellogenin: identification, purification, and immunoassay. Comp. Biochem. Physiol. C, Pharma col. Toxicol. Endocrinol. 128, 237-245. Turner, C. L., 1941. Gonopodial characteristics produced in the anal fins of female Gambusia affinis affinis by treatment with ethyl-test osterone. Biol. Bull. 80, 371383. Uzumcu, M., Suzuki, H., Skinner, M. K., 2 004. Effect of the anti-androgenic endocrine disruptor vinclozolin on embryonic testis cord formation and postnatal testis development and function. Reprod. Toxicol. 18, 765-774. Van den Belt, K., Wester, P. W., Van der Ve n, L. T. M., Verheyen, R., Witters, H., 2002. Effects of ethynylestradiol on the re productive physiol ogy in zebrafish (Danio rerio): time dependency and reversibilit y. Environ. Toxicol. Chem. 21, 767-775. Vanin, A. F., Mordvintcev, P. I., Hauschild t, S., Mulsch, A., 1993. The relationship between L-arginine-dependent nitric oxide s ynthesis, nitrite rel ease, and dinitrosyliron complex formation by activated macrophages. Biochim. Biophys. Acta. 1177, 37. Vanvoorhis, B. J., Dunn, M. S., Snyder, G. D., Weiner, C. P., 1994. Nitric-oxide — an autocrine regulator of human granulosaluteal cell steroidogenesis. Endocrinology. 135, 1799-1806.

PAGE 184

169 Vargas, M. J., de Sostoa, A., 1996. Life history of Gambusia holbrooki (Pisces, Poeciliidae) in the Ebro Delta (NE Iberia n Peninsula). Hydrobiologia. 341, 215224. Verslycke, T., Vandenbergh, G. F., Vers onnen, B., Arijs, K., Janssen, C. R., 2002. Induction of vitellogenesis in 17-alpha -ethinylestradiol-exposed rainbow trout (Oncorhynchus mykiss): a method comparison. Com p. Biochem. Physiol. CToxicol. Pharmacol. 132, 483-492. Vizziano, D., LeGac, F., Fostier, A., 1996. Eff ect of 17-beta-estradiol, testosterone, and 11-ketotestosterone on 17,20 beta-dihydr oxy-4-pregnen-3-one production in the rainbow trout testis. Gen. Comp. Endocrinol. 104, 179-188. Walters, D. M., Freeman, B. J., 2000. Distribution of Gambusia (Poeciliidae) in a southeastern river system and the use of fin ray counts for species determination. Copeia. 2, 555-559. Weitzberg, E., Lundberg, J. O. N., 1998. No n-enzymatic nitric oxide production in humans. Nitric Oxide-Biol. Chem. 2, 1-7. Willey, J. B., Krone, P. H., 2001. Effects of endosulfan and nonylphenol on the primordial germ cell population in pre-larv al zebrafish embryos. Aquat. Toxicol. 54, 113-123. Willingham, E., Crews, D., 2000. The red-eared slider turtle: An animal model for the study of low doses and mixtures. Am. Zool. 40, 421-428. Wine, R. N., Li, L. H., Barnes, L. H., Gu lati, D. K., Chapin, R. E., 1997. Reproductive toxicity of di-n-butylphthalate in a con tinuous breeding protocol in sprague-dawley rats. Environ. Health Perspect. 105, 102-107. Winemiller, K. O., 1993. Seasonality of repro duction by livebearing fish in tropical rainforest streams. Oecologia. 95, 266-276. Woodward, A. R., Jennings, M. L., Percival H. F., Moore, C. T., 1993. Low clutch viability of American alligators on Lake Apopka. Fl. Sci. 56, 52-63. Wooten, M. C., Scribner, K. T., Smith, M. H., 1988. Genetic variability and systematics of Gambusia in the southeastern United States. Copeia. 2, 283-289. Wu, R. S. S., Zhou, B. S., Ra ndall, D. J., Woo, N. Y. S., Lam, P. K. S., 2003. Aquatic hypoxia is an endocrine disruptor and im pairs fish reproduction. Environ. Sci. Technol. 37, 1137-1141. Yamagata, Y., Nakamura, Y., Sugino, N., Hara da, A., Takayama, H., Kashida, S., Kato, H., 2002. Alterations in nitr ate/nitrite and nitric oxide synthase in preovulatory follicles in gonadotropin-primed immature rat. Endocr. J. 49, 219-226.

PAGE 185

170 Zaki, A., Chaoui, A. A., Talibi, A., Derouiche, A. F., Aboussaouira, T., Zarrouck, K., Chait, A., Himmi, T., 2004. Impact of nitrate intake in drinking water on the thyroid gland activity in male rat. Toxicol. Lett. 147, 27-33. Zane, L., Nelson, W. S., Jones, A. G., Avis e, J. C., 1999. Microsatellite assessment of multiple paternity in natural populations of a live bearing fish, Gambusia holbrooki. J. Evol. Biol. 12, 61-69. Zar, J. H., 1999. Biostatistical analysis, 4th edition. Prentice Hall. 663 pp. Zini, A., O’Bryan, M. K., Magid, M. S., Sc hlegel, P. N., 1996. Immunohistochemical localization of endothelial ni tric oxide synthase in hu man testis, epididymis, and vas deferens suggests a possible role for nitric oxide in spermatogenesis, sperm maturation, and programmed cell death. Biol. Reprod. 55, 935-941. Zraly, Z., Bendova, J., Svecova, D., Faldikova, L., Veznik, Z., Zajicova, A., 1997. Effects of oral intake of nitrates on reproduc tive functions of bulls. Vet Med-Czech. 42, 345-354. Zulian, E., Bisazza, A., Marin, G., 1993. De terminants of size in male eastern mosquitofish (Gambusia holbrooki) — inheritance and plasticity of a sexual selected character. Boll. Zool. 60, 317-322. Zulian, E., Bisazza, A., Marin, G., 1995. Va riations in male body size in natural populations of Gambusia holbrooki. Ethol. Ecol. Evol. 7, 1-10. Zweier, J. L., Samouilov, A., Kuppusamy, P., 1999. Non-enzymatic nitric oxide synthesis in biological systems. Biochim. Biophy. Acta. 1411, 250-262.

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171 BIOGRAPHICAL SKETCH Thea M. Edwards was born on June 30, 1970 in Harare, Zimbabwe. She spent her early childhood in the African great outdoors Starting at the age of 8, she accompanied her adventurous parents on th ree trans-Atlantic voyages a nd an extended tour of North America. While in the United States, her fa mily lived in Pennsylvania, New Jersey, and Virginia. In 7th grade, she dissected a clam, and deci ded that biology was for her. In 1988, Thea graduated from James Madison High School in Vienna, Virginia. After high school, Thea attended Virgin ia Tech in Blacksburg, Virginia. She majored first in wildlife conservation, and then in horticulture, graduating with her bachelor’s degree in 1991. In 1993, she wa s accepted to the graduate program in horticulture at the University of Florida, where she completed her master’s degree on the hormonal effects of ethylene on miniature rose s. She then completed a second, nonthesis master’s degree in botany, with a minor in education. In 1998, after extended hard thinking and soul search ing for the next step, Thea became acquainted with the research of Dr Louis Guillette. Dr. Guillette’s research encompassed several areas that she wished to include in the developm ent of her scientific career: comparative reproduction, mechanis tic endocrinology, nont raditional species, conservation, and human impacts on the environment. During her 6 years as a graduate student in zoology, Thea developed four majo r research projects, wrote and received a grant to fund her research from the Flor ida Department of En vironmental Protection, taught two courses independently, served as a teaching assistant for 9 semesters, and

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172 mentored 22 undergraduate researchers, tw o of whom completed honors theses. She completely enjoyed her experience in graduate school and looks forward to a professional scientific and academic career.


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ENVIRONMENTAL INFLUENCES ON MOSQUITOFISH REPRODUCTION


By

THEA M. EDWARDS













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005





























Copyright 2005

by

Thea M. Edwards





























To my parents, Margaret and Peter















ACKNOWLEDGMENTS

During the process of creating my dissertation, I depended on the help and support

of a large number of people. First and foremost, I thank my advisor, Lou Guillette for

sharing with me his wealth of experience and knowledge. I also thank my supervisory

committee, who offered me their time and access to their labs: Lauren Chapman guided

me in developing my statistical abilities; Dave Evans and his graduate students offered

me insight and ideas on nitrate physiology and gave me free access to their wonderful

plate reader; Tom Frazer gave me access to his lab for nitrate measurements of countless

water samples; Taisen Iguchi read my manuscripts and gave opinions and suggestions.

Frank Nordlie was an honorary member of my committee by attending my qualifying

exam, lending me his hematocrit reader, and helping me think about Gambusia. I also

thank Dr. Mari Carmen Uribe for helping me interpret Gambusia histology, Neal Benson

and Melissa Chen for their assistance with my sperm assay, and Stephanie Keller for

measuring the nitrate in my water samples.

I was accompanied on this journey through graduate school by my fellow graduate

students in the Guillette lab. They are: Brandon Moore, my inspiration; Tam Barbeau

and Teresa Bryan, who helped me hang up the estrogen molecule; D. Bermudez, who

threw some of the most memorable parties in my graduate school career; Mark

Gunderson, who taught me how to do RIAs and draw blood from alligators; Heather

Hamlin, whose enthusiasm for research knows no bounds; Krista McCoy, to whom I owe

all that I know about frogs; Matt Milnes, with whom I shared many scientific discussions









and many beers, sometimes together; Ed Orlando, who showed me how to dissect my

first mosquitofish and enthusiastically engages in Gambusia conversations that would be

impossible with anyone else; and Gunnar Toft, whose motivation got my Gambusia

projects started.

Although I worked hard at my research, its completion would have been impossible

without the spectacular help of the 22 undergraduate students who joined me at different

times during my projects. In particular, I thank Lisa Choe, Rebecca Emrich, Annie

Heffernan, Hilary (Thompson) Miller, Maria Paredes, and John Matt Thro, who formed

the long-term core of "Team Gambusia." In addition, I thank Kelly April, Tricia Bardis,

Donnell Bowen, Koo Chung, Chi Chi Echeazu, Jaime Joyce, Elisabeth Kuehlem, Gina

Long, Rich Lufkin, Chad Mackman, Fred Norris, Laura Patterson, Scott Schultz, Paree

Taslimi, Sarah Tynes, and Scott Watson.

Finally, I thank my family for their love, encouragement, faith, comfort, advice,

and willingness to learn about the science that is such an important part of my life.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S .............................................................................................. x

LIST OF FIGURES ......... ......................... ...... ........ ............ xi

ABSTRACT ........ .............. ............. ...... ...................... xiv

CHAPTER

1 OVERVIEW OF Gambusia BIOLOGY, STUDY SITES, AND
CONTAM INANTS OF INTEREST ........................................ ........................

Study Overview ..................................... .......................... .. ......................
G am busia L ife H history ........................................... ....... .... ...... .. .. ........ .. 3
Taxonomy and Distribution of Gambusia holbrooki .........................................3
H ab itat an d D iet................................................. ................ .. 4
Fem ale R productive Cycle ........................................... ........................... 5
M ale R productive Cycle ........................................................ ..................... 6
Gambusia holbrooki as a Sentinel Species............... ...................................9
Overview of Reproductive Endocrinology in Fishes..................................................11
L ak e s ...................................... ..................................................... 12
L ak e A p o p k a .................................................... ................ 13
L ake W oodruff ... ............................................................. ........ .. ..... 14
F lorida Springs and N itrate.................................................................. ...... ........... 15
O v erv iew .............................. ........................................................ ....... 15
Nitrogen Cycling, In Vivo Nitrogen Metabolism, and Effect of Nitrate on
Steroidogenesis ............................ .................................... ................. 16
Effects of Nitrates on Sperm Motility and Viability .......................................18
Summary of Nitrate's Effects on Reproduction................................................18
Review of Endocrine Disruption in Fishes............................................................19
Hypotheses and Goals................... ................................19

2 TEMPORAL REPRODUCTIVE PATTERNS FOR FEMALE MOSQUITOFISH
CAPTURED FROM TWO FLORIDA LAKES.............. .................................. 24

Introduction ........................... ........................................................ 24
M eth o d s ..............................................................................2 7









Field and Tissue C collections ........................................ .......... ............... 27
M uscle Estradiol M easurem ents................................................... ...............29
S statistics ...................................................................................................3 0
Gonadosom atic Index ........................................ ....... ........................
R e su lts ................... ............ ...... ..... ....... ................... ...................... 3 1
Environmental Differences between Lakes..................................................... 31
Temporal and Lake-Associated Variation in Response Variables Related to
Reproduction ...................................... .................... 32
B ody size .................. ....... ........ ........... .......... ........... 32
Temporal changes in reproductive activity ...............................................32
Embryo number, size, and stage of development............... ... ........... 33
Hepatosom atic index ................................... .....................................34
E stra d io l ................................................................................................. 3 4
D isc u ssio n ...................... .. ............. .. ...................................................... 3 5

3 SEASONAL SPERM QUALITY IN MALE Gambusia Holbrooki (EASTERN
MOSQUITOFISH) COLLECTED FROM TWO FLORIDA LAKES ..................... 48

Introduction .............. ...... ............. ......... .. ................ .......... 48
M eth o d s .............................................................................. 5 1
Field C ollections............................................. 51
T esticular H istology ........................ .. ...................... .... ....... .... ..... ...... 52
Sperm Collection ........ ........................................ ................... 52
S p erm Stain in g .............................................................53
Sperm C ounts and V ability ........................................ .......................... 53
C calculations ........... ......... ....... ........... ...... .. ........ ..... ....... 54
S ta tistic s ................................................... ................. ................ 5 5
R e su lts .........................................................................................................................5 5
S p erm V iab ility ............................................................................................. 5 6
S p erm C ou nts ...............................................................57
D isc u ssio n .............................. .. .....................................................5 8
Seasonal Variation in Sperm Counts .................................... ........ .......... ..58
Lake-Associated Variation in Sperm Counts and Quality ................................60
C o n c lu sio n s ..................................................................... 6 3

4 SEASONAL VARIATION IN BODY SIZE, MUSCLE ANDROGEN
CONCENTRATIONS, AND TESTICULAR AND HEPATIC WEIGHTS
AMONG MALE MOSQUITOFISH FROM TWO LAKES IN CENTRAL
F L O R ID A ...................................... ................................................. 7 2

Introduction..................................... .................................. .......... 72
M eth o d s ..............................................................................7 4
F field C collections ............................................... ...................74
M uscle Androgen M easurements ............................................... ...................76
Statistics ...................................... ............................... .......... ...... 77
R e su lts ...................................... .......................................................7 8
A biotic F actors .....................................................................78









B o d y S iz e .......................................................7 8
G on op odiu m L ength ......................................... .............................................79
A ndrogens .............................................. 79
Testicular W eight ...................................... ............... .... ....... 80
H ep atic W eight ............................................................................. .............. 8 1
D isc u ssio n ............................................................................................................. 8 2
B o d y S iz e .......................................................8 2
G on op odiu m L ength ......................................... .............................................84
A ndrogens .............................................. 84
Testicular W eight ...................................... ............... .... ....... 85
H epatic W eight ................................................................. .. ... ............ 86
S u m m ary ............................................................................... 8 7

5 WATER QUALITY INFLUENCES REPRODUCTION IN FEMALE
MOSQUITOFISH (Gambusia Holbrooki) FROM EIGHT FLORIDA SPRINGS ....95

In tro du ctio n ....................................... ................... ............................ 9 5
M eth o d s .................................................................. ................ 9 8
Field Collections and W after Quality ....................................... ............... 98
Body Size and D issections ............................................................................ 99
Estradiol Concentration ......... ...... ......... ...... ............... 100
Statistics ............... ................................................................................... 10 1
Relationships among reproductive variables...........................................101
Relationships among water quality parameters and reproductive
variables ................................... .......................... ... ....... 102
O utliers ............................................................... ... ........ 103
R results .......................... ... ............. ........................................... ........ 103
Relationships among Reproductive Variables...............................103
W after Quality .......................................................... ... ................ 104
Relationships between Water Quality and Reproduction............................... 105
D isc u ssio n ........................................................................................................... 1 0 6
M atrotrop hy .................................................................. 10 8
Conclusion................... ..................109

6 WATER QUALITY INFLUENCES REPRODUCTION IN MALE
MOSQUITOFISH (Gambusia Holbrooki) FROM EIGHT FLORIDA SPRINGS.. 118

Introdu action ...................................... ............................... ......... ...... 118
M eth od s ................................................................. ................. 12 0
Field Collections and W ater Quality .... .......... ....................................... 120
B ody Size and D issections ........................................ .......................... 121
Muscle Androgen Measurements ............................................................122
Sperm Counts and Sperm Viability..... .......... ........................................ 123
Statistical Analysis .................................... .... ............ ....125
Relationships among reproductive and morphometric variables .............125
Relationships among water quality parameters and reproductive
variables ................................... .......................... ... ....... 126


viii









R results ..................127................................................
W after Q quality ............... ...... ........ ......................127
Relationships among Reproductive and Morphometric Variables..................128
Relationships among Water Quality Parameters and Reproductive Variables .128
Discussion ...................... .......... ......... ................ ... ..... ......... 130
Relationships between Nitrate and Reproduction ..........................................130
G onopodium L ength .......................... ................ ............... ... 131
Testicular Hypertrophy and Muscle 11-KT Concentrations .............................131
Sperm atogenesis .................. ...................................... .. ...... .... 132
C onclu sion s ......................................................................133

7 SUMMARY AND FUTURE DIRECTIONS..........................................................142

O v e rv ie w ............................................................................................................. 1 4 2
Sum m ary ............................ ..... ....................... .......... ............. ............... 142
New Hypotheses and Development of Gambusia as a Model Species ..................143

LIST OF REFEREN CES ......... ......................................................... 151

B IO G R A PH ICA L SK ETCH ............ .................................................... .....................171
















LIST OF TABLES


Table pge

1-1 Endocrine disruption in fishes.......................................... ............................ 21

2-1 Additional water quality information for Lake Apopka and Lake Woodruff ..........38

5-1 Relationships of reproductive response variables measured in adult female
Gambusia holbrooki collected from eight Florida springs...................................110

5-2 Florida collection sites for female Gambusia holbrooki ............ .................. 110

5-3 Relationships of water quality parameters and response variables measured in
adult female Gambusia holbrooki collected from eight Florida springs ..............111

6-1 Florida collection sites for male Gambusia holbrooki ......................................134

6-2 Male mosquitofish sample sizes..................................................... .............. 134

6-3 Significant relationships among water quality parameters from fish-collection
sites and response variables measured in adult male Gambusia holbrooki
collected from eight Florida springs............................................ ............... 135

7-1 Results, integrated with other studies of endocrine disruption in fishes..............147

7-2 Summary of new hypotheses suggested by dissertation results........................ 150
















LIST OF FIGURES


Figure pge

2-1 Stages of embryonic development for eastern mosquitofish .................................39

2-2 Percentage of female mosquitofish with broods at different stages of embryonic
d ev elo p m en t ....................................................... ................ 4 0

2-3 Seasonal changes in water temperature for Lake Apopka and Lake Woodruff.......41

2-4 Temporal variation in body size of adult female mosquitofish from Lake
Apopka and Lake Woodruff ............ ..... ................................... 42

2-5 Temporal variation in mean embryo number and percentage of sampled females
from each lake .........................................................................43

2-6 Embryo number observed for female mosquitofish of different standard lengths...44

2-7 Mosquitofish embryonic wet weight at different developmental stages..................45

2-8 Mean adjusted hepatic weight of mosquitofish with embryos at different stages ...46

2-9 Temporal variation in muscle estradiol concentrations of adult female
mosquitofish from Lake Apopka and Lake Woodruff.................................. 47

3-1 Testicular histology of Gambusia holbrooki ...................................... .................64

3-2 Sperm methods for Gambusia holbrooki ...................................... ............... 66

3-3 Gambusia sperm counts flow cytometry printout................... ..................67

3-4 Water temperature and daylength data for the collection period ...........................68

3-5 Mean percent live sperm observed among adult male Gambusia holbrooki
collected from two lakes in central Florida..................... ..... .. ............. 69

3-6 Mean sperm count per spermatozeugma observed among adult male Gambusia
holbrooki collected from two lakes in central Florida .......................................... 70

3-7 Mean live sperm count observed among adult male Gambusia holbrooki from
Lake Apopka and Lake W oodruff. ... ................................ ......................... ............... 71









4-1 Water temperature for Lake Apopka and Lake Woodruff, shown with ambient
photoperiod for each collection date ............................................. ............... 88

4-2 Mean standard length of adult male mosquitofish from Lake Apopka and Lake
W o o d ru ff ...............................8.................................................... ... 9

4-3 Body weight of adult male mosquitofish from Lake Apopka and Lake Woodruff .90

4-4 Mean gonopodium length adjusted for standard length, of adult male
mosquitofish from Lake Apopka and Lake Woodruff.................................. 91

4-5 Mean muscle androgen concentrations for adult male mosquitofish from Lake
Apopka and Lake Woodruff.................. ......... .... ............... 92

4-6 Adjusted testicular weight of adult male mosquitofish from Lake Apopka and
L ake W oodruff ............................................... ........................................ .... 93

4-7 Adjusted hepatic weights of adult male mosquitofish from Lake Apopka and
L ak e W oodruff ................. .................................................... .............. ........ .... 94

5-1 Adjusted hepatic weight, embryo wet weight; and embryo dry weight plotted by
em b ry onic stag e................................................ ................. 112

5-2 Percentage of non-reproductive, mature females sampled from Florida springs
with varying nitrate concentrations ............................................. ................... 113

5-3 Adjusted mean embryo number for females captured in Florida springs with
varying tem peratures .................. .............................. .... .. .. .. ...... .. .. 114

5-4 Embryo dry weight for embryos taken from females captured in Florida springs
w ith varying concentrations of nitrate.................................................................115

5-5 Adjusted hepatic weight for females captured in Florida springs with varying
dissolved oxygen concentrations............... ........... ......... ...................... 116

5-6 Muscle estradiol concentrations for females from each spring ............................117

6-1 Relationship between water nitrate concentrations and water pH among eight
Florida springs ............ ... ..................... ................. .. ... ......... 136

6-2 Muscle androgen concentrations for adult male mosquitofish collected from
eight Florida springs ............................................. ............... ..... 137

6-3 Linear relationships between water nitrate concentrations and several
reproductive response variables of adult male mosquitofish captured from eight
F lorida spring s ........... ............................ ................................. ...... 138









6-4 Linear relationships between water temperature and several reproductive
response variables of adult male mosquitofish captured from eight Florida
sp rin g s .......................................................................... 1 3 9

6-5 Linear relationship between muscle 11-KT concentrations and water pH for
adult male mosquitofish captured from eight Florida springs.............................140

6-6 Mean muscle 11-KT concentrations of adult male mosquitofish captured from
high or low nitrate springs...................................... ......... ................ 141















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ENVIRONMENTAL INFLUENCES ON MOSQUITOFISH REPRODUCTION

By

Thea M. Edwards

August 2005

Chair: Louis J. Guillette, Jr.
Major Department: Zoology

Reproduction in all organisms is regulated by a wide range of environmental

factors. In fishes, these include (among other factors): temperature, photoperiod,

nutrition, and social interactions. In the past 50 years, an additional factor has emerged:

anthropogenic pollution. Many widely used contaminants (PCBs, pesticides, fertilizers,

plasticizers) are now distributed throughout our environment, particularly in aquatic

systems. Several of these have been shown to disturb normal development, growth, and

reproduction of vertebrates through disruptive interactions with the endocrine system.

In this extended seasonal study, we evaluated reproductive parameters in adult

Gambusia holbrooki captured from two central Florida lakes: Lake Apopka (with a

documented history of organochlorine contamination) and Lake Woodruff Wildlife

Refuge (reference site). Relative to the Lake Woodruff population, males and females

from Lake Apopka exhibited increased hepatosomatic indices; females also exhibited

altered estradiol patterns and an unexpected increase in fecundity. Males from Lake









Apopka exhibited increased testicular size, but decreased sperm counts and sperm

viability in some months, particularly at the end of the reproductive season.

In a second suite of studies, we assessed reproduction, at one point during the

breeding season, among adult Gambusia, captured from eight Florida springs with

varying concentrations of nitrate. Nitrate contamination of Florida springs is a growing

concern. In fact, nitrate concentrations in some springs exceed the EPA-established

concentration limit for drinking water (10 mg/L N03-N). Nitrate exposure is associated

with altered development, reduced steroidogenesis, and diminished reproductive success

in a number of species. In male mosquitofish exposed to elevated nitrate concentrations

(4 to 5 mg/L N03-N), we observed increased gonadosomatic index, reduced 11-

ketotestosterone concentrations, and reduced sperm counts. Females from springs with

elevated nitrate concentrations exhibited reduced embryo dry weights and a decreased

rate of reproductive activity, based on presence or absence of vitellogenic oocytes.

Taken together, our results suggest that long-term exposure to environmental

contaminants, specifically organochlorines and nitrate, is associated with altered

reproductive outcomes. In addition, the temporal nature of our research greatly expands

and integrates our knowledge of Gambusia reproduction in terms of seasonal variation

and basic life history.














CHAPTER 1
OVERVIEW OF Gambusia BIOLOGY, STUDY SITES, AND CONTAMINANTS OF
INTEREST

Study Overview

Aquatic environments are inherently variable and often exhibit pronounced

temporal changes that influence the biology and ecology of their resident flora and fauna.

Many changes are natural in that they are non-anthropogenic. Some are also repetitively

cyclical and represent phenomena to which the animals in the system are adapted.

Mosquitofish, for example, become reproductively active in the spring, when water

temperatures exceed 20 to 220C, and they end activity in the fall when daylength shortens

to less than 12 h (Chapters 1 and 2 of our study; Fraile et al., 1994; Koya and Kamiya,

2000). It is likely that this seasonal pattern evolved around practical considerations of

prey abundance, mate readiness, and larval survivorship; factors that affect fitness, and

that generally vary with seasonal environmental change (Winemiller, 1993).

In addition to the natural factors that regulate reproduction, aquatic animals are

increasingly subject to "un-natural" regulation by anthropogenic endocrine-disrupting

contaminants (EDCs). Widespread use of pesticides, fertilizers, plastics, and other

industrial chemicals has increased exponentially over the past 50 years (Danielopol et al.,

2003). The effect is so dramatic that epidemiologists can track the related rise in human

reproductive disorders that are causally linked to contaminant exposure (Carlsen et al.,

1992; Skakkebaek et al., 1998).









Contaminant-induced reproductive changes in the human population probably

occur after similar changes in wildlife. This is because most wildlife species are smaller

than humans and potentially sensitive to lower doses. Those with shorter generation

times could be more susceptible to cumulative, cross-generational changes. In addition,

some species possess a degree of phenotypic plasticity that makes them more likely to

exhibit overt symptoms. For example, in a wide variety of fishes, sexual differentiation

is highly labile, and exposure of differentiating fishes to estrogens (regardless of

genotypic sex) can produce morphologically female monocultures (Piferrer, 2001).

Therefore, studies of wild animals are useful in risk assessment and should be used to

guide environmental policy to improve conservation, ecological sustainability, and

human health and welfare.

Our study investigated temporal patterns of reproduction in wild adult Gambusia

holbrooki (eastern mosquitofish) captured from Lake Woodruff and Lake Apopka in

central Florida. These lakes were sampled because other biological data on fishes

(Gallagher et al., 2001; Toft et al., 2003) and alligators (Guillette et al., 2000) from these

lakes suggest that they provide a comparative system in which to study the impact of

EDCs on reproductive variables. In addition to sampling these lakes, we conducted a

single-month reproductive study of Gambusia holbrooki captured from eight artesian

springs, located along the Suwannee and Santa Fe Rivers in north Florida. These springs

represent (at present) a gradient of nitrate contamination and provided a natural

experimental opportunity to assess the potential relationship between nitrate

concentration and variation in mosquitofish reproduction. The remainder of this chapter

details mosquitofish life history, clarifies why I chose to work with mosquitofish, and









presents an overview of reproductive endocrinology in fishes. This is followed by

descriptions of the two lakes and eight springs sampled in our study, along with a review

of the effects of pesticides and nitrate on fish reproduction. Finally, I present the

hypotheses that guided our study.

Gambusia Life History

Taxonomy and Distribution of Gambusia holbrooki

Within the genus Gambusia (family Poeciliidae), there are approximately 45

species with a native range that extends from New Jersey, west to Iowa and the

Mississippi River drainage; and south to the Gulf coast, the Caribbean, and Mexico.

From Mexico, the native range continues south through Central America, to Columbia

(reviewed by Parenti and Rauchenberger, 1989). Of these 45 species, Gambusia

holbrooki (eastern mosquitofish) and Gambusia affinis (western mosquitofish) are the

most widely studied. In addition to their native ranges in the southern United States,

these two species have been introduced for biological control of mosquito larvae and are

now found on all of the continents except Antarctica (reviewed by Courtenay and Meffe,

1989). Thus, their functional range is substantially larger than their native range.

The focus of our study is Gambusia holbrooki, which is distinguished from its

western sister species (Gambusia affinis), by a thin geographic and biological line, across

which hybridization does occur (Walters and Freeman, 2000). Morphologically, the two

species are differentiated by fin ray counts. Gambusia holbrooki possess 8 dorsal and 11

anal fin rays, whereas G. affinis have 7 dorsal and 10 anal fin rays (Walters and Freeman,

2000).

In their native range, G. affinis are found west of Mobile Bay in Alabama; whereas

G. holbrooki occur to the east, and the zone of sympatry lies within the Mobile River









Basin (Wooten et al., 1988). Successful interspecific hybridization is possible between

G. holbrooki females and G. affinis males, while the alternative hybridization of G.

affinis females and G. holbrooki males does not result in viable offspring (Black and

Howell, 1979). This one-way incompatibility is probably caused by species differences

in the sex chromosomes (Black and Howell, 1979). Gambusia holbrooki do not exhibit

heteromorphic sex chromosomes in either sex, although inheritance patterns of male-

linked melanism suggest an XY system of sex determination (Angus, 1989; Black and

Howell, 1979). Conversely, among G. affinis, females possess heteromorphic sex

chromosomes and males possess homomorphic sex chromosomes indicative of ZW sex

determination (Black and Howell, 1979). According to Scribner and Avise (1994), in

areas where the two species co-exist, G. holbrooki will quickly (within 4 generations)

out-compete G. affinis in terms of mitochondrial and nuclear allele frequency, an

outcome that is likely to be related to the one-way chromosomal incompatibility

described above. Scribner et al. (1999) concluded that offspring from G. holbrooki

females also have some selective advantage. Because G. holbrooki and G. affinis do

hybridize where they co-occur, they were originally considered two subspecies of

Gambusia affinis (thus G. affinis holbrooki and G. affinis affinis). For the reasons above,

the two are now considered separate species (Wooten et al., 1988), but pre-1988 literature

often refers to Gambusia affinis, without indicating the subspecies.

Habitat and Diet

Gambusia holbrooki are small, sexually dimorphic, viviparous omnivores that

inhabit fresh and brackish waterways ranging from ephemeral ponds and ditches, to rice

fields; to streams, ponds, rivers, lakes, springs, estuaries, and marshes (Daniels and

Felley, 1992; McKinsey and Chapman, 1998; Porte et al., 1992; Toft et al., 2003; Vargas









and de Sostoa, 1996). Adult sizes range from 13 to 32 mm for males and 17 to 63 mm

for females (Chapters 2 to 6 of our study; Vargas and de Sostoa, 1996). Although they

will eat algae and detritus, mosquitofish prefer animal prey, including insects, anuran

eggs, crustaceans, rotifers, Daphnia, and round worms (Meffe and Snelson, 1989). The

life expectancy of mosquitofish is generally no more than 2 years (Vargas and de Sostoa,

1996). Although females mature about 20 days later than males (Koya et al., 2003), they

typically grow faster, achieve a larger size, and outlive males (Vargas and de Sostoa,

1996).

Female Reproductive Cycle

Mosquitofish are reproductively active in the spring, summer, and fall in Florida

(our study), Japan (Koya et al., 1998; Koya and Iwase, 2004), and Spain (Vargas and de

Sostoa, 1996); or all year round in Hawaii (Haynes and Cashner, 1995). Female

Gambusia mature about 110 days after they are born (Koya et al., 2003), and produce

sequential, synchronized broods throughout their breeding season: as one brood nears

parturition, the next cohort of oocytes are accumulating yolk (Koya and Kamiya, 2000).

Females store sperm and thus can produce multiple broods, even in isolation from males,

assuming they have mated once before (Hubbs, 1999). This feature contributes to their

success as founder species. Fertilization of oocytes occurs after yolk accumulation, when

eggs exceed 1.7 mm in diameter (Koya et al., 2000). Yolked oocytes become atretic if

they do not reach maturity with their cohort (Koya et al., 2000). Litter size ranges from 1

to 245 precocious offspring, depending on female size and time of year (Chapters 1 and 4

of our study; Vargas and de Sostoa, 1996). Gestation (which takes place within the

single, fused ovary) lasts 22 to 39 days, depending on temperature and photoperiod

(reviewed by Koya et al., 1998). After birth, the larvae live independently of the parents.









Koya and Kamiya (2000) showed experimentally that (in Gambusia affinis

collected from an irrigation canal in central Japan) ovarian recrudescence begins with the

rise in springtime temperature, regardless of daylength. In that population, they found

that vitellogenesis occurred at 140C and pregnancy at 180C. In the fall, vitellogenesis

ended when daylength was less than 12.5 h, regardless of temperature. The final brood

finished its development at a temperature-dependent rate and ovarian regression occurred

after final parturition.

Mosquitofish are typically classified as lecithotrophs because they produce large

yolky eggs (Haynes, 1995). However, lecithotrophy implies that embryo nutrition is

limited to the yolk placed in the oocyte before fertilization. By definition, lecithotrophs

lose weight during gestation because of respiratory losses. However, several studies,

including ours, show that embryos gain in diameter and wet and dry weight during

gestation (Chapters 1 and 4 of our study; Vargas and de Sostoa, 1996). This process is

supported by appropriate changes in liver size that are presumably related to

vitellogenesis (Chapters 1 and 4 of our study). Furthermore, (using Gambusia geiseri)

Marsh-Matthews et al. (2001) showed that maternal transfer of tritiated leucine to

embryos was measurable within 2 hours of injection. Their findings suggest that (in

addition to yolk provisioning) mosquitofish provide directly for their embryos during

gestation via matrotrophy.

Male Reproductive Cycle

Mature poeciliid males are readily identified by their gonopodium (a grooved bony

modification of the anal fin used to transfer spermatozeugmata to the genital opening of

the female). The gonopodium forms during puberty by elongation of anal fin

lepidotrichia and fusion of pterygiophores 3, 4, and 5, with a species-specific









arrangement of hooks at the tip (Rosa-Molinar et al., 1994). This is in contrast with

females, which have a fan-shaped anal fin without fusion of the pterygiophores.

The structure and mechanical control of the gonopodium are supported by the

internal skeleton, which (like the anal fin) becomes masculinized in response to

androgens during puberty (Ogino et al., 2004; Rosa-Molinar et al., 1994, 1996).

Specifically, in males (but not females), the hemal spine at vertebra 13 is resorbed; and

the hemal spines at vertebrae 14, 15, and 16 thicken, elongate, and swing anteriorally

(swing caudally in females). Spines 14, 15, and 16 are connected by interosseal

ligaments (poorly developed in females) to the fused proximal pterygiophores of the anal

fin. Therefore, as the hemal spines bend forward, they pull the anal fin in an anterior

direction. This movement aligns the gonopodium with the male's urogenital opening so

that sperm can be delivered via the gonopodium during mating. The movement also

places the anal fin and its appendicular support at the fish's center of gravity (Rosa-

Molinar et al., 1996). This improves fine-motor control of the gonopodium, which the

male must abduct across his body with enough precision to insert the hooked tip briefly

into the female. The entire copulatory event takes less than a second and is performed

while both fish are in motion (Rosa-Molinar et al., 1996). Lastly, the mature male

skeleton provides support for the large muscle used to maneuver the gonopodium (Rosa-

Molinar et al., 1996).

Males copulate with females frequently, making attempts even before they are fully

mature (Bisazza et al., 1996). Males possess mature spermatozoa about 90 days after

birth (Koya et al., 2003), although some variation among individuals or populations is

likely. In mosquitofish, the testes are fused into a single, round, white-colored organ that









is located centrally in the abdomen, dorsal to the origin of the gonopodium (Fraile et al.,

1992). A single vas deferens connects the gonopodium to the efferent ducts that coalesce

within the central lumen of each testis (Fraile et al., 1992).

The outer wall of the testis is lined with spermatogonia (Fraile et al., 1992). In

spring through fall, spermatogonia proliferate in successive waves of mitosis, forming

nests (cysts) of primary spermatocytes bounded by Sertoli cells (Fraile et al., 1992). In a

process that takes approximately 30 days, spermatocytes within a single cyst undergo

synchronized meiosis and differentiation to produce spermatids and ultimately tailed

spermatozoa (Fraile et al., 1992; Koya and Iwase, 2004). As the cysts mature, they move

from the periphery of the testis to the center, where they are released to the efferent

sperm ducts as spherical aggregates of sperm (spermatozeugmata), with tails in the center

and heads on the periphery (Fraile et al., 1992). At this point, Sertoli cells no longer

surround the spermatozeugmata; instead, they hypertrophy and become part of the

efferent duct tubule (Fraile et al., 1992). The tubules secrete a gelatinous matrix that

holds the spherical structure of the spermatozeugma together until it reaches the oviduct

of a female (reviewed by Meffe and Snelson, 1989). As winter approaches, production of

new spermatocytes ceases. Through the winter, stored cysts of mature spermatozoa

occupy most of the testicular volume, and will be used during early spring copulation,

which occurs before the first wave of spring spermatogenesis is complete (Koya and

Iwase, 2004).

Like most teleosts, male mosquitofish exhibit seasonal variation in sperm

production that is regulated by changes in temperature and photoperiod (Fraile et al.,

1994). However, factors that initiate mosquitofish spermatogenesis are not yet fully









understood. Males that have recently entered testicular quiescence cannot be stimulated

to produce new sperm by increasing temperature or photoperiod (Fraile et al., 1993).

However, fish captured at the end of their quiescent period will proliferate

spermatogonia, even if temperatures remain low and days are short (Fraile et al., 1994).

Increasing ambient temperatures are required for differentiation of spermatogonia to

spermatocytes, and photoperiod must lengthen for spermatocytes to enter meiosis (Fraile

et al., 1994; De Miguel et al., 1994).

Gambusia holbrooki as a Sentinel Species

I chose this prolific species for their small size, wide availability, rapid time to

maturity (about 3 months)(Koya et al., 2003), and viviparity. In addition, endocrine-

disrupting contaminants (EDCs) that impact reproduction are often lipophilic and

therefore bioaccumulate in the fatty tissues of exposed animals (Porte et al., 1992).

Mosquitofish are intermediate in the food web, and are thus likely to bioaccumulate

contaminants absorbed by their prey. Maternal provisioning of the egg, and later the

embryo (in viviparous species like Gambusia) involves transfer of stored lipids from the

mother to the offspring (Meffe and Snelson, 1993). For individuals with high body loads

of accumulated contaminants, this provisioning process can result in an exceptionally

high dose of contaminants (relative to ambient concentrations) to the offspring during the

period of sexual differentiation (Harding et al., 1997).

Developmental exposure to EDCs can permanently alter the organization of the

reproductive system (Guillette et al., 1995). This could result in significant reproductive

problems at the population level. According to Nakamura (1978) and Sone et al. (2005),

Gambusia affinis larvae are sexually differentiated by the time they are born. Although

in another study, Koya et al. (2003) report that, at two days before birth, all Gambusia









affinis embryos are female, with oocytes present in their gonads. At birth, some of the

offspring had developed testes instead. Thus, Koya et al. (2003) suggest that G. affinis

exhibit embryonic protogyny; however, after birth, these fish are gonochoristic.

In addition to certain convenient aspects of their biology (noted above), I chose to

study Gambusia because they are already the subject of a few endocrine disruption

studies. For example, abnormal vitellogenin production occurs in males after exposure to

estrogenic compounds (Tolar et al., 2001). Angus et al. (2002) observed enlarged testes

and livers among male Gambusia inhabiting water contaminated with treated sewage

effluent. Note that the presence of excreted ethynylestradiol, derived from birth control

pills gives sewage effluent an estrogenic character (Schultz et al., 2003). Dreze et al.

(2000) observed skewed sex ratios that favored females, and/or delayed sexual

development in male Gambusia exposed to estrogenic 4-nonylphenol. Orlando et al.

(2002) showed increased ovarian and brain aromatase (converts androgens to estrogens)

activity among adult female mosquitofish caught from the Fenholloway River in Florida.

The Fenholloway River is polluted with paper mill effluent, which in other studies has

been shown to masculinize the anal fins of female mosquitofish (Bortone and Cody,

1999; Howell et al., 1980; Parks et al., 2001). This last example of masculinized anal

fins is one of the more famous early cases of endocrine disruption in fishes and is

described in greater detail below.

In 1978, masculinized female mosquitofish, Gambusia affinis holbrooki, were

discovered in Elevenmile Creek (Escambia County, Florida), downstream from a paper

mill (Howell et al., 1980). The females possessed partially to fully formed gonopodia,

complete with the hooks, spines, and fusion of fin rays 3, 4, and 5. There was no









evidence of gonadal masculinization: masculinized females possessed only ovarian

tissue. In addition, Howell et al. (1980) observed the masculinized sexual behavior of

these females, who pursued normal and masculinized females while swinging and

thrusting their gonopodia. Immature males (12 to 13 mm standard length) from the same

collection sites (downstream from the paper mill) displayed precocious gonopodial

development and more aggressive courtship behavior compared to normal males (Howell

et al., 1980).

Howell et al. (1980) hypothesized that the masculinizing effect of paper-mill

effluent was due to androgenic chemicals in the effluent. This hypothesis is supported by

Turner (1941) and Angus et al. (2001), who induced gonopodial development in

immature female Gambusia affinis with ethynyl testosterone and 11-ketotestosterone,

respectively. Androstenedione, a precursor to testosterone, was later identified in

Fenholloway water (Durhan et al., 2002; Jenkins et al., 2001). However, after running in

vitro tests of androgenic activity, Durhan et al. (2002) concluded that the isolated

androstenedione was not responsible for the androgenic character of Fenholloway water.

Howell et al. (1980) made the interesting observation that female Gambusia are

genetically capable of gonopodial development, but the potential remains dormant in the

normal absence of male androgen levels. This suggestion makes sense in light of the

sexual bipotentiality of mosquitofish embryos described above.

Overview of Reproductive Endocrinology in Fishes

Although gonadal activity is often the focus of teleostean fish reproduction studies,

reproduction really begins in the hypothalamus. In response to environmental stimuli

such as photoperiod, the hypothalamus secretes gonadotropin releasing hormone

(GnRH), which causes the anterior pituitary to release two gonadotropic hormones









(GTH) (Norris, 1997). These are sometimes called GTH-I, similar to mammalian

follicle-stimulating hormone (FSH); and GTH-II, similar to mammalian luteinizing

hormone (LH). FSH stimulates spermatogenesis and oogenesis; LH causes final gamete

maturation and ovulation or sperm release (Norris, 1997). In both ovaries and testes, LH

affects gametes by stimulating the synthesis of progesterone-derived 17,20p-dihydroxy-

4-pregnen-3-one (17,20p-P) (Kobayashi et al., 1993; 2002).

In addition to gamete production, LH and FSH also stimulate gonadal

steroidogenesis in teleosts (Norris, 1997; Schulz and Miura, 2002). The three steroids

relevant to our study are estradiol-170, testosterone, and 11-ketotestosterone (11-KT).

Although all three hormones occur in both sexes, we focused on estradiol in females and

testosterone and 11-KT in males. In females, estradiol promotes sexual maturation,

gonadal growth, hepatic vitellogenesis (yolk precursor production), and oogenesis

(Kobayashi et al., 2002; Norris, 1997). In males, testosterone promotes sexual

maturation, development of secondary sex characters, spermatogenesis (particularly

toward the end), sperm quality, spawning, and sexual behavior (Norris, 1997, Ogino et

al., 2004; Toft et al., 2003, Wu et al., 2003). It is likely that 11-KT also participates in

these processes; it is best known for its ability to induce spermatogonial proliferation,

which usually is not accomplished by testosterone (Schulz and Miura, 2002).

Lakes

For the studies described in Chapters 1 through 3, we sampled fish from Lake

Apopka and Lake Woodruff in central Florida. These lakes differ in terms of their

ecology and contaminant loads; some of the main differences are highlighted below. We

selected these lakes because they are geographically close, and thus subject to the same

photoperiod. Furthermore, our lab has previously published detailed contaminant data









coupled with comparative reproduction studies of alligators from these two lakes

(Guillette et al., 1999; Guillette et al., 2000). These earlier findings stimulated the

hypotheses tested in our study.

Lake Apopka

Lake Apopka is impacted by a nearby (1 mile away) EPA-designated Superfund

site. Superfund status indicates that the area is contaminated with uncontrolled hazardous

waste and poses a recognized risk to the environment or human health. Main pollutants

in Lake Apopka include polychlorinated biphenyls (PCBs) and several organochlorine

pesticides, including Dicofol, DDT and its metabolites p,p'-DDD and p,p'-DDE,

Dieldrin, Endrin, Mirex, Methoxychlor, Chlordane, Toxaphene, and trans-Nonachlor

(Guillette et al., 2000). These chemicals were washed into the lake from agricultural

lands, or the Superfund site, where a Dicofol (15% DDT) spill occurred in 1980. The

chemicals listed here have been identified and measured in both alligator eggs and

alligator serum taken from animals living in Lake Apopka (Guillette et al., 2000; Heinz et

al., 1991). In addition, elevated concentrations of several of these compounds have been

measured in the tissues of mosquitofish (U.S. Fish and Wildlife Service, unpubl. data)

and brown bullheads (Gallagher et al., 2001) from Lake Apopka.

In mosquitofish, contaminant concentrations are around 0.17 mg/kg for DDT, 9.2

mg/kg for Toxaphene, 1.1 mg/kg for p,p'-DDD and 0.54 mg/kg for trans-Nonachlor

(Greg Masson, U.S. Fish and Wildlife Service, pers. comm.). Alligator egg dosing

studies of 0.1 to 10 mg/kg of DDE, DDD, or trans-Nonachlor have caused alterations in

sex determination, endocrine function, secondary sex characteristics and/or gonadal

anatomy (Crain, 1997; Matter et al., 1998; Rooney, 1998). Compared to cohorts from

Lake Woodruff, female alligators from Lake Apopka exhibit above-normal plasma









estradiol concentrations and abnormal ovarian morphology (with large numbers of

polyovular follicles and polynuclear oocytes). Male alligators have lower-than-normal

plasma testosterone concentrations, poorly organized testes, and abnormally small phalli

(Guillette et al., 1994). In addition, alligator eggs from Lake Apopka exhibit low

hatchability; and neonates show increased rates of mortality, poor motor coordination,

changes in metabolism (particularly in liver and steroidogenic enzymes), and altered gene

expression (reviewed in Guillette et al., 2000; Guillette and Gunderson, 2001). Given

these previous studies, it is reasonable to expect that Gambusia in Lake Apopka are

subject to reproductive alterations in association with contaminant exposure.

Lake Woodruff

Lake Woodruff is a National Wildlife Refuge. While not contaminant-free,

alligators captured from Lake Woodruff (relative to Lake Apopka) have fewer chemicals

in their body tissues; and those chemicals occur at lower concentrations (Guillette et al.,

1999). For this reason, our laboratory has traditionally used Lake Woodruff as a

reference site. In addition to contaminant concentrations, other water-quality measures

also distinguish the two lakes. Based on our data (in conjunction with average data for

2000 to 2003, taken from EPA's STORET public-access database,

http://www.epa.gov/storet/dbtop.html), Lake Woodruff has a different seasonal

temperature profile that favors cooler temperatures in fall and spring, lower nitrogen and

phosphorus concentrations, greater water clarity (Secchi depth), and lower turbidity and

total suspended solids relative to Lake Apopka. In evaluating the results of our study, we

considered these ecological differences, and also considered lake-associated variation in

contaminant concentrations.









Florida Springs and Nitrate

Overview

Over the past 40 years, concentrations of nitrate (NO3-N) in several of Florida's

artesian springs have increased from less than 0.1 mg/L to more than 5 mg/L (Katz,

2004). The highest measured concentration was 38 mg/L NO3-N in a small spring along

the Suwannee River in northern Florida (Katz et al., 1999). This is almost four times the

EPA drinking-water standard of 10 mg/L NO3-N. Most of the nitrate comes from

inorganic fertilizers applied to land, ultimately leaching through the ground to recharge

Florida's aquifer (Katz, 2004). A potentially important ecological problem often caused

by increased nitrates is eutrophication, which can increase algal and plant growth. The

excessive flora can cause fluctuations in aquatic light levels and dissolved oxygen

concentrations, thus affecting survival and diversity of aquatic organisms and overall

community structure (Attayde and Hansson, 1999; Capriulo et al., 2002; Irfanullah and

Moss, 2004).

Apart from the negative ecological effects of eutrophication, nitrate can also

directly harm animals living in affected aquatic systems. These effects range from gross

toxicity to subtle, but equally alarming, changes in physiology and development

(Guillette and Edwards, 2005). For example, mortality of the larvae of cutthroat trout,

Chinook salmon, and rainbow trout occurs at NO3-N concentrations ranging from 2.3 to

7.6 mg/L (Kincheloe et al., 1979). Survival of chorus frog and leopard frog tadpoles

decreased significantly after exposure to 10 mg/L NO3-N (Hecnar, 1995). This

concentration is considered the upper limit for safety in drinking water (EPA, 1996). On

the other hand, the 96 h median lethal concentration (LC50) for fathead minnow larvae is

1,341 mg/L NO3-N; while it is 462 mg/L NO3-N for adult Daphnia magna (an









invertebrate) (Scott and Crunkilton, 2000). These examples show that sensitivity to

nitrate varies greatly among species, and often depends on the stage of development at

the time of exposure.

The best-known human health effect of nitrate is methemoglobinemia (blue-baby

syndrome) (Gatseva et al., 1996; Scott and Crunkilton, 2000). This condition occurs

when nitrate interacts with hemoglobin in the blood, causing it to crystallize. A similar

condition, called brown blood disease, occurs in fishes exposed to high nitrite levels.

Tissue hypoxia and cyanosis result because the crystallized hemoglobin cannot function

as an oxygen carrier. Risks associated with methemoglobinemia are the reason

maximum nitrate concentration in drinking water is regulated at 10 mg/L N03-N (EPA,

1996). In addition to methemoglobinemia, nitrate and nitrite have been implicated in

mild hepatic degeneration in rats (Gatseva et al., 1999); reduced steroidogenesis in rats

(Panesar, 1999; Panesar and Chan, 2000), frogs (Barbeau, 2004), and alligators (Guillette

and Edwards, 2005); decreased human sperm motility; and increased human sperm

mortality (Rosselli et al., 1995).

Nitrogen Cycling, In Vivo Nitrogen Metabolism, and Effect of Nitrate on
Steroidogenesis

Nitrogen is naturally cycled in terrestrial and aquatic ecosystems. For example,

ammonia excreted by fishes is converted to nitrite (NO2) by aerobic nitrifying bacteria

(Nitrosomonas sp.), and then oxidized to more stable nitrate (NO3) by Nitrobacter

bacteria. Nitrate is assimilated by plants as a nutrient, or can be converted back to nitrite

and then atmospheric nitrogen (N2) by anaerobic denitrifying bacteria. In anoxic

environments, or when the nitrifying or denitrifying activity of bacterial populations is









overwhelmed (as can result from overfeeding in aquaculture systems), nitrite levels can

spike, placing fish populations at immediate risk for brown blood disease.

In vivo, conversions between nitrate and nitrite also occur. Nitrate and nitrite enter

the bodies of freshwater animals by crossing the gill epithelia and accumulating in

extracellular fluid (Jensen, 1995). In crustaceans, nitrate and nitrite are transported

against the concentration gradient by substituting for chloride in the bicarbonate-chloride

exchange mechanism that normally participates in the osmoregulatory and respiratory

functions of the gill ( Jensen, 1995; Lee and Pritchard, 1985). In crayfish, nitrate uptake

is pH dependent with uptake increasing as water pH declines (Jensen, 1995). In vivo,

NO3 can be converted to nitrite (NO2), and then nitric oxide (NO) (Panesar and Chan,

2000; Samouilov et al., 1998). Nitrite is converted to NO in various ways, including

endogenous nitrite reductase (NR), nitric oxide synthase (NOS), various non-NOS

enzymatic and non-enzymatic mechanisms, and low pH (Cadenas et al., 2000; Doblander

and Lackner, 1996; Kozlov et al., 1999; Lepore, 2000; Meyer, 1995; Nohl et al., 2001;

Panesar and Chan, 2000; Samouilov et al., 1998; Stuehr and Marletta, 1985; Vanin et al.,

1993; Weitzberg and Lundberg, 1998; Zweier et al., 1999). Nitric oxide is a gas that

diffuses through tissues, playing diverse roles in vasodilation, cell-to-cell signaling,

neurotransmission, and immunity. One specific action of NO is the inhibition of steroid

hormone synthesis (DelPunta et al., 1996; Kostic et al., 1998; Panesar, 1999; Panesar and

Chan, 2000; Vanvoorhis et al., 1994; Weitzberg and Lundberg, 1998).

In normal steroidogenesis, free cholesterol is taken into the mitochondria and

converted to progesterone (the precursor of testosterone, 11-ketotestosterone, and

estradiol). This involves steroidogenic acute regulatory protein (StAR) and cytochrome









P450 enzymes such as P450-sidechain cleavage enzyme (SCC) and 3P-hydroxysteroid

dehydrogenase (33HSD). Nitric oxide inhibits these enzymes, with the result that steroid

hormone production is reduced (Panesar and Chan, 2000). This has serious implications

for reproduction and development, since both directly depend on appropriate hormone

levels.

Effects of Nitrates on Sperm Motility and Viability

Effects of nitrate or nitric oxide (NO) on sperm motility and viability have been

investigated only recently, and results are conflicting. Certainly, if steroidogenesis is

inhibited by nitrate, nitrite, or NO, then spermatogenesis could be affected since both

androgens and estrogens are required for spermatogenesis (Cochran, 1992; Hess et al.,

1997; Miura et al., 1991; Vizziano et al., 1996). Additionally, NOS and NO are

associated with activation of eggs and sperm (acrosome reaction) (Kuo et al., 2000;

Revelli et al., 2001). Rosselli et al. (1995) found that human sperm incubated with NO

have decreased motility and increased mortality. The percentage of immotile and dead

sperm also correlated positively with nitrite-nitrate levels in the seminal plasma of the

sperm donors (Rosselli et al., 1995). Furthermore, bulls exposed orally to nitrates (100 to

250 g/day/animal) showed reduced sperm motility, increased sperm abnormalities, and

degenerative lesions in the spermatocyte and spermatid germ layers of the testis (Zraly et

al., 1997).

Summary of Nitrate's Effects on Reproduction

Evidence in the literature shows that nitrate can affect sperm quality and the

synthesis of sex steroid hormones, with possible deleterious down-stream effects on other









reproductive variables, which rely on appropriate hormone concentrations. Hypotheses

based on this evidence are investigated in the 4th and 5th chapters of our study.

Review of Endocrine Disruption in Fishes

Table 1-1 shows known effects of endocrine disruption in fishes. To date, more

data are available regarding effects on males than on females. Although numerous

studies of nitrate-mediated endocrine disruption are published for mammals, this area is

understudied in fishes.

Hypotheses and Goals

Based on the review presented here, we hypothesized that adult female and male

Gambusia holbrooki, captured from sites with high organochlorine concentrations (Lake

Apopka) or high nitrate (springs), would exhibit altered reproductive parameters

compared with reference or low nitrate sites. Based on previous studies (Table 1-1), we

expected to observe increased hepatosomatic index, reduced gonadosomatic index,

altered steroid hormone profiles (estradiol in females; testosterone and 11-

ketotestosterone in males), reduced embryo number, decreased embryo weights; poor

sperm quality; and diminished gonopodial length among fish from contaminated sites. In

addition to our assessment of possible endocrine disruption in the sampled populations,

we expected to observe seasonal variation in reproduction related to changes in

temperature and photoperiod.

Overall, our goal was to understand the potential for endocrine disruption in our

study systems in context of the seasonal reproductive cycle and some aspects of

ecological variation. We measured variables that are informative in terms of basic

reproductive biology of Gambusia, are likely targets of endocrine disruption (Table 1-1:

note parameters highlighted in gray), and that, if disrupted, could affect fitness. Field






20


studies such as mine are useful because they measure reproductive characteristics in the

wild, under natural conditions that are impossible to replicate in the lab. Thus, they can

be used to generate hypotheses regarding any observed variation in reproduction, and

suggest causal mechanisms. However, it is understood that, without complementary

experimental studies that test field-generated hypotheses, field studies are correlative:

they suggest, but do not show cause and effect.










Table 1-1. Endocrine disruption in fishes
Class of Sample Compounds Observed Alterations in Reproduction References
Endocrine Caused By or Associated With
Disruptorst Endocrine Disruptor Exposure


Estrogen


Estradiolghrst"
4-Tert-
pentylphenolfgh
4-Tert-octylphenolsx
Octylphenolt
P-nonylphenola
4-Nonylphenolde
Nonylphenol"
Endosulfanw
b
Keponeb
DDDbyz
Bisphenol A1
PCBs'yz
Treated sewage
effluentklm P
Chlordaneqyz
Chronic hypoxia (1 +
0.2 mg/L)v
Toxapheney"
DDEyz
Organochlorine
III\tILII'


TPlasma vitellogenin ghlm"ps
tHepatosomatic indexit 6
TOvotestes/intersexaghk"qs"
tOviduct formationgfkm
,Delayed puberty/persistent
immature tested 5
,Gonadosomatic indextux
<->Sertoli cell structuret 5
4Gonadal development $
,Diameter of seminiferous tubulesh 5
TAtrophy of germinal epitheliumh 5
,Primordial germ cell numberfg 5
*->PCG distribution in developing
gonadw
TMalformed germ cells' (intersex
fish)
,Delayed spermatogenesisghn 5
TLoss of spermatogenic cystsh 5
,Sperm Countsknz 1
,Milt volumek"t 5 (intersex fishes)
,Sperm motilitykv" (intersex fishes)
TOccluded reproductive ductsnt 5
(intersex fishes)
,<->Delayed gonopodial
developmentcdo
,Genital papilla length' 5
,Adult coloration 5
,Courtship behavior'
,Fertilization successk' 5 (intersex
fishes)
,Embryonic/larval survivale"uv Y
$Oocyte maturationb
TOocyte atresia"
tEmbryo growths 9
,Ca", amino acid availability to
fetuses during gestations
,Delayed hatching"v Y
1E2 binding in livers
$Serum E2, T
tPlasma T, 11-KTY
-> Plasma E2,T" (intersex fish)
tSerum E2vy
,Serum T, 11-KTPuv
117, 20-DHP"u


aBamhoom et al., 2004
bDas and Thomas, 1999
cDoyle and Lim, 2002
dDreze et al., 2000
eFairchild et al., 1999
fGimeno et al., 1997
gGimeno et al., 1998a
hGimeno et al., 1998b
'Haubruge et al., 2000
'Nakayama et al., 2005
kJobling et al., 2002b
1Lye et al., 1997
mRodgers-Gray et al.,
2001
"Jobling et al., 2002a
oBatty and Lim, 1999
PFolmar et al., 1996
'Harshbarger et al.,
2000
rKirby et al., 2003
sRasmussen et al., 2002
tRasmussen and
Korsgaard, 2004
"Schultz et al., 2003
VWu et al., 2003
wWilley and Krone,
2001
xToft and Baatrup,
2001
YGallagher et al., 2001
'Toft et al., 2003









Table 1-1 Continued
Class of Sample Compounds Observed Alterations in Reproduction References
Endocrine Caused By or Associated With
Disruptorst Endocrine Disruptor Exposure
Anti- Vinclozolinabc" 4Sperm countab"ce "Baatrup and Junge,
Androgen DDEabde Fertilization success" 2001
Flutamideab ,Adult colorationabc" bBayley et al., 2002
,GSIa "cBayley et al., 2003
,Courtship behaviorabc" dGallagher et al., 2001
DDelayed maturation eToft et al. 2003
,Gonopodial development o
1Serum E2d
1Plasma T, 11-KTd
Androgen 11-ketotestosteroneb tGonopodial d I, i 'p lii 9 aHowell et al., 1980
Paper mill Male biased sex ratio bAngus et al., 2001
effluentacdef Male colorationf 9 Bortone and Cody,
Methyl-testosteronefg Number of reproductive femalesf 1999
lIntersexg Y o dJenkins et al., 2001
eLarsson and Forlin,
2002
fLarsson et al., 2002
gHahlbeck et al., 2004
Aromatase* Tributyltinabcd Male-biased sex ratiod $5 aHaubruge et al., 2000
Inhibition $Sperm counts $ bRurangwa et al., 2002
1Sperm lacking flagellad "cNakayama et al., 2005
,ATP content of sperm 5 dMcAllister and Kime,
,Lactate dehydrogenase activity in 2003
sperm
,Sperm motilitybd
,Fertilization success" $
,Hatchabilityc 9$
,Embryo survivorship" 9
Other Landfill leachateab Male-biased sex ratioab aNoaksson et al., 2001
,GSIab bNoaksson et al., 2004
,Brain aromatase activity
,Plasma T, E2a
dDelayed vitellogenesisb
Nitratec ,Spawningc" cShimura et al., 2002
,Egg number
tFertilization rate" 9
,Delayed hatching time" 9
,Hatching rate of the eggs" 9










Table 1-1 Continued
tEndocrine disruptor classification is general and non-exclusive. Several chemicals operate through a
number of mechanisms that vary with context. For example, DDE has been called an estrogen, an
anti-estrogen, and an anti-androgen. Classifications presented here depend on the papers cited.
Organochlorine mixture refers to the chemical mixture detected in the plasma of juvenile alligators
from Lake Apopka. The mix includes PCBs, DDE, DDD, mirex, endrin, dieldrin, trans-nonachlor,
and oxychlordane. These chemicals cause male to female sex reversal of reptile embryos (reviewed
by Guillette et al., 2000)
Aromatase catalyzes the conversion of testosterone to estradiol, and androstenedione to estrone
(Johnson and Everitt, 1995).
Italicized descriptors refer to papers on Gambusia species. Superscripts match sample compounds,
observed effects, and author citations. Highlighted descriptors are related to hypotheses tested in our
study. = female. = male. (Intersex fish(es)) = the sex of the fish(es) in the cited study was an
abnormal mix of female and male. = increase. 4 = decrease or inhibition. <- = altered. 11-KT 11-
ketotestosterone. ATP = adenosine triphosphate. DDD, DDE = metabolites of the insecticide DDT.
GSI = gonadosomatic index. E2 = estradiol. HSI = hepatosomatic index. PCBs = polychlorinated
biphenyls. PGC = primordial germ cell. T = testosterone. T3 = tri-iodothyronine. 17, 20-DHP = 17a-
2013-dihydroxy-progesterone.














CHAPTER 2
TEMPORAL REPRODUCTIVE PATTERNS FOR FEMALE MOSQUITOFISH
CAPTURED FROM TWO FLORIDA LAKES

Introduction

In the field of endocrine disruption, Howell et al. (1980) first recognized

mosquitofish as a model species when they reported that females living downstream from

paper mills exhibited masculinized anal fin development. Since then, a handful of other

studies have been published (i.e., Dreze et al., 2000; Porte et al., 1992; Toft et al., 2003;

Toft and Guillette, 2005), suggesting that mosquitofish are a valuable model.

In part, the value of mosquitofish (Gambusia holbrooki and their western sister,

Gambusia affinis) as sentinel species is associated with their ubiquitous global

distribution, which has resulted from their widespread use as a biological control agent

for mosquitoes (Courtenay and Meffe, 1989). This practice continues today (based on a

May 2005 internet search of government pest control programs), despite the fact that

most countries with introduced populations have found them to be ineffective,

undesirable, or both (Courtenay and Meffe, 1989). The "undesirability" is due to

Gambusia's extraordinary ability to adapt and proliferate in new environments

(Courtenay and Meffe, 1989). Introduced Gambusia can have broad negative impacts on

the biota of aquatic ecosystems. They harass or out-compete native fishes, and eat a wide

variety of aquatic invertebrates, as well as the eggs, larvae, or adults of many anuran and

fish species, including food and sport fishes (reviewed by Arthington and Lloyd, 1989).

Outside their native range, these activities disturb habitats, cause economic losses, and,









ironically, can eliminate other native mosquito predators, which are often more

efficacious (Courtenay and Meffe, 1989; Danielson, 1968).

Mosquitofish are successful because they breed continuously during the

reproductive season, producing several sequential and slightly overlapping broods of 1 to

245 precocious offspring at approximately 22-39 day intervals, depending on

environmental temperature (reviewed by Koya et al., 1998). A second reason for their

success is their ability to tolerate poor water quality, high levels of pollution, and ongoing

habitat disturbance by humans (Courtenay and Meffe, 1989). In addition, their generalist

diets (which expose mosquitofish to a variety of potential contaminant sources) and

intermediate position in the food web suggest that they bioaccumulate high

concentrations of lipophilic contaminants (like PCBs) relative to their environment. This

aspect of their biology is useful in biomonitoring programs that assess biological effects

of anthropogenic contaminants. For example, Porte et al. (1992) detected seasonal

variation in PCBs and organophosphate (OP) pesticides in Gambusia muscle tissue that

reflected seasonal inputs of these chemicals to the Ebro Delta in Spain, an area that is

heavily used for rice farming. In that study, the authors noted that PCB concentrations

were low in females sampled bi-monthly during the April November breeding season,

but increased significantly in December and February, when females are reproductively

quiescent. Given that mosquitofish produce large (1.5 to 2 mm (Vargas and de Sostoa,

1996)), yolky oocytes; this indirect evidence suggests that female mosquitofish can

lighten their body contaminant loads by offloading contaminants as they lose fat reserves

to yolk production. Moreover, during gestation, female Gambusia have been shown to

transfer radiolabeled leucine and 4-nonylphenol (a xenoestrogen) to their offspring









following maternal exposure (Marsh-Matthews et al., 2001; Thibaut et al., 2002). In

other fish species, when females bioaccumulate contaminants, they typically pass those

contaminants to their offspring in doses that are concentrated, relative to ambient

concentrations (Harding et al., 1997; Nakayama et al., 2005). This can affect maternal

fitness by impacting offspring survival, development, or fertility (Black et al., 1998;

Nakayama et al., 2005; Rasmussen et al., 2002; Saiki and Ogle, 1995).

Given the global potential of Gambusia as model organisms for examining the

effects of environmental contaminants on reproduction, coupled with their ecological and

economic impacts, as described above, our study was undertaken to learn more about

seasonal/temporal patterns of reproduction among female Gambusia holbrooki in their

native range. We collected mosquitofish from two lakes in central Florida: Lake Apopka

is eutrophic, with a 50-year history of agricultural, municipal, and industrial

contamination, whereas Lake Woodruff National Wildlife Refuge is oligotrophic, is less

impacted by human activities, and served as a reference site for a number of related

studies (e.g. Guillette et al., 2000). Mosquitofish exhibit great genetic diversity coupled

with a potential for phenotypic plasticity in life history characters (Downhower et al.,

2000; Haynes and Cashner, 1995; Meffe et al., 1995; Stockwell and Vinyard, 2000).

These features contribute to the success of Gambusia as an introduced species (Greene

and Brown, 1991). Therefore, the second goal of our study was to assess reproductive

variation between the two lake populations in context of lake-associated environmental

differences.









Methods

Field and Tissue Collections

Between March 2001 and June 2002, 16 monthly collections of adult female

Gambusia holbrooki were made from Lake Apopka (north shore, near Beauclair Canal)

and Lake Woodruff Wildlife Refuge (northwest shore, Spring Garden Lake) in central

Florida, USA. Fish of mature size (> 1.7 cm, based on our experience) were captured

using a 3-mm mesh dip net. Fish maturity was verified in the laboratory from the

presence of clearly differentiated white or yolked oocytes in the ovary (Haynes, 1995).

Fish were considered reproductively quiescent (mature, but not pregnant) at stage 0,

when no yolk was visible in the oocytes (Fig. 2-1). Each lake collection took 1 day, plus

additional days to process fish. Thus monthly collections from the two lakes were made

on different days (average of 4 days apart).

Each monthly sample, ranging from 39 60 fish per lake, was divided into two

subsets. The first subset was used to obtain ovarian and hepatic weight, embryo number,

and embryo stage. These fish (n = 23 31 per month, per lake) were held live in aerated

coolers filled with lake water, fed flake fish food ad libitum, and processed within 1-2

days of capture. Before necropsy, fish were over-anesthetized in room-temperature water

containing 0.1% MS222 (3-aminobenzoic acid ethyl ester, methanesulfonate salt, Sigma

#A5040). The ovary (0.1 to 322 mg) and liver (0.1 to 31.5 mg) were removed and

weighed to the nearest 0.1 mg. Embryos (stage 3 and greater) were separated from the

ovarian stroma, counted, and staged according to Haynes (1995) (Fig. 2-1). Embryos

were staged at half stages (e.g., 4.5) if they were transitional between two stages.

Unfertilized oocytes that were younger than stage 3 were staged, but not counted. Note

that, within a brood, embryos are generally synchronized in their development. Average









embryo wet weight for each brood was calculated as ovarian weight divided by embryo

number. Note that this value is exaggerated slightly for larvae just before birth (stage 9 -

10) because, at that time, oocytes for the next brood have started to accumulate yolk, thus

adding weight to the ovary that is not related to the counted embryos.

The second subset of captured fish was used for measurement of muscle estradiol

concentrations. These fish (n = 7 12 per month, per lake; mean = 10) were frozen

immediately at capture and held on ice while in the field. Upon return to the laboratory,

the caudal fin was removed, and all caudal peduncle tissue posterior to the gonad was cut

from the fish, weighed (average = 71 mg), and frozen (-800C). Estradiol was measured

on lipid extracts of these peduncle tissues, as described below. Because the caudal

peduncle is primarily muscle, we will refer to it as muscle for the remainder of the

chapter. Heppell and Sullivan (2000) showed that seasonal patterns of muscle and

plasma estradiol concentrations were comparable in female gag grouper, although actual

concentrations in muscle (measured as pg/g) were an order of magnitude lower than

plasma concentrations (measured as ng/ml).

For all fish (both subsets), standard length (SL) was measured to the nearest 0.01

cm from the snout tip to the caudal peduncle using calipers. Fish were blotted dry and

weighed with an electronic balance to the nearest milligram.

Water temperature, pH, and conductivity data were obtained at the time and

location where fish were sampled, using a handheld Ultrameter (Model 6P, Myron L

Company, Carlsbad, CA). To describe additional water quality parameters for the two

lakes, we referred to STORET, a public-access EPA database of environmental

measurements (http://www.epa.gov/storet/dbtop.html). We gathered data from 2000 -









2003 on nitrogen and phosphorus concentrations, secchi depth, turbidity, and total

suspended solids. It should be noted that the usefulness of this database is limited by the

relatively few sampling dates recorded each year: Lake Apopka was sampled 5 times in

2000, and once in each of 2001-2003; Lake Woodruff was sampled 6 times in 2000,

twice in 2001, once in 2002, and 3 times in 2003. Our lake comparison based on

STORET data reflects the average values for measurements made in 2000 2003 and

thus provides a helpful, but generalized view of water quality in each lake.

Muscle Estradiol Measurements

For measurement of muscle concentrations of 17-0-estradiol, frozen peduncle

tissues were thawed in glass tubes, on ice, and homogenized in 1 ml 65mM borate buffer

(pH 8.0). Homogenate was extracted twice with 5 ml diethyl ether. For each extraction,

ether and homogenate were mixed together for two minutes using a multi-tube vortex

mixer. For the first extraction, tubes were allowed to settle for three minutes to separate

phases. For the second extraction, phases were separated by centrifugation for two

minutes. The aqueous phase was frozen in a methanol bath chilled to -250C with dry ice.

The ether from both extractions was combined in a second glass tube and evaporated

under dry forced air.

Hormone concentrations were determined using validated enzyme immunoassay

(EIA) kits (Cat No. 582251) purchased from Cayman Chemical Company (Ann Arbor,

Michigan). Dry extract was reconstituted in 500 [tl EIA buffer so that samples would fall

within the range of the standard curve. EIAs were run as recommended by Cayman with

an 18 h refrigerated incubation to increase sensitivity. Data were quantified against a

standard curve linearized using a logit transformation of B/Bo (bound sample/maximum









bound). Duplicate or triplicate interassay variance (IAV) samples at two dilutions were

included with each plate. The coefficient of variance among all plates, averaged for the

two dilutions was 22.6%. To normalize sample estradiol concentrations across assays,

we multiplied by a correction factor derived from the relationship between individual

plate IAV values and the mean IAV values for all plates.

Statistics

Estradiol data points lying more than three standard deviations from the mean for

all samples (n = 9, 3%) were excluded from the data set. Using a correlation matrix, data

were screened for correlations among standard length (SL), body weight, ovarian and

hepatic weight, embryo number, embryo wet weight, and embryo stage. We also looked

for correlations between maternal body size and muscle estradiol concentrations.

Apparent correlations were visualized using linear regression following logo

transformation of the variables. Embryo number was positively related to maternal SL

(r2 = 0.44, p < 0.0001). Likewise, hepatic weight was positively related to maternal body

weight (r2 = 0.57, p < 0.0001). Therefore, mean embryo number and hepatic weight were

adjusted for body size (using ANCOVA) when appropriate.

We used ANOVA or ANCOVA to compare mean response variable values

between consecutive months within a lake and to compare mean response variable values

between lakes in each month. Changes in embryo wet-weight and adjusted hepatic

weight during gestation were similarly compared between stages within a lake, and

between lakes at each stage. Correlation, regression, and ANOVA analyses were

completed using Statview 5.0. ANCOVA analyses and calculations of adjusted means

were completed using SPSS 13.0. Results were considered significant at p < 0.05.









Gonadosomatic Index

Gonadosomatic index (GSI) is a traditional measurement of fish ovarian weight

relative to body size. Mean GSI is often used to indicate temporal changes in female (or

male) reproductive status. However, in viviparous mosquitofish, GSI is not an accurate

measure, particularly in a seasonal study that invokes mean GSI values to describe

reproductive trends. This is because females are not reproductively synchronized as a

group: in any given month during the reproductive season, females exhibit all stages of

embryonic development (Fig. 2-2) (although within a female, the embryos are

synchronized). Furthermore, ovarian weight is determined in part by embryo number,

which varies with maternal body size. For these reasons, we do not consider GSI a valid

measurement for female mosquitofish, particularly when evaluated as an average value,

and thus have not included it as a response variable.

Results

Environmental Differences between Lakes

Water temperature, which exhibited a seasonal pattern, was similar between the

two lakes, except at the end of fall and beginning of spring, when the temperature of Lake

Apopka was about 5C warmer (Fig. 2-3). Mean conductivity, which differed slightly

between lakes, ranged from 430 to 766 [tS (mean = 591 tS) in Lake Apopka and from

700 to 1800 [tS (mean = 1231 [tS) in Lake Woodruff. However, this difference is

unlikely to be biologically relevant to osmotic balance (1800 [tS < 4% seawater). Lake

pH fluctuated monthly at both sites, ranging from 6.78 to 8.73 (mean = 7.62) in Lake

Apopka and 6.47 to 8.47 (mean = 7.22) in Lake Woodruff. Fluctuations in pH did not

follow a predictable pattern in either lake, and thus we could not detect any particular

effect of pH on the measured variables relevant to our study. Additional descriptive









information on lake water quality is shown in Table 2-1. On average (2000 to 2003),

Lake Woodruff has lower nitrogen and phosphorus concentrations than the more

eutrophic Lake Apopka. Lake Woodruff also has greater water clarity (Secchi depth) and

lower turbidity and total suspended solids. Our visual observations of water clarity in the

two lakes corroborate these data.

Temporal and Lake-Associated Variation in Response Variables Related to
Reproduction

Body size

In general, the adult female mosquitofish collected from Lake Apopka were bigger

than those from Lake Woodruff (Fig. 2-4). Mean body size of captured females from

both lakes was higher at the beginning of the reproductive season, and lower at the end of

the year. This change could reflect fall recruitment of newly matured fish born earlier in

the year (which, although mature, will not finish growing until the next spring). We

observed that 1.7 cm was the smallest size for mature females from either lake,

suggesting that this is the minimum size for female reproductive activity. However, all

fish at this size need not be mature, as we did observe immature fish that were up to 2.0

cm in standard length.

Temporal changes in reproductive activity

Females collected from both lakes were reproductively active (> 90% pregnant)

during the spring summer and fall, and quiescent for at least two months during the

winter (Figs. 2-2 and 2-5). The timing of spring recrudescence was different between the

two sample populations, with females from Lake Woodruff being delayed by 2 to 3

months (Fig. 2-5). In addition, spring reproductive recrudescence was more

synchronized in females from Lake Apopka compared to those from Lake Woodruff,









which were more variable in their timing of reproductive onset (Fig. 2-5). This

difference in timing is probably due to spring temperature differences between the lakes

(Fig. 2-3), as Lake Apopka warms up more rapidly in the spring. The observed pattern

suggests that temperature, rather than photoperiod, is the cue responsible for spring onset

of reproductive activity in mosquitofish.

Embryo number, size, and stage of development

In sample populations from both lakes, embryo number was significantly (p <

0.0001) and positively related to female standard length (SL), although the strength and

slope of this relationship was different between females from the two lakes (Apopka: r2

= 0.54; Woodruff: r = 0.14; ANCOVA indicated a significant interaction between lake

and SL, p < 0.0005 ) (Fig. 2-6). In Lake Apopka, large females produced many more

offspring than small females, a trend that was not as strong among females from Lake

Woodruff (Fig. 2-6). On average, females from Lake Apopka produced 12.5 embryos

per brood, whereas females form Lake Woodruff produced only 5.1 embryos per brood.

Mean litter size (adjusted for maternal standard length) was typically larger among

females from Lake Apopka, except at the beginning and end of the reproductive season,

when litter sizes were similar in the two lakes (Fig. 2-5).

Because litter sizes were larger among females from Lake Apopka, it is reasonable

to hypothesize that a tradeoff between embryo number and embryo size exists such that

Apopka embryos might be smaller than Woodruff embryos. This, however, was not the

case. Although all embryos gained wet weight as they developed, there was no lake-

associated difference in embryo wet-weight at birth, based on ANOVA (Fig. 2-7). We

did observe lake related differences in embryo wet weight between stages 9 10,









although this could be an artifact of the method of wet weight calculation, as explained

above in Methods.

Hepatosomatic index

We observed that mean hepatic weight (adjusted for female body weight) was

dependent on embryonic stage, an association that is probably related to vitellogenesis.

For maximum sample size, we combined fish from both lakes and noted that adjusted

hepatic weight increased from stage 0 to stage 2.5, declined gradually through stage 6 -

8, and rose again between stages 8 and 10.5 (Fig. 2-8A). Note that females in transition

between broods do not exhibit stage 0 (reproductive quiescence, no yolked oocytes

present) as broods overlap slightly (Fig. 2-1, see photographic panel for stage 2). When

HSI data were split between lakes, we observed a similar gestation-related pattern in HSI

among females from both lakes, although adjusted hepatic weight was often higher

among females from Lake Apopka (Fig. 2-8B).

Estradiol

Temporal variation in female muscle estradiol (E2) concentrations was evident and

often differed between the two lake populations (Fig. 2-9). Females from Lake Apopka

exhibited a significant decline in E2 from September through December, followed by

three significant peaks in February, April, and June. Females from Lake Woodruff also

exhibited significant fluctuations in E2 throughout the year, but the amplitude of the

fluctuation was less than for females from Lake Apopka. In addition, females from Lake

Woodruff exhibited a fall peak in muscle E2 that was significantly higher than E2

concentrations in Apopka cohorts.









Discussion

Female mosquitofish in central Florida begin reproductive activity in spring when

water temperatures exceed 220C, and end activity in fall when daylength shortens to

between 12 and 11 h, after which females no longer produce new broods, but presumably

complete gestation of broods already in progress. Interestingly the influence of

temperature in spring overrides short photoperiods, because we observed reproductive

females in the late January collection from Lake Apopka, when daylength was 10.5 h, but

water temperature reached 27C. Similarly, decreasing photoperiod in the fall overrides

temperature, because fewer than 25% of Apopka females were pregnant in late October

(daylength = 11 h), even though daytime water temperatures could still exceed 250C.

These observations are similar to those of Koya and Kamiya (2000), who observed that,

in a Japanese population of Gambusia affinis, spring vitellogenesis occurred when

temperature exceeded 140C, and pregnancy proceeded when temperature exceeded 180C.

In the same study, Koya and Kamiya (2000) reported that sexually active females ceased

vitellogenesis when photoperiod shortened to 12.5 h.

Previously published data, and data presented here, indicate that Lake Apopka is

more eutrophic in terms of nitrogen and phosphorus content and contains more estrogenic

or antiandrogenic endocrine-disrupting compounds when compared to Lake Woodruff

(EPA STORET database, http://www.epa.gov/storet/dbtop.html; Guillette et al., 1999).

Moreover, Lake Apopka has lower water clarity as described by Secchi depth, turbidity,

and total suspended solids. Exposure to estrogenic chemicals is often related to reduced

fecundity. For example, dosing of female medaka or fathead minnows with estradiol or

bisphenol A (weakly estrogenic) has been shown to increase vitellogenesis and, at higher









doses, reduce or abolish egg production (Palace et al., 2002; Patyna et al., 1999; Scholz,

and Gutzeit, 2000; Sohoni et al., 2001). Likewise, estradiol exposure arrests embryo

development in zebra Danios (Kime and Nash, 1999). In addition, reduced water clarity

in Lake Apopka might be expected to reduce reproductive output of females because of

the lower light availability, as shown experimentally for G. affinis by Hubbs (1999).

Despite these data, which would predict lower fecundity among Apopka females relative

to females from Lake Woodruff, we found the opposite. In our study, females from Lake

Apopka were significantly larger and more fecund, even when fecundity is adjusted for

maternal body size. Moreover, their increase in fecundity was not accompanied by a

decrease in embryo size, suggesting that Apopka females genuinely have greater

reproductive output compared to Woodruff females. The larger size of Apopka females

suggests that they grow faster and/or live longer than females from Lake Woodruff. This

may be due to higher primary productivity in Lake Apopka, driven by increased nutrient

loads, which results in greater food availability with respect to mosquitofish. In a field

study of Poeciliid growth rates, Grether et al. (2001) reported that guppy females and

juveniles grew faster in rainforest streams with higher primary productivity.

As with fecundity, adjusted hepatic weight was higher among Apopka females

relative to females from Lake Woodruff. It is likely that fecundity and hepatic weight are

causally related, but unclear in which direction. For example, with their higher fecundity,

Apopka females probably require increased yolk production. In mosquitofish, yolk is

derived, in part, from vitellogenin produced in the liver in response to circulating

estrogens (Tolar et al., 2001). Thus, the increased need for vitellogenesis could explain

the increased hepatic weight observed among Apopka fish. Alternatively, the estrogenic









contaminants that characterize Lake Apopka (Guillette et al., 1999) could stimulate

vitellogenesis (Palace et al., 2002; Sohoni et al., 2001). In an unexpected twist,

contaminant-stimulated vitellogenesis could actually permit the increased fecundity of

Apopka females. However, offspring survivorship remains to be tested.

In the first half of 2002, we noted a significant rise in muscle estradiol

concentrations among females from Lake Apopka compared to females from Lake

Woodruff. Gallagher et al. (2001) reported estradiol concentrations for female bullheads

caught in January and July from Lake Apopka and Lake Woodruff, but they observed no

lake-associated differences. However, their limited number of collection dates could

explain their non-detection of changes in endogenous estradiol. In other studies of

female fishes captured from sites affected by estrogenic pollution, like Lake Apopka,

increased estradiol concentrations have been reported. This is true, for example, of

female walleye, captured from water contaminated with estrogenic treated sewage

effluent (Folmar et al., 2001).

Conclusions and additional hypotheses. Data presented here indicate that female

mosquitofish in central Florida exhibit a well-defined reproductive cycle. In addition, we

observed lake-associated variation in hepatosomatic index and muscle estradiol

concentrations that suggest a subtle level of estrogenic endocrine disruption among

Apopka females, consistent with other studies. However, in conjunction with these

observations, we also report that females from Lake Apopka exhibit greater reproductive

output in terms of embryo number, compared to females from Lake Woodruff. Although

relative survivorship of larval and juvenile fish remains to be established, our present data









suggest that chemical pollution in Lake Apopka might not disrupt resident mosquitofish

at the population level.

In comparison to other species, mosquitofish are recognized for their ability to

adapt to variable or polluted environments (Courtenay and Meffe, 1989). This ability is

marked by increased heterozygosity and overall genetic diversity among exposed

individuals (Downhower et al., 2000; Stockwell and Vinyard, 2000; Theodorakis and

Shugart, 1997). Genetic diversity is supported by Gambusia's mating system, in which

females often mate with more than one male, resulting in broods characterized by

multiple paternity (Zane et al., 1999). Greene and Brown (1991) observed that larger

female Gambusia affinis were more likely than small females to mate with multiple

males. They also observed that multiply inseminated females were more heterozygous

than singly mated females, and, that the offspring of multiply inseminated females

exhibited greater genetic diversity. Females from Lake Apopka are larger than females

from Lake Woodruff, and it would be interesting to know if their degrees of

heterozygosity and genetic diversity were also greater. If that was the case, Gambusia

could provide an informative model for the evolution of characters that are adaptive in

polluted environments.

Table 2-1. Additional water quality information for Lake Apopka and Lake Woodruff
Water Parameter Apopka* Woodruff*
Phosphorus as P (mg/L) 0.11 + 0.03 0.07 + 0.01
Nitrite (NO2) + Nitrate (NO3) as N (mg/L) 1.15 + 0.71 0.05 0.02
Nitrogen, Kjeldahl (sum of free ammonia and 17 0
organic nitrogen) (mg/L)____________
Secchi disk depth (m) 0.39 + 0.09 0.76 + 0.06
Turbidity (NTU) 12.24 + 2.66 3.27 + 0.69
Total suspended solids (TSS) (mg/L) 50.50 + 7.12 8.53 + 3.45
*Mean value for lake taken from sampling dates in 2000 to 2003; data retrieved from EPA's
public-access STORET database (http://www.epa.gov/storet/dbtop.html).













































Stages of embryonic development for eastern mosquitofish (Gambusia
holbrooki). Stage 0 consists of small, white oocytes (black arrows),
indicating a mature, but reproductively quiescent ovary. Stages 1 and 2
indicate progressive yolking of oocytes (black arrows). Note that broods
overlap. Stage 3 is the first stage at which all yolked oocytes are similar in
size. Stage 4 is distinguished by presence of the blastodisk (black arrow).
Stage 5 embryos have elongated, and optic discs (black arrow) are visible but
unpigmented. Stage 6 embryos exhibit pigmented optic discs (black arrow).
Stage 7 embryos are enlarged, have some skin pigmentation and advanced,
but incomplete eye development. Stage 8 embryos exhibit fully formed and
pigmented eyes, pigmented skin, but tail does not overlap head. Stage 9
embryos retain a large yolk sac but tail overlaps head. Stage 10 embryos
have absorbed most their yolk sacs and often survive if removed from the
ovary. Stage 11 embryos have absorbed their yolk sacs and are ready for
birth. Staging is based on Haynes (1995).


Figure 2-1.


,,








Not Pregnant a Stage 0.5 3.5 m Stage 4 6.5 E Stage 7 8.5 E Stage 9 11


100%
90%
80%
70%
60%
50%
40%
30%
20%
10%


1 111I


1


Not Pregnant


Collection Date Lake Apopka
I Stage 0.5 3.5 m Stage 4 6.5 m Stage 7 8.5 m Stage 9 11


100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%


Collection Date Lake Woodruff


Percentage of female mosquitofish with broods at the indicated stages of
embryonic development. A) Females from Lake Apopka. B) Females from
Lake Woodruff. Embryos are developmentally synchronized within a brood,
but, as this graph shows, they are not synchronized across broods. Females
presenting as "not pregnant" were mature, but reproductively quiescent.
Note that the first May 2002 collection is missing from Lake Apopka:
drought conditions on the lake prevented boat access for two weeks.


Figure 2-2.










--Apopka Woodruff


40

" 35
30
0 30

| 25

20

S15


14:00
13:30
13:00
12:30
12:00
11:30
11:00
10:30
10:00


Collection Date


Figure 2-3. Seasonal changes in water temperature for Lake Apopka and Lake Woodruff,
shown with ambient photoperiod for each collection date


Daylength






42



-*-Apopka Woodruff


- -


Collection Date


-- Apopka -m- Woodruff


700 -

600 -

0 500

F 400

- 300
0
200

100
S>


Figure 2-4.


Collection Date


Temporal variation in body size of adult female mosquitofish from Lake
Apopka and Lake Woodruff. A) Standard length (SL). B) Body weight.
Graphs show means +1 SE. *Months in which the mean body size offish
from the two lakes are significantly different (ANOVA, p < 0.05). A heavier
line between data points indicates a significant temporal change within a lake
(ANOVA, p < 0.05).


S2.6

2.4

2.2

2






43


-*- Apopka Woodruff AP % preg WR % preg

16 100
14- 90
12 80




6 406
30
4 -20
2 10
0 0
0 , 0



Collection Date

Figure 2-5. The right hand y-axis shows the percentage of sampled females from each
lake that were pregnant (contained yolked oocytes) at each collection date.
The left hand y-axis quantifies temporal variation in mean embryo number
(litter size), adjusted for standard length, of adult female mosquitofish from
Lake Apopka and Lake Woodruff. Graph shows means +1 SE. *Months in
which the mean adjusted embryo number of fish from the two lakes are
significantly different (ANCOVA, p < 0.05). A heavier line between data
points indicates a significant seasonal change in embryo number within a
lake (ANCOVA, p < 0.05).










* Apopka 0 Woodruff


-o 40

0 30

S20


2 2.5 3 3.5
Female Standard Length (cm)


4.5 5


Embryo number (litter size) observed for female mosquitofish of different
standard lengths. Data for fish from Lake Apopka and Lake Woodruff are
shown separately. Regression is significant for both populations
(p < 0.0001).


10

0-
1.5


Figure 2-6.


r2 opka =0






r2 oodruf .1










--Apopka -- Woodruff


0 1 2 3 4 5 6 7 8 9 10 11


Embryonic Stage


Mosquitofish embryonic wet weight at different developmental stages. Data
for fish from Lake Apopka and Lake Woodruff are shown separately. Graph
shows means +1 SE. *Stages at which the mean embryo wet weight was
significantly different between females from the two lakes (ANOVA,
p < 0.05). A heavier line between data points indicates significant stage-to-
stage variation within a lake (ANOVA, p < 0.05).


Figure 2-7.











6.0

5.5

,5.0

.2P 4.5

S4.0

S3.5

S3.0
<


Embryonic Stage

* Apopka U Woodruff

















Embryonic Stage


Figure 2-8.


Mean hepatic weight (+1 SE), adjusted for female body weight of
mosquitofish with embryos at different stages. A) Fish from Lake Woodruff
and Lake Apopka combined. B) Fish from Lake Woodruff and Lake Apopka
shown separately. Curved lines in (A) indicate significant changes in hepatic
weight across stages (ANCOVA, p < 0.05). Similar significant trends were
observed in (B) (not shown for graph clarity). *Indicates that adjusted mean
hepatic weight at a given stage of embryonic development was different
between lake populations (ANCOVA, p < 0.05).











-- Apopka Woodruff


Collection Date


Temporal variation in muscle estradiol concentrations of adult female
mosquitofish from Lake Apopka and Lake Woodruff. Graph shows means +1
SE. *Months in which the mean estradiol concentrations of fish from the two
lakes are significantly different (ANOVA, p < 0.05). A heavier line between
data points indicates a significant seasonal change within a lake (ANOVA, p <
0.05).


4.


Figure 2-9.


I


>b
^ ^ > C/3














CHAPTER 3
SEASONAL SPERM QUALITY IN MALE Gambusia holbrooki (EASTERN
MOSQUITOFISH) COLLECTED FROM TWO FLORIDA LAKES

Introduction

Sperm count and sperm viability are important measures of male fertility. In

several human populations, sperm counts have decreased over the past 50 years, an

observation that is related to increased subfertility and infertility among men (Carlson et

al., 1992; Swan et al., 2000; Jensen et al., 2002). The underlying causes of reduced

human sperm counts remain controversial. However, a variety of studies in non-human

animals, particularly fishes, suggest a link with environmental contaminants that have

been shown to disrupt endocrine function and/or reproductive development (Gray, 1998;

Toft and Guillette, 2005; Jobling et al., 2002). For example, in sexually developing and

adult guppies, decreases in spermatogenesis and stripped sperm counts have been

observed after exposure to vinclozolin (fungicide) or p,p'-DDE (DDT metabolite)

(Baatrup and Junge, 2001; Bayley et al., 2002). Similar observations have been reported

in swordtails exposed to nonylphenol plasticizerr) (Kwak et al., 2001); goldfish or

zebrafish treated with estradiol or ethynylestradiol (associated with sewage effluent),

respectively (Schoenfuss et al., 2002; Van den Belt et al., 2002); and in adult Japanese

medaka exposed to 4-tert-octylphenol (estrogen mimic) (Gronen et al., 1999).

In addition to these experimental studies, field studies have shown similar effects

among wild English flounder and roach captured from waterways contaminated with

treated sewage effluent (Lye et al., 1998; Jobling et al., 2002b). Toft et al. (2003)







49

previously reported reduced sperm counts among male mosquitofish collected from Lake

Apopka in Florida (USA). Over the three months during which sampling occurred, males

from Lake Apopka exhibited an average of 47% fewer stripped sperm cells per mg testis

compared with cohorts captured from Lake Woodruff (reference lake). Unlike Lake

Woodruff, Lake Apopka has a history of contamination by p,p'-DDE and other

endocrine-disrupting contaminants with estrogenic or antiandrogenic activity (Guillette et

al., 1999). In a follow-up study, Toft and Guillette (2005) observed reduced sperm

counts among reference mosquitofish exposed for 1 month to water from Lake Apopka.

The annual spermatogenic cycle of mosquitofish has previously been described for

populations sampled in Le6n Province, Spain, and central Japan (Fraile et al., 1992; Koya

and Iwase, 2004). Based on these reports, the cycle involves a continuous period of

spermatogenesis (spring through fall), followed by a shorter period of winter quiescence.

In mosquitofish, the testes are fused into a single, round, white-colored organ that is

located centrally in the abdomen, dorsal to the origin of the gonopodium (Fraile et al.,

1992). The grooved gonopodium is used by the male to transfer spermatozeugmata to the

genital opening of the female. This structure forms during puberty by fusion and

modification of the anal fin rays under stimulation by endogenous androgen (Angus et

al., 2001; Ogino et al., 2004). A single vas deferens connects the gonopodium to the

efferent ducts that coalesce from within the central lumen of each testis (Fraile et al.,

1992). The outer wall of the testis is lined with spermatogonia (Fraile et al., 1992). In

spring through fall, spermatogonia proliferate in successive waves of mitosis, forming

nests (cysts) of primary spermatocytes bounded by Sertoli cells (Fraile et al., 1992). In a

process that takes approximately 30 days, spermatocytes within a single cyst undergo







50

synchronized meiosis and differentiation to produce spermatids and ultimately tailed

spermatozoa (Fraile et al., 1992; Koya and Iwase, 2004). As the cysts mature, they move

from the periphery of the testis to the center, where they are released to the efferent

sperm ducts as spherical aggregates of sperm (spermatozeugmata), with tails in the center

and heads on the periphery (Fraile et al., 1992). At this point, Sertoli cells no longer

surround the spermatozeugmata, but rather, they hypertrophy and become part of the

efferent duct tubule (Fraile et al., 1992). The tubules secrete a gelatinous matrix that

holds the spherical structure of the spermatozeugma together until it reaches the oviduct

of a female (reviewed by Constantz, 1989). As winter approaches, production of new

spermatocytes ceases. Through the winter, stored cysts of mature spermatozoa occupy

most of the testicular volume, and will be used during early spring copulation, which

occurs before the first wave of spring spermatogenesis is complete (Koya and Iwase,

2004).

Like most teleosts, mosquitofish exhibit seasonal variation in sperm production that

is regulated by changes in temperature and photoperiod (Fraile et al., 1994). However,

factors that initiate mosquitofish spermatogenesis are not yet fully understood. Males

that have recently entered testicular quiescence cannot be stimulated to produce new

sperm by increasing temperature or photoperiod (Fraile et al., 1993). However, fish

captured at the end of their quiescent period will exhibit proliferation of spermatogonia,

even if temperatures remain low and days are short (Fraile et al., 1994). Increasing

ambient temperatures are required for differentiation of spermatogonia to spermatocytes,

and photoperiod must lengthen in order for spermatocytes to enter meiosis (Fraile et al.,

1994; De Miguel et al., 1994).







51

We conducted our study from May 2001 to September 2002 to extend our

understanding of seasonal sperm production in mosquitofish populations from Lakes

Apopka and Woodruff in central Florida. In addition, we tested our hypothesis that low

sperm counts per mg testis, previously reported for fish from Lake Apopka, are related to

reduced sperm counts per spermatozeugma. The resulting data will help identify possible

mechanisms by which sperm count can be affected among males from Lake Apopka. In

addition to sperm counts, we measured seasonal changes in sperm viability among fish

from both lakes.

Methods

Field Collections

Between May 2001 and September 2002, 15 monthly collections of adult male

Gambusia holbrooki were made from Lake Apopka (north shore, near Beauclair Canal)

and Lake Woodruff Wildlife Refuge (northwest shore, Spring Garden Lake) in central

Florida, USA. Mature fish (identified by their hooked gonopodium) were captured using

a 3-mm mesh dip net. Average monthly sample size for the sperm study was 15 fish per

lake, with a range of 5 to 21. Concurrently, we captured additional fish for testicular

histology (n = 3 tolO per month, per lake). Water temperature, pH, and conductivity data

were obtained at the time and location where fish were sampled, using a handheld

Ultrameter (Model 6P, Myron L Company, Carlsbad, CA). Fish were held live in aerated

coolers filled with lake water, fed flake fish food ad libitum, and processed 1 to 2 days

after capture. Fish were over-anesthetized immediately before sperm collection in room-

temperature 0.1% MS222 (3-aminobenzoic acid ethyl ester, methanesulfonate salt, Sigma

#A5040). Standard length (SL) was measured to the nearest 0.01 cm from the snout tip

to the caudal peduncle using calipers. Gonopodium length was measured using an ocular







52

micrometer mounted on a dissecting microscope. Fish were blotted dry and weighed

with an electronic balance to the nearest milligram (range was 56 to 463 mg).

Testicular Histology

Fish captured for testicular histology were anaesthetized in 0.1% MS222. Testes

were removed and fixed in Smith's fixative (aqueous solution with 0.2% potassium

dichromate, 12% acetic acid, and 47% neutral buffered formalin) for 24 h, followed by

storage in 75% ethanol. Testes were dehydrated overnight, by transferring tissues to

progressively drier ethanol solutions, infused with and embedded in paraffin, and

sectioned at 10 im. Mounted sections were stained using a trichrome procedure with

Harris' hematoxylin, fast green, and Biebrich scarlet orange. Testes were considered

reproductively active when spermatocytes and/or spermatids (in addition to

spermatogonia and spermatozoa), were present, and quiescent when only spermatozoa

and spermatogonia were present (Fraile et al., 1992; Koya and Iwase, 2004) (Fig. 3-1).

The stages of spermatogenesis are shown in Figure 3-1.

Sperm Collection

Each fish was rinsed in distilled water and placed on its side, in a small petri dish

containing enough 150 mM KC1 to barely cover the fish. With the gonopodium

abducted, spermatozeugmata (szm) were stripped from the fish by gently pressing down

on the abdomen anterior to the gonopodium and sweeping caudally, using the smooth,

rounded end of large forceps with the two tips taped together (Fig. 3-2A). Typically,

hundreds of variably sized szm are ejected from the fish after 1-2 sweeps (Figs. 3-2B, C,

D). The fish was removed from the dish, and duplicate or triplicate sub-samples of a

known number of szm (average = 23) were randomly drawn up in 300 pl KC1 using a

micropipette. This collection step must be done rapidly because the KC1 activates the







53

spermatozoa, causing them to disperse from the szm in a matter of minutes (Figs. 3-2E,

F). We collected and counted only intact szm. The KC1 containing the sperm was placed

in a 12 x 75mm polystyrene culture tube and kept on ice until the samples were counted

(up to 3.5 h). Based on validation tests, sperm maintained on ice remain viable for at

least 4 h (0 to 5% change in the number of live sperm in 9 replicate samples measured

repeatedly over 5 h).

Sperm Staining

All sperm samples were stained at the same time using the Live/Dead Sperm

Viability Kit available from Molecular Probes, Inc. (Cat. #L-7011). Samples containing

300 [il sperm were first stained with 15 [l florescent SYBR Green, diluted 500 fold from

kit stock. Samples were vortexed and incubated on ice for 10 minutes, after which 1.5 il

propidium iodide (PI) stock were added. Samples were again vortexed and incubated on

ice for at least 10 minutes more. SYBR Green and PI are nucleic acid stains that

differentially stain live sperm green and dead sperm orange, respectively. Sperm are

characterized as dead if their cell membrane is compromised, allowing the rapid entry of

propidium iodide. To protect florescent stains from light, tubes and stains were handled

under low light conditions and tubes were incubated in a covered cooler containing ice.

Sperm Counts and Viability

To obtain absolute sperm numbers (expressed as sperm number per

spermatozeugma (szm)), we needed to quantify the volume of sample processed by the

flow cytometer. This volume was measured indirectly by adding a known number of

florescent particles (average of 23,000 particles per 300[l sperm sample) to each tube

and then counting the number of particles processed with the sperm sample. We used

AccuCount 5.2 micron florescent particles (Spherotech Inc., Cat. #ACFP-50-5) because







54

they were similar in size to the sperm, and their red fluorescence spectra allowed them to

be distinguished from SYBR Green and PI. Particles were suspended in a small buffer

volume (average = 21 .il) that was added to individual samples. Sperm samples were

vortexed immediately before counting to ensure homogeneous suspension of the

particles.

Flow cytometric analysis was performed on a FACSort flow cytometer (BD

Biosciences, San Jose, CA). This instrument uses an argon-ion laser emitting 15 mW of

488 nm light to illuminate the cells. Data for 10,000 to 20,000 particles were collected

and analyzed using CellQuest 3.3 software (BD Biosciences). Forward and side light

scatter measurements and green (530 +/- 15nm), orange (585 +/- 21 nm), and red (> 650

nm) fluorescence measurements were collected for each sample. The instrument

threshold was set on forward light scatter. Sperm cells were identified using a gate on the

forward vs. side light scatter dot plot (Fig. 3-3). The contents of this gate were displayed

in a second plot that gated green (SYBR Green) and orange (PI) fluorescing particles

separately (Fig. 3-3). Cells emitting only green florescence were counted as live, and

cells emitting any orange florescence (indicating a breach in the cell membrane) were

counted as dead cells. Calibration particle counts were quantified from their peak on a

separate red-fluorescence histogram (Fig. 3-3). Our methods are similar to those used for

Nile tilapia (Segovia et al., 2000)

Calculations

Absolute sperm count per spermatozeugma was calculated using the formula:

(L+D)(B)/(b)(S), where L = number of live sperm counted; D = number of dead sperm

counted; B = number of calibration particles added per sample; b = number of calibration

particles counted; and S = number of spermatozeugmata originally added to the tube.







55

Sperm viability was measured as the percentage of live sperm: L/(L+D). Live sperm

count per szm was calculated based on this percentage.

Statistics

Data points lying more than three standard deviations from the mean value for

sperm count or percentage of live sperm were excluded from the data set (applied to 6

fish or 1.5% of the total sample). Combined data were screened for correlations among

SL, body weight, gonopodium length (adjusted for SL), sperm count per szm, percentage

of live sperm, and live sperm count per szm using a correlation matrix. Gonopodium

length was adjusted for SL (using ANCOVA) because the two were significantly

correlated (r2 = 0.70; p < 0.0001). Relationships between mean sperm parameters and

water temperature, pH, and conductivity measured each month were assessed using linear

regression. Differences in sperm data between lakes in any given month were tested

using ANOVA. Monthly variation in sperm data within each lake was tested using

Fisher's PLSD post-hoc test for ANOVA, with capture date as the independent variable.

Results

Sperm count per spermatozeugma (szm), percent live sperm, and live sperm count

per szm did not correlate with SL, body weight, or adjusted gonopodium length (r2 <

0.01). There also was no correlation between sperm count per szm and percent live

sperm (r2 < 0.0001). In addition, sperm count and viability were not significantly related

to monthly water temperature, pH, or conductivity (r2 < 0.14; p > 0.06), although the

lowestp-value obtained (p = 0.06) suggests a seasonal effect of water temperature on

percent live sperm (Figs. 3-4 and 3-5). Figure 3-4 illustrates seasonal change in water

temperature and photoperiod during the study. In addition, 2002 was characterized by

significant drought, particularly through the summer, and the water level of Lake Apopka







56

was more affected than that of Lake Woodruff in the areas where Gambusia were

captured.

Sperm Viability

In 2001, percent live sperm remained stable at about 85% (with a brief drop to 75%

noted in July) from May through the beginning of December among males from Lake

Woodruff (reference lake) (Fig. 3-5). From December through mid-March 2002, mean

viability dropped to 45%, followed by steady recovery to previous levels through

mid-May. Among Woodruff fish, viability was maintained from May through the last

collection in early September (Fig. 3-5). Based on testicular histology obtained from

cohorts captured at the same time, male Gambusia from Lake Woodruff reduced

production of new spermatocytes in early October, with reinitiation observed in early

January. Through the winter, the fish stored spermatozoa, which progressively lost

viability until gonadal recrudescence in the spring (Fig. 3-5).

Overall, sperm viability (percent live) among fish from Lake Apopka followed a

similar trend to those from Lake Woodruff, with spring recrudescence occurring at a

similar time (Fig. 3-5). However, the winter decline in viability started 1 month earlier

(November) among males from Lake Apopka (Fig. 3-5). In addition, from June through

September 2002, there was a significant decline in sperm viability (percent viability =

50%) among fish from Lake Apopka relative to cohorts from Lake Woodruff. This

decline was not expected based on summer data from 2001 (Fig. 3-5). Testicular

histology revealed a decrease in spermatogenesis in September, but not in June. Note

that no collections were made between June and September.









Sperm Counts

During the collection period, mean sperm counts varied from 3400 7200 sperm

per spermatozeugma (szm) among male mosquitofish collected from the two lakes (Fig.

3-6). In late August 2001, males from Lake Woodruff exhibited significantly higher

sperm counts per spermatozeugma (szm) relative to males from Lake Apopka, although

the opposite trend was observed 1 year later, in early September 2002 (Fig. 3-6).

Woodruff males also had higher sperm counts in late January 2002 due to a third seasonal

peak in sperm count that was not observed among fish from Lake Apopka (Fig. 3-6). The

other two seasonal peaks observed among males from both lakes occurred in July and

October-November 2001 (Fig. 3-6). Note that this second seasonal peak in sperm counts

occurred 1 month earlier among Apopka males, compared to Woodruff males (late

October versus late November 2001) (Fig. 3-6).

Although cyclic variation in sperm counts was observed during 2001, sperm counts

apparently remained constant from mid March through September 2002 (Fig. 3-6).

However, no samples were collected between mid June and September 2002, when the

first seasonal peak should occur, as predicted by data from 2001. Therefore the apparent

lack of periodicity observed in 2002 may be an artifact of sampling date. In addition,

drought conditions in 2002 may account for additional variation between the two

sampling years.

As a measure of fertility, live sperm count per szm is the most relevant of the sperm

measures presented here (Fig. 3-7). For both lakes, live sperm count (2000 to 5800 live

sperm per szm) followed the same seasonal pattern described above for total sperm

counts (Fig. 3-7). However, in late August 2001 and throughout the winter, males from

Lake Woodruff exhibited higher live sperm counts relative to cohorts from Lake Apopka







58

(Fig. 3-7). We observed the same effect in September 2002. At no time did live sperm

counts from Apopka males exceed those observed among males from Lake Woodruff.

Discussion

Male mosquitofish from both Lake Woodruff and Lake Apopka exhibited cyclic

spermatogenesis, with the number of sperm per spermatozeugma rising and falling two to

three times per year. In addition, sperm viability followed a distinct seasonal pattern,

particularly among fish from Lake Woodruff, with viability maintained at 75 to 95%

during the breeding season, when sperm are continuously released and replenished, but

dropping to as low as 45% during winter quiescence, when stored sperm presumably

degrade over time. We observed that spring testicular recrudescence was related to

increasing temperature followed by increasing photoperiod. This observation is similar

to previous reports of mosquitofish described by Fraile et al. (1994) and De Miguel et al.

(1994).

Seasonal Variation in Sperm Counts

In our previous study (Toft et al., 2003), we observed that mosquitofish from Lake

Apopka exhibited an average of 47% fewer stripped sperm cells per mg testis compared

with cohorts captured from Lake Woodruff. In our study, we report sperm counts as

sperm number per spermatozeugma. Based on our study, the lake-associated difference

in total ejaculated sperm number observed by Toft et al. (2003) is probably not due to

reduced sperm counts per spermatozeugma, at least not during most of the year (late

January 2002 was an exception). However, the fact that we observed temporal variation

in the number of sperm per spermatozeugma is an interesting finding in itself, because it

suggests that the rates of spermatogonial mitosis or apoptosis vary on a seasonal basis.

Our finding adds to previous mosquitofish studies, which describe monthly variation in







59

the number of cysts, or volume of testis, devoted to a given stage of sperm cell

development (Fraile et al., 1992; Koya and Iwase, 2004). These authors attributed most

of the variation to seasonal changes in copulatory behavior, and differences in the time

required for each stage of sperm cell development. Copulatory behavior could also

regulate the number of sperm per spermatozeugma. For example, since most

mosquitofish broods exhibit multiple paternity (Zane et al., 1999), suggesting that sperm

competition affects individual mosquitofish fitness (Evans et al., 2003), it is possible that

males increase the number of sperm per spermatozeugma when competition is high. To

our knowledge, this hypothesis has not been tested in Gambusia. Evans et al. (2003)

found that male Gambusia housed with 3 females and another male for eight days (high

risk of sperm competition) mated more often and used more sperm when placed with a

novel female, relative to males housed with females alone for eight days (low risk of

sperm competition). In this system, the housing regime (eight days with or without other

males) did not affect the number of stripped sperm retrieved from treated males that were

not mated to novel females. However, the authors did not test the effects of mating

frequency on sperm production rate. In addition, eight days is too short a time frame to

test effects on spermatogenesis, which typically requires 30 days in Gambusia for a

single sperm cycle (Koya and Iwase, 2004).

As mentioned above, temporal variation in sperm count per spermatozeugma could

be due to changing rates of spermatogonial mitosis. Spermatogonia undergo mitosis for

two reasons. The first is to maintain a population of spermatogonial stem cells; the

second is to produce nests (called cysts in Gambusia) of primary spermatocytes that

undergo meiosis (reviewed by Miura and Miura, 2003). Some of the factors that regulate







60

spermatogonial mitosis have been determined for Japanese eels and were recently

reviewed by Miura and Miura (2003). Briefly, spermatogonial stem cell production is

regulated by estradiol, which, in eels, stimulates the expression of a protein called "eel

spermatogenesis related substance" (eSRS34). Interestingly, estradiol and eSRS34

stimulation result only in renewal of germ cells; they do not promote spermatocyte

proliferation and meiosis. For these events to occur, the testis must synthesize 11-

ketotestosterone (11-KT), which it does in response to gonadotropins released from the

pituitary. 11-KT works in conjunction with IGF-1 and activin B, both secreted by Sertoli

cells, to promote spermatogonial proliferation and meiosis. Additional regulation of any

step in these pathways would affect spermatogonial mitosis and possibly explain

temporal changes in sperm counts per spermatozeugma. For example, environmental

cues, such as temperature and female reproductive pheromones, have been shown to

stimulate gonadotropin release in male goldfish (Kobayashi et al., 2002).

Lake-Associated Variation in Sperm Counts and Quality

In the months when lake related variation was observed (August to September

2001, November 2001 to February 2002, June to September 2002), male mosquitofish

from Lake Woodruff exhibited significantly higher live and/or total sperm counts per

spermatozeugma and/or greater viability relative to males from Lake Apopka. An

exception occurred in early September 2002, when males from Lake Apopka exhibited

higher total sperm counts, but their live sperm counts were still significantly lower than

males from Lake Woodruff

The differences in sperm quality between the two lake populations are due to three

main observations. First, Woodruff males exhibited three seasonal peaks in sperm count,

in July, late November, and late January, relative to two seasonal peaks in July and







61

October among fish from Lake Apopka. Second, while fish from both lakes exhibited

decreased sperm viability during the winter, the period of low viability began 1 month

earlier (and was therefore 1 month longer) among fish from Lake Apopka. Third, in June

through September 2002, sperm viability among fish from Lake Apopka was

significantly reduced relative to cohorts from Lake Woodruff, and to patterns established

by both populations in 2001. The unpredicted drop may be related to the drought

conditions, which severely affected Lake Apopka's water levels in summer 2002,

particularly in the shallow periphery where mosquitofish are found. Low water levels,

especially in combination with warm temperatures and the eutrophic conditions of Lake

Apopka are likely to be associated with reduced dissolved oxygen concentrations

(unfortunately, daily data on oxygen concentrations are not available for the study

period). In carp, chronic hypoxia has been shown to reduce serum concentrations of

testosterone and estradiol, impede gonadal development, and reduce spawning success,

sperm motility, and fertilization rate (Wu et al., 2003).

Interestingly, the January peak in sperm count observed among Woodruff males

coincides with the time of declining winter sperm viability. This January peak may be

adaptive in that it dilutes the impact of lower viability, allowing males to maximize their

live sperm counts well into January or even February, when some females might be

entering spring ovarian recrudescence (Chapter 2). If this is the case, then the lack of a

January peak in sperm counts among males from Lake Apopka could be deleterious.

Alternatively, the January strategy among males from Lake Woodruff may be a plastic

and possibly costly response that is cued to female reproductive activity. Females from

Lake Woodruff, collected at the same time as males in our study, produced vitellogenic







62

oocytes 6 to 7 weeks later in the spring, compared to females from Lake Apopka.

Therefore the third peak in sperm count observed among Woodruff males may be a

response to delayed recruitment among female cohorts. Koya and Iwase (2004) observed

similar synchrony between the sexes.

In addition to these social factors, the higher concentrations of estrogenic or anti-

androgenic contaminants in Lake Apopka (Guillette et al., 1999) could explain the

reduced sperm quality observed among Apopka mosquitofish in some months. This

hypothesis is supported by the fact that elevated concentrations of p,p'-DDE

(antiandrogenic) and toxaphene estrogenicc) have been measured in the body tissues of

mosquitofish collected from Lake Apopka (US Fish and Wildlife Service, unpubl. data).

Estrogenic and antiandrogenic molecules, including 4-tert-pentylphenol, 4-tert-

octylphenol, nonylphenol, p,p'-DDE, bisphenol A, and estradiol have been shown to

cause reduction of primordial germ cell numbers or spermatogenic cysts, progressive

disappearance of spermatozoa and spermatogenic cysts, degeneration of sperm cells, or

inhibition of spermatogenesis in male carp, guppies, and platyfish (Gimeno et al., 1998a,

b; Kinnberg et al., 2000; Kinnberg and Toft, 2003). Additionally, vinclozolin, a

fungicide with anti-androgenic activity has been shown to reduce testis cord number,

increase germ cell apoptosis, and reduce sperm motility among rats exposed during

embryonic development (Uzumcu et al., 2004).

One additional hypothesis is that mosquitofish are more sensitive to endocrine

disruption in some months, particularly if the amplitude of endogenous signals is already

low. For example, alligators exhibit seasonal variation in testicular response to

gonadotropin (Edwards et al., 2004), suggesting that dosage of components in a signaling







63

pathway is important and seasonally variable. Presence of endocrine disruptors may

attenuate the efficacy of a signaling system, and this may be especially disruptive at

either the end or beginning of the breeding season, when signal concentrations may not

be optimized. This hypothesis could explain the lack of a third January peak in sperm

count per spermatozeugma and the early drop in winter sperm viability observed among

fish from Lake Apopka.

Conclusions

Overall, we observed temporal variation in both sperm counts per spermatozeugma

and sperm viability. Mosquitofish from Lake Apopka exhibited reduced total sperm

counts, live sperm counts, and sperm viability at several points during the 15 months of

collections. Taken together with previous studies from our laboratory, in which we

reported reduced total sperm counts per mg testis among mosquitofish captured from

Lake Apopka and among reference mosquitofish exposed to water from Lake Apopka for

1 month (Toft et al., 2003; Toft and Guillette, 2005), we conclude that chemical

components in Lake Apopka are a likely, but not sole, cause of reduced sperm quality

among resident mosquitofish. The underlying causes of reduced sperm quality could

include increased apoptosis or degeneration of germ cells and sperm cells, reduced

mitosis of germ cells leading to germ cell renewal (estradiol mediated), and reduced

efficacy of signaling pathways associated with spermatogenesis, particularly during

periods of transition between breeding and testicular quiescence.









Figure 3-1. Testicular histology of Gambusia holbrooki, showing breeding (main
picture) and quiescent (inset, lower left) states. SC = spermatocytes, SG =
spermatogonia, ST = spermatids, SZ = spermatozoa, SZM =
spermatozeugmata. Inset on lower right shows Sertoli cells (small arrows)
surrounding spermatozeugmata and lining efferent ducts. Sertoli cells enclose
all spermatogenic cysts, but are most visible around spermatozoa because they
hypertrophy immediately before releasing spermatozeugma into an efferent
duct. Large arrow indicates the line along which the testes fused during
development.


























.*:

E

icar., **';


-S4













































Figure 3-2. Sperm methods for Gambusia holbrooki. Spermatozeugmata (szm) are
stripped from an anaesthetized male mosquitofish by gently pressing down on
the abdomen anterior to the gonopodium and sweeping caudally, using the
smooth, rounded end of large forceps with the two tips taped together. A) The
fish is held in place by a second pair of forceps. B) Hundreds to thousands
(number is highly variable) of szm are retrieved from a single fish. C) Szm
vary in shape and size. D) Each szm contains a few thousand spermatozoa,
arranged with heads toward the outside of the szm, and tails in the center. E)
Once szm are removed from the male, they disperse in a few minutes from the
gelatinous matrix that holds them together. F) Gambusia sperm exhibit an
elongated head and single flagellum.














ul yIcIU
Two views oftotal
v cell data (scatter
"-' and topographic).
Population of
Sperm cells is
,,. .. gated by oval
10) 10 104
Side Scatter

Gate Statitics Two views of gated
File17e 6 sperm cells (scatter
Sample ID: 2c-20r and topographic).
Acquisition Date. 17-May-02
Gate: G1
Gated Events: 9617
TotalEvens 200Dead cells (stained with
Gate Events GatedWoll propidium iodide (PI))
G1 9617 100.00 48.09
G3 512 5.32 2,56
G4 7491 77.89 37.45
SLive cells (stained
with SYBR green)


EEE E I Non-spermparticles


Nile red bead count,
gated as 'M1"


File: TE020517.066
Acquisition Date: 17-May 02
Gated Events: 20000
Marker Left. Right Events


Sample ID: 12c-20r
Gate. No Gate
Total Events: 20000
%'Gated %Total Mean


Geo Mean CV Median Peak Ch


All 1. 9647 20000 10000 100,00 29,19 5.07 276.25 6.04
M1 172, 379 1701 8.51 8.51 260.08 258.56 10.82 264.16
M2 407, 649 39 0.19 0.19 490.41 486,52 12.83 486.97


Figure 3-3.


Page I


Gambusia sperm counts flow cytometry printout. The top panel shows two
views of the total sample, gated within the oval. The middle panel shows
two views of three particle populations (dead sperm cells, live sperm cells,
and non-sperm particles) that are distinguished by their respective
wavelength of florescence. The last panel indicates the gated bead count,
used to calibrate the volume of sample taken up by the flow cytometer.










Apopka --Woodruff


22o~oo2~o' o
Collection2) D)a
Collection Date


Water temperature and daylength data for the collection period. Daylength
data source: Astronomical Applications Dept., U. S. Naval Observatory,
Washington, D.C. 20392-5420. This graph duplicates Figure 2-2, but is
presented again here because it is highly relevant to the interpretation of data
in this chapter.


S30
- 25
S20
15


14:00
13:30
13:00
12:30
12:00
11:30
11:00
10:30
10:00


Figure 3-4.


Daylength










SApopka Woodruff


Collection Date


Mean percent live sperm (+1 SE) observed among adult male Gambusia
holbrooki collected from two lakes in central Florida. Collections were made
between May 2001 and September 2002. Significant variation over time
within a lake is indicated by bolded trend lines (ANOVA, p < 0.05).
*Indicates a significant lake effect within a given month (ANOVA, p < 0.05).


Figure 3-5.










-- Apopka -- Woodruff


8000
7000

6000
5000
4000
3000

2000
1000


Mean sperm count per spermatozeugma (szm) (+1 SE) observed among adult
male Gambusia holbrooki collected from two lakes in central Florida.
Collections were made between May 2001 and September 2002. Significant
variation over time within a lake is indicated by bolded trend lines (ANOVA,
p < 0.05). *Indicates a significant lake effect within a given month
(ANOVA, p < 0.05). Note that no collections were made between June and
September 2002, which may explain the lack of periodicity otherwise
predicted by data from 2001.


Collection Date


Figure 3-6.










-- Apopka -- Woodruff


S8000
0 7000
S6000
U 5000
0 4000
0 3000
2000
, 1000


Mean live sperm count per spermatozeugmatum (szm) (+1 SE) observed
among adult male Gambusia holbrooki collected from two lakes in central
Florida. Collections were made between May 2001 and September 2002.
Significant variation over time within a lake is indicated by bolded trend
lines (ANOVA, p < 0.05). *Indicates a significant lake effect within a given
month (ANOVA, p < 0.05). Note that no collections were made between
June and September 2002, which may explain the lack of periodicity
otherwise predicted by data from 2001.


Collection Date


Figure 3-7.


I














CHAPTER 4
SEASONAL VARIATION IN BODY SIZE, MUSCLE ANDROGEN
CONCENTRATIONS, AND TESTICULAR AND HEPATIC WEIGHTS AMONG
MALE MOSQUITOFISH FROM TWO LAKES IN CENTRAL FLORIDA

Introduction

Fish reproduction is regulated by a wide variety of abiotic and biotic environmental

factors. These include temperature and photoperiod (Fraile et al., 1994), nutrition (Cech

et al., 1992), and behavioral interactions between the sexes and among individuals of the

same sex (Bisazza et al., 2001; McPeek, 1992). In addition to these natural factors, most

aquatic systems are now impacted by anthropogenic contaminants, which also have the

potential to disrupt reproductive function of fishes (Baatrup and Junge, 2001; Jobling et

al., 2002a).

Many of the chemicals that now pollute aquatic systems have been shown to affect

male fish reproduction by changing steroid hormone action or metabolism. For example,

estradiol and estrogenic chemicals like nonylphenol, octylphenol, pentylphenol, and the

chemical mixture found in treated sewage effluent, are associated with delayed puberty,

persistently immature testes, or ovotestes, a condition in which males develop ovarian

tissue within their testes (Barnhoorn et al., 2004; Dreze et al., 2000; Gimeno et al., 1998a;

Toft and Baatrup, 2001). Ovotestes are also associated with abnormal presence of an

oviduct and reduced semen quality in terms of volume, morphology, and fertilization

success (Jobling et al., 2002b).

In a previously published study, conducted in conjunction with the first three

months of this project, we reported reduced sperm counts, slightly shorter gonopodia,









higher whole-body testosterone concentrations, and increased testicular and hepatic

weights among adult male mosquitofish from Lake Apopka in central Florida (Toft et al.,

2003). The Lake Apopka population was compared to those from a nearby reference

lake, Lake Woodruff. Unlike Lake Woodruff, Lake Apopka has a history of chemical

and nutrient contamination from agricultural, industrial, and municipal sources (Guillette

et al., 2000). Previously, the contamination in Lake Apopka has been characterized as

estrogenic and anti-androgenic, based on the chemicals measured in the serum and eggs

of resident alligators and body tissues of resident mosquitofish (Guillette et al., 1999;

Guillette et al., 2000; Heinz et al., 1991; U.S. Fish and Wildlife Service, unpubl. data).

Measured contaminants include several pesticides, like dieldrin, endrin, mirex,

oxychlordane, trans-nonachlor, DDT, p,p'-DDE and DDD (DDT metabolites), and

toxaphene (Guillette et al., 1999; Heinz et al., 1991; U.S. Fish and Wildlife Service,

unpubl. data). These chemicals have been experimentally shown to alter steroid and

thyroid hormone synthesis and degradation, cause male to female sex reversal of

alligators and red-eared slider turtles, and agonize or antagonize steroid receptor binding

and activity (for reviews, see Akingbemi and Hardy, 2001; Guillette et al., 2000;

Guillette and Gunderson, 2001; Willingham and Crews, 2000).

In a follow-up to the mosquitofish study cited above, Toft and Guillette (2005)

observed reduced sperm counts and altered sexual behavior among mosquitofish after a

one-month exposure to water from Lake Apopka versus water from two reference sites.

In that study, fish originated from one of the reference sites. In another paper, Gallagher

et al. (2001) reported elevated levels of plasma estrogens among male bullhead catfish

from Lake Apopka as compared with male bullheads from Lake Woodruff. These studies









expanded upon the more established Apopka-Woodruff alligator literature. That is,

relative to alligators from Lake Woodruff, alligators from Lake Apopka exhibit poor

hatching success (Woodward et al., 1993), changes in gonadal morphology (Guillette et

al., 1994), altered hepatic enzyme expression and activity (Gunderson et al., 2001), and

reduced plasma testosterone and phallus size in males (Guillette et al., 1999). The

gonadal and steroid abnormalities are observable at hatching suggesting that changes

occur during development (Guillette et al., 1995). Given the observed relationship

between environmental contaminants and altered reproductive variables among fishes

and alligators, we designed our study to further examine possible impacts of

contaminants on mosquitofish reproduction. Here, we focus on male mosquitofish

(Gambusia holbrooki), captured monthly for 17 months from the Apopka-Woodruff

model system. The seasonal component of our study is intended to place any observed,

lake-associated alterations in reproduction in context of the seasonal cycle and "normal"

environmental variation like temperature or photoperiod.

Methods

Field Collections

Between March 2001 and September 2002, 17 monthly collections of adult male

Gambusia holbrooki were made from Lake Apopka (north shore, near Beauclair Canal)

and Lake Woodruff Wildlife Refuge (northwest shore, Spring Garden Lake) in central

Florida, USA. Fish were captured using a 3-mm mesh dip net and those with a well-

developed and hooked gonopodium were considered mature (Angus et al., 2001). Each

lake collection took 1 day, plus additional days to process fish. Thus monthly collections

from the two lakes were made on different days (average of 4 days apart). For 12 of the

17 months, Lake Woodruff was sampled first.









Water temperature, pH, and conductivity data were obtained at the time and

location where fish were sampled, using a handheld Ultrameter (Model 6P, Myron L

Company, Carlsbad, CA). Each monthly sample, ranging from 37 to 67 fish per lake,

was divided into three subsets. The first subset was used to obtain testicular and hepatic

weight. These fish (n = 6 to 21 per month, per lake; mean = 18) were held live in aerated

coolers filled with lake water, fed flake fish food ad libitum, and processed within 1 to 2

days of capture. Before necropsy, fish were over-anesthetized in room-temperature 0.1%

MS222 (3-aminobenzoic acid ethyl ester, methanesulfonate salt, Sigma #A5040). The

testis (0.4 to 9.9 mg) and liver (0.1 to 8.0 mg) were removed and weighed to the nearest

0.1 mg.

The second subset was used for the measurement of androgen (testosterone and 11-

ketotestosterone) concentrations in the caudal peduncle tissues. Since the peduncle is

primarily muscle, we will refer to it as muscle for the remainder of the chapter.

Androgen concentrations were measured in muscle rather than plasma because Gambusia

are too small to yield an adequate amount of plasma. These fish (n = 6 to 12 per month,

per lake; mean = 10) were frozen immediately at capture and held on ice while in the

field. Upon return to the laboratory, the caudal fin was removed, and all peduncle tissue

posterior to the gonad was cut from the fish, weighed (average = 60 mg), and frozen at -

800C. Androgens were measured on lipid extracts of these peduncle tissues (referred to

as "muscle androgens"), as described below.

The third subset was used for sperm analysis, as reported in Chapter 3. For all fish,

standard length (SL) was measured to the nearest 0.01 cm from the snout tip to the caudal

peduncle using calipers. Fish were blotted dry and weighed with an electronic balance to









the nearest milligram. Gonopodium length was measured to the nearest 0.05 mm, using

an ocular micrometer mounted on a dissecting microscope. In mosquitofish, the

gonopodium is a grooved structure, formed by the fusion of several anal fin rays, and

used to transfer sperm to the female, as fertilization is internal in this viviparous species

(Batty and Lim, 1999). We have included measurement of the gonopodium because fin

ray fusion and elongation are androgen dependent, and the development of this secondary

sex character can be perturbed by exposure to antiandrogens such as p,p'-DDE (present

in Lake Apopka) and flutamide (Bayley et al., 2002; Ogino et al., 2004).

Muscle Androgen Measurements

For measurement of muscle concentrations of testosterone and 11-ketotestosterone,

frozen muscle pedunclee) tissues were thawed in glass tubes, on ice, and homogenized in

750 [tl 65 mM borate buffer (pH 8.0). Homogenate was extracted twice with 5 ml diethyl

ether. For each extraction, ether and homogenate were mixed together for two minutes

using a multi-tube vortex mixer. For the first extraction, tubes were allowed to settle for

three minutes to separate phases. For the second extraction, phases were separated by

centrifugation for two minutes. The aqueous phase was frozen in a methanol bath chilled

to -250C with dry ice. The ether from both extractions was combined in a second glass

tube and evaporated under dry forced air.

Hormone concentrations were determined using validated enzyme immunoassay

(EIA) kits (Cat No. 582701 (T); 582751 (11-KT) purchased from Cayman Chemical

Company (Ann Arbor, Michigan). Dry extract was reconstituted in 600-800 p.L EIA

buffer so that samples would fall within the range of the standard curve. EIAs were run

as recommended by Cayman with an 18 h refrigerated incubation to increase sensitivity.









Data were quantified against a standard curve linearized using a logit transformation of

B/Bo (bound sample/maximum bound). Duplicate interassay variance (IAV) samples at

two dilutions were included with each plate. The coefficient of variance among all

plates, averaged for the two dilutions was 18.5% for T, and 11.7% for 11-KT. To

normalize sample androgen concentrations across assays, we multiplied by a correction

factor derived from the relationship between individual plate IAV values and the mean

IAV values for all plates.

Statistics

Androgen data points lying more than three standard deviations from the mean (n =

3 for T assays (1%); 7 for 11-KT assays (2%)) were excluded from the data set. Data

were screened for correlations between standard length (SL) or body weight, and

gonopodium length, testicular and hepatic weight, and androgen concentrations using a

correlation matrix. Apparent correlations were visualized using linear regression. For

variables that scaled significantly with body size, body size was entered as a covariate

when ANCOVAs were performed.

Monthly means for each lake were compared using ANOVA or ANCOVA. To

improve homogeneity of variance, all variables (with the exception of SL and body

weight, when analyzed alone) were logo transformed. Androgen concentrations were

logo (y+1) transformed to avoid negative log values (Zar, 1999). A priori pairwise LSD

comparisons were made between lakes for each month and between consecutive months

within each lake. This latter analysis was used to describe seasonal variation within a

lake. Correlation, regression, and ANOVA analyses were completed using Statview 5.0.

ANCOVA analyses and calculations of adjusted means were completed using SPSS 13.0.









Results

Abiotic Factors

Seasonal variability in water temperature (120C in January to about 350C during

May to August) was similar between the two lakes, with the exception that Lake Apopka

exhibited an early warming period ( about 70C warmer) between February and March of

both years (Fig. 4-1). Photoperiod varied seasonally from 10.5 to 14 h (Fig. 4-1). Water

pH varied from 6.5 to 8.5, with a mean of 7.2 in Lake Woodruff, and from 6.8 to 8.7,

with a mean of 7.5 in Lake Apopka. Conductivity was generally higher in Lake

Woodruff, ranging from 700 to 1807 tS/cm, with a mean of 1196 tS/cm (peaks occurred

in March/April both years). Conductivity in Lake Apopka ranged from 433 to 765

jS/cm, with a mean of 583 jS/cm.

Body Size

Body weight and standard length (SL) were significantly related (r = 0.74; p <

0.0001). In 2001, males from Lake Apopka were significantly larger in six out of nine

months, in terms of SL (Fig. 4-2) and body weight (Fig. 4-3), than males from Lake

Woodruff. In the first half of 2002, fish from both lakes were similar in SL and weight

(Figs. 4-2 and 4-3), but in late summer, fish from Lake Woodruff were longer and heavier

(Figs. 4-2 and 4-3). As a general pattern, average male body size appears to decline

during the breeding season, beginning sometime in March to May, and reaching a

seasonal low in September to October, quickly rising again by October or November. In

2001, the spring decline among males from Lake Apopka was delayed by 2 months

compared to males from Lake Woodruff (Figs. 4-2 and 4-3). However, in 2002, mean

body size of males from both lakes began to decline at about the same time.









Gonopodium Length

Gonopodium length was positively related to SL (r = 0.67, p < 0.0001). In some

months, in both 2001 and 2002, we observed a lake-associated difference in adjusted

gonopodium length (Fig. 4-4). However, there was no pattern with regards to which lake

had fish with longer or shorter gonopodia. In addition, the maximum lake-associated

difference in adjusted gonopodium length was about 0.25 mm (Fig. 4-4). Seasonally,

adjusted gonopodium length appeared to fluctuate greatly, particularly during the winter

months (Fig. 4-4). This is likely due to variation in the body size of sampled males.

Androgens

Testosterone concentrations were negatively correlated with SL (r2 = 0.12;

p < 0.0001). Seasonally, there was substantial lake-associated variation in androgen

concentrations (Fig. 4-5). For 11-KT, peak seasonal concentrations represent an increase

of 2 tolO fold (Woodruff), or 2 to 5 fold (Apopka) relative to seasonal lows (Fig. 4-5A).

For adjusted testosterone, peak seasonal concentrations represent an increase of 2 to 5

fold (Woodruff), or 4 to 6 fold (Apopka) relative to seasonal lows (Fig. 4-5B). Males

from Lake Woodruff exhibited two annual peaks of 11-KT, one in March-April-May and

one in September-October (Fig. 4-5A). This second peak occurred in 2001, but was not

apparent by September 2002 when the last samples were collected. The annual low in

11-KT concentrations occurred in January among fish from Lake Woodruff. Like males

from Lake Woodruff, a spring peak in 11-KT among males from Lake Apopka occurred

between March and May (Fig. 4-5A). However, males from Lake Apopka did not exhibit

a significant rise in 11-KT in September, although they did exhibit a small peak in June

2002, a peak that was not observed in 2001 (Fig. 4-5A). Finally, males from Lake









Apopka exhibited an additional peak in 11-KT in January, the time that coincided with

the lowest annual 11-KT concentrations among males from Lake Woodruff (Fig. 4-5A).

Like muscle 11-KT concentrations, muscle testosterone (T) concentrations varied

both seasonally and between lakes. In 2002, the March-April peak in T coincided with

the spring peak in 11-KT among fish from both lakes (Fig. 4-5B). However, this pattern

was not observed in 2001. Likewise, fish from both lakes exhibited high T

concentrations in September-October 2001 (Fig. 4-5B). As with 11-KT concentrations,

this pattern had not yet developed by September 2002. Fish from Lake Woodruff

exhibited a third peak in T in December just before the annual low in January-February

(Fig. 4-5B). This peak was not observed among fish from Lake Apopka. However, fish

from Lake Apopka exhibited a third rise in muscle T concentrations in June (2002 only),

which coincided with the June 2002 rise in muscle 11-KT concentrations (Fig. 4-5B).

This June 2002 peak in T was not observed among fish from Lake Woodruff

Testicular Weight

Testicular and hepatic weights were positively related to body weight (r2 = 0.35, p

< 0.0001; r = 0.46, p < 0.0001, respectively). Except during November 2001 and March

2002, adjusted testicular weights were significantly higher among males from Lake

Apopka compared to males from Lake Woodruff (Fig. 4-6). The difference in actual

adjusted testicular weight in most months is substantial. For example, the greatest lake-

associated difference was observed at the end of April 2001, when adjusted testicular

weight among fish from Lake Apopka was 145% greater than that for fish from Lake

Woodruff (Fig. 4-6). Among fish from Lake Woodruff, testicular weight peaked in late

March, early August (2001), and late October (2001) (Fig. 4-6). The March peak was

consistent between 2001 and 2002 and coincided with the beginning of the spring period









of elevated androgen (both T and 11-KT) concentrations (Figs. 4-5 and 4-6). Likewise,

the early August peak precedes the elevated androgen concentrations observed in late

August-September, and the late October peak in testicular size precedes elevated

testosterone concentrations observed in late November. No testicular measurements were

made in August or October 2002, so a year-to-year comparison of this variable is not

available.

As observed among fish from Lake Woodruff, males from Lake Apopka exhibited

similar seasonality in testicular size; with increased size often preceding observed

elevations in androgen concentrations (Figs. 4-5 and 4-6). In 2001 and 2002, testicular

size peaked in April, coinciding with elevated 11-KT concentrations during both years.

Similarly, testicular weights were increased in early August, preceding high testosterone

concentrations in late September. It should be noted that testicular size data are missing

for fish from Lake Apopka in late August. The testosterone data suggest that testicular

weight remained high throughout August and early September among these fish. Finally,

testicular size peaked in late January, possibly explaining the high 11-KT concentrations

also observed at that time. This late January peak in testicular weight also precedes

elevated concentrations of both 11-KT and T in March.

Hepatic Weight

The most notable lake associated differences in adjusted hepatic weight occurred in

May June 2001 and 2002, when mean hepatic weight was significantly greater among

fish from Lake Apopka (Fig. 4-7). Temporal fluctuation occurred throughout the study

period among fish from both lakes, although the patterns on each lake are dissimilar and

frequently oppose each other (Fig. 4-7). Fish from Lake Woodruff exhibited temporal

peaks in early August 2001, November 2001, and March/late April 2002. Each peak in









hepatic weight was followed by a seasonal low, suggesting a pulsatile pattern. Fish from

Lake Apopka exhibited seasonal peaks in June 2001 and 2002, and in late January 2002.

Except in March 2002, hepatic weights among fish from Lake Apopka remain elevated

year-round with respect to seasonal lows observed among fish from Lake Woodruff (Fig.

4-7).

Discussion

Body Size

Size at maturity among male mosquitofish is highly variable, being affected by

photoperiod, mating strategy, female preference, and sex ratio (Bisazza et al., 1996;

Zulian et al., 1993). Although females appear to prefer larger males, small males

generally have a mating advantage, possibly because they can approach females without

detection (Bisazza et al., 2001). Small size reduces mating success only when the sex

ratio is male-biased, as is often the case at the end of the reproductive season (Bisazza

and Marin, 1995; Zulian et al., 1995). Male body size is not a strongly heritable trait.

Zulian et al. (1993) report that, regardless of paternal size, length at maturity is greater for

males raised in groups, and this larger body size sometimes occurs in conjunction with

delayed maturation. In the same study, they report that mosquitofish reared under short

photoperiods (9 h of light) matured earlier and at a smaller size. Taken together, these

studies predict that male body size should be small at the beginning of the breeding

season, and larger at the end. Our data did not follow this pattern. Instead, we observed

a general decline in body size among sampled fish during the breeding season, until

October, when females become reproductively quiescent (Chapter 2). Although we did

not collect sex ratio data, our body size data suggest that, in Lake Apopka and Lake

Woodruff, the sex ratio does not become male biased as the breeding season progresses.









Declining body size during the breeding season could reflect increasing recruitment of

small males that were born in the same year that they were sampled. The increase in

body size during reproductive quiescence probably reflects continued winter growth by

the established male population.

Although the sampled males from both lakes exhibited declining body size during

the breeding season in 2001, we noted that the spring decline among males from Lake

Apopka was delayed by 2 months compared to males from Lake Woodruff

Interestingly, females from Lake Apopka become reproductively active in late January,

about 1.5 months earlier than those from Lake Woodruff (Chapter 2). If the Apopka

breeding season begins early, and males mature rapidly at a small size because

photoperiod is short and the sex ratio is female biased (as suggested by the studies

described above), then we would expect mean male body size to decline earlier in the

breeding season rather than later. The observed delay in body size decline suggests that

maturation was delayed among males from Lake Apopka, relative to those from Lake

Woodruff, at least in 2001. Possible causes for delayed maturation include an overall

higher density of males year-round (Zulian et al., 1993) or exposure to an antiandrogen

such as p,p'-DDE (Bayley et al., 2002). DDE makes up most of the contaminant load

measured in the serum of alligators from Lake Apopka (Guillette et al., 1999).

Furthermore, elevated concentrations of p,p'-DDE have been measured in mosquitofish

from Lake Apopka (5300 mg/kg) (US Fish and Wildlife Service, unpubl. data). Although

delayed maturation among males from Lake Apopka is a likely explanation for

differences in body size between the two lake populations in 2001, the effect did not

persist in 2002.









Gonopodium Length

Lake associated differences in adjusted gonopodial length were maximized at about

0.25 mm or 4%. However, fish with shorter (or longer) gonopodia did not always come

from the same lake. We reported similarly low variation in gonopodial length in a

previous paper (Toft et al., 2003). To our knowledge, no data are available to indicate if

this degree of variation is large enough to confer any functional changes.

Androgens

Both 11-ketotestosterone (11-KT) and testosterone (T) exhibited temporal

variation. We previously reported whole-body testosterone concentrations of 1000 -

1600 pg/g for male mosquitofish captured from Lake Apopka and Lake Woodruff in

March May 2001 (Toft et al., 2003). Muscle testosterone concentrations reported here

for the same period are about 150 pg/g for fish from both lakes. These data suggest that

testosterone concentrations in the testis and possibly the brain, which are included in the

whole-body measurement, are higher than circulating testosterone concentrations, as

measured in the muscle. Our measured concentrations of muscle 11-KT were similar to

muscle 11-KT concentrations measured in male gag grouper (Heppell and Sullivan,

2000).

In African catfish and Japanese eels, 11-KT, rather than T, is implicated in the

stimulation of spermatogenesis (Cavaco et al., 1998; Miura and Miura, 2001). Based on

sperm count and viability data (Chapter 3), male mosquitofish exhibit active

spermatogenesis by May, after a period of winter quiescence. Our data suggest that 11-

KT, which rises in March-April, is associated with spermatogenic activation among fish

from both lakes. A small peak in 11-KT in September among fish from Lake Woodruff,

but not Lake Apopka may explain why males from Lake Woodruff have more viable









sperm later in the year (Chapter 3). Interestingly, the January peak in 11-KT among fish

from Lake Apopka was not associated with any observable effects on spermatogenesis,

even up to three months later (Chapter 3). It is likely that during winter quiescence,

testes are not responsive to androgen stimulation in terms of spermatogenesis, thus the

reason for the January 11-KT peak among fish from Lake Apopka is not clear.

In 2002, males from Lake Apopka exhibited a large rise in muscle 11-KT

concentrations in April, preceding an expected rise in sperm viability in May (Chapter 3).

However, viability dropped substantially and unexpectedly in June, in association with a

large rise in muscle T concentrations. In African catfish, the stimulatory effect of 11-KT

on spermatogenesis can be blocked by co-treatment with T (Cavaco et al., 2001).

Although we cannot explain the June rise in muscle T concentrations, which did not

occur in 2001, nor among males from Lake Woodruff, the June rise in muscle T

concentrations may explain the concomitant decrease in sperm viability observed among

fish from Lake Apopka in 2002 (Chapter 3).

Testicular Weight

Among males from both lakes, temporal variation in testicular size was often

positively related to changes in muscle androgen concentrations. The period of increased

testicular size, implying increased testicular activity, ranged from March to November,

followed by a period of winter quiescence. This observation is supported by our data on

sperm quantity and viability (Chapter 3). It is also similar to previously published

observations examining Gambusia reproduction in Japan, although the reproductive

season for the Japanese populations was shorter (Koya and Iwase, 2004). Although the

pattern among fish from the two lakes was similar, testicular weights were significantly

higher among males from Lake Apopka compared to males from Lake Woodruff. On the