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Parasitological and Osmoregulatory Evaluations of the Seminole Killifish, Fundulus seminolis, a Candidate Species for Ma...

Permanent Link: http://ufdc.ufl.edu/UFE0022722/00001

Material Information

Title: Parasitological and Osmoregulatory Evaluations of the Seminole Killifish, Fundulus seminolis, a Candidate Species for Marine Baitfish Aquaculture
Physical Description: 1 online resource (74 p.)
Language: english
Creator: Dimaggio, Matthew
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: baitfish, fundulus, george, istat, killifish, lake, osmoregulation, salinity, seminole, seminolis
Fisheries and Aquatic Sciences -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: U.S. baitfish production had a 2005 farm gate value of $38 million. Freshwater species currently comprise the majority of all cultured baitfish. Aquaculture of marine baitfish species is still in its relative infancy and the increasing value of coastal property is forcing marine aquaculture inland. Fundulus seminolis, a freshwater species endemic to Florida, has shown economic potential for use as a marine baitfish, with a small number of commercial operations currently in production. The objectives of this study were a parasitological survey of wild F. seminolis broodfish, characterization of the salinity tolerance of the species, evaluation of a point-of-care blood analyzer for use with F. seminolis, and elucidation of the physiology associated with their gradual seawater acclimation. In adherence with responsible aquaculture practices, a parasitic survey of the wild caught broodstock from Lake George, Florida was conducted to identify potential health problems with the species. This is the first comprehensive parasitic survey of F. seminolis and the Lake George region. Thirteen distinct taxa were identified as parasites of F. seminolis. Eight parasitic taxa were elucidated which had never before been recorded on F. seminolis. Two separate acute acclimation experiments, natural seawater and sodium chloride, were carried out to determine if survival was influenced by the salinity source. F. seminolis were able to tolerate acute transfer to 0, 8, and 16 g/L using both salinity sources but only specimens in natural seawater were able to survive in 24 g/L. No survival was observed in either salt source at 32 g/L. A gradual seawater acclimation was also investigated to examine survival at various acclimation rates. A survival rate of 100% was achieved when salinity was changed from 0 to 32 g/L over 24, 48, 72, and 96 h. The i-STAT? point-of-care blood analyzer was evaluated against conventionally accepted instrumentation for determination of hematocrit, sodium, potassium, and chloride in F. seminolis. Whole blood and heparin diluted whole blood aliquots were analyzed. Results analyzed by t-test, correlation coefficients, and the Bland-Altman method all indicated results obtained with the i-STAT? unit were unreliable when compared with accepted conventional methodologies. A gradual seawater acclimation from 0 to 32 g/L over 24, 48, 72, and 96 h was conducted. Body weight, muscle water content, hematocrit, sodium, potassium, chloride, and plasma osmolality were analyzed. Generated data revealed physiological stress manifested in multiple variables analyzed after acclimation times of 24, 48 and 72 h. Generally, data gathered from the 96 h acclimation suggest the initiation of physiological acclimation as select analytes began to migrate back towards reference values derived from controls. Results of these experiments provide information pertinent to the fields of physiology, ecology, and aquaculture regarding this rarely studied species. Additionally, experimental outcomes will help to diversify aquaculture within Florida and shape the marketing and distribution strategies for this economically valuable killifish.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Matthew Dimaggio.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Ohs, Cortney.
Local: Co-adviser: Petty, Denise.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022722:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022722/00001

Material Information

Title: Parasitological and Osmoregulatory Evaluations of the Seminole Killifish, Fundulus seminolis, a Candidate Species for Marine Baitfish Aquaculture
Physical Description: 1 online resource (74 p.)
Language: english
Creator: Dimaggio, Matthew
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: baitfish, fundulus, george, istat, killifish, lake, osmoregulation, salinity, seminole, seminolis
Fisheries and Aquatic Sciences -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: U.S. baitfish production had a 2005 farm gate value of $38 million. Freshwater species currently comprise the majority of all cultured baitfish. Aquaculture of marine baitfish species is still in its relative infancy and the increasing value of coastal property is forcing marine aquaculture inland. Fundulus seminolis, a freshwater species endemic to Florida, has shown economic potential for use as a marine baitfish, with a small number of commercial operations currently in production. The objectives of this study were a parasitological survey of wild F. seminolis broodfish, characterization of the salinity tolerance of the species, evaluation of a point-of-care blood analyzer for use with F. seminolis, and elucidation of the physiology associated with their gradual seawater acclimation. In adherence with responsible aquaculture practices, a parasitic survey of the wild caught broodstock from Lake George, Florida was conducted to identify potential health problems with the species. This is the first comprehensive parasitic survey of F. seminolis and the Lake George region. Thirteen distinct taxa were identified as parasites of F. seminolis. Eight parasitic taxa were elucidated which had never before been recorded on F. seminolis. Two separate acute acclimation experiments, natural seawater and sodium chloride, were carried out to determine if survival was influenced by the salinity source. F. seminolis were able to tolerate acute transfer to 0, 8, and 16 g/L using both salinity sources but only specimens in natural seawater were able to survive in 24 g/L. No survival was observed in either salt source at 32 g/L. A gradual seawater acclimation was also investigated to examine survival at various acclimation rates. A survival rate of 100% was achieved when salinity was changed from 0 to 32 g/L over 24, 48, 72, and 96 h. The i-STAT? point-of-care blood analyzer was evaluated against conventionally accepted instrumentation for determination of hematocrit, sodium, potassium, and chloride in F. seminolis. Whole blood and heparin diluted whole blood aliquots were analyzed. Results analyzed by t-test, correlation coefficients, and the Bland-Altman method all indicated results obtained with the i-STAT? unit were unreliable when compared with accepted conventional methodologies. A gradual seawater acclimation from 0 to 32 g/L over 24, 48, 72, and 96 h was conducted. Body weight, muscle water content, hematocrit, sodium, potassium, chloride, and plasma osmolality were analyzed. Generated data revealed physiological stress manifested in multiple variables analyzed after acclimation times of 24, 48 and 72 h. Generally, data gathered from the 96 h acclimation suggest the initiation of physiological acclimation as select analytes began to migrate back towards reference values derived from controls. Results of these experiments provide information pertinent to the fields of physiology, ecology, and aquaculture regarding this rarely studied species. Additionally, experimental outcomes will help to diversify aquaculture within Florida and shape the marketing and distribution strategies for this economically valuable killifish.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Matthew Dimaggio.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Ohs, Cortney.
Local: Co-adviser: Petty, Denise.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022722:00001


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PARASITOLOGICAL AND OSMOREGULATORY EVALUATIONS OF THE SEMINOLE KILLIFISH, Fundulus seminolis, A CANDIDATE SPECIES FOR MARINE BAITFISH AQUACULTURE By MATTHEW A. DIMAGGIO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Matthew A. DiMaggio 2

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To my father, who fostered my love of learning. To my mother, who has gi ven me new perspective. I love you both. 3

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ACKNOWLEDGMENTS The following study would not have been possible without the help of numerous individuals. I thank Edsel E. Redden and Greg Harris for their help in procuring research specimens and Dr. Ilze Berzins and the Universi ty of Florida Tropical Aquaculture Laboratory for use of vital instrumentation. I thank Scott Grabe, Shawn DeSa ntis, John Marcellus, and Tina Crosby for their tireless assistan ce throughout the course of numer ous experiments. I recognize Dr. Pamela Schofield for her statistical assistance and Dr. Andrew Rhyne for his helpful suggestions and innovative solutions. I express my gratitude to committee member, Dr. Jeff Hill, for his time and suggestions; and to my cochair, Dr. Denise Petty, whose thirst for knowledge and desire to impart it to others has been an inspiration. I would like to esp ecially thank my advisor Dr. Cortney Ohs for countless hours spent analyzing data and editing manuscripts. His guidance and example have helped me to appreciate the nuances of research. To my late grandfather, a man who inspired my love for the ocean and taught me to fish, I will forever be grateful. I also thank my parents for their encouragement in my pursuit of higher education. My success is a direct reflection of their self sacrifice and dedication to their family. Finally, I thank my wife, whose loyalty and devotion led her across the country with me. Without her continued love and support, my as pirations might never have been realized. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT.....................................................................................................................................9 CHAPTER 1 INTRODUCTION................................................................................................................. .11 2 PARASITIC FAUNA OF THE SEMINOLE KILLIFISH Fundulus seminolis FROM LAKE GEORGE, FLORIDA.................................................................................................14 Introduction................................................................................................................... ..........14 Methods..................................................................................................................................15 Results.....................................................................................................................................16 Discussion...............................................................................................................................17 3 EXPERIMENTAL SALINITY TOLE RANCE DETERMINATIONS FOR THE SEMINOLE KILLIFISH, Fundulus seminolis .......................................................................22 Introduction................................................................................................................... ..........22 Methods..................................................................................................................................25 Sodium Chloride Acute Salinity Tolerance.....................................................................25 Natural Seawater Acute Salinity Tolerance....................................................................26 Natural Seawater Gradual Acclimation Survival............................................................27 Statistical Analysis..........................................................................................................2 9 Results.....................................................................................................................................29 Sodium Chloride Acute Salinity Tolerance.....................................................................29 Natural Seawater Acute Salinity Tolerance....................................................................30 Sodium Chloride vs. Natural Seawater...........................................................................31 Natural Seawater Gradual Acclimation Survival............................................................31 Discussion...............................................................................................................................31 5

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4 EVALUATION OF A POINT OF CARE BLOOD ANALYZER FOR USE IN DETERMINATION OF SELECT HE MATOLIGICAL INDICES IN Fundulus seminolis .................................................................................................................................36 Introduction................................................................................................................... ..........36 Methods..................................................................................................................................37 Whole Blood Analysis.....................................................................................................38 Whole Blood Heparin Dilution Analysis........................................................................39 Statistical Analysis..........................................................................................................4 0 Results.....................................................................................................................................40 Whole Blood Analysis.....................................................................................................40 Whole Blood Heparin Dilution Analysis........................................................................41 Discussion...............................................................................................................................41 5 PHYSIOLOGICAL EVALUATION OF Fundulus seminolis FOLLOWING FOUR SEAWATER ACCLIMATION PROTOCOLS.....................................................................48 Introduction................................................................................................................... ..........48 Methods..................................................................................................................................50 Natural Seawater Acclimation.........................................................................................50 Statistical Analysis..........................................................................................................5 3 Results.....................................................................................................................................53 Discussion...............................................................................................................................55 6 CONCLUSION................................................................................................................... ....63 REFERENCES..............................................................................................................................66 BIOGRAPHICAL SKETCH.........................................................................................................74 6

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LIST OF TABLES Table page 2-1 Classification of parasite numbers per fiel d of view at predetermined magnifications on the skin, fin, gill and intestine of Fundulus seminolis. .................................................20 2-2 Intestinal biopsy observations from 100 Fundulus seminolis ............................................20 2-3 Skin biopsy observations from 100 Fundulus seminolis. ..................................................20 2-4 Gill biopsy observations from 100 Fundulus seminolis. ...................................................21 2-5 Fin biopsy observations from 100 Fundulus seminolis. ....................................................21 3-1 Kaplan-Meier survival analys is for acute NaCl transfer....................................................35 3-2 Log-Rank analysis among treatm ents for NaCl acute transfer..........................................35 3-3 Kaplan-Meier survival anal ysis for acute NSW transfer...................................................35 3-4 Log-Rank analysis among treatm ents for NSW acute transfer..........................................35 4-1 Mean SD values for analyzed hema tological parameters using both the POC analyzer (i-STAT) and conventionally accepted instrumentation (CAI) generated from whole blood aliquots.................................................................................................45 4-2 Mean SD values for analyzed hema tological parameters using both the POC analyzer (i-STAT) and conventionally accepted instrumentation (CAI) generated from heparin diluted whole blood aliquots........................................................................45 5-1 Mean SD hematological parameters of F. seminolis acclimated to control (0 g/L) or NSW (32 g/L) over experimental time periods of 24, 48, 72, and 96 h........................62 7

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LIST OF FIGURES Figure page 4-1 Bland-Altman plots of blood parameter va lues generated from whole blood aliquots.....46 4-2 Bland-Altman plots of blood parameter va lues generated from heparin diluted whole blood aliquots.....................................................................................................................47 5-1 Mean plasma sodium by treatment over experimental acclimation periods......................59 5-2 Mean plasma chloride by treatment over experimental acclimation periods....................59 5-3 Mean plasma osmolality by treatment over experimental acclimation periods.................60 5-4 Mean percent body weight change by treatment over experimental acclimation periods................................................................................................................................60 5-5 Mean percent muscle water content by treatment over experimental acclimation periods................................................................................................................................61 5-6 Mean plasma potassium by treatment over experimental acclimation periods.................61 5-7 Mean hematocrit by treatment ove r experimental acclimation periods.............................62 8

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PARASITOLOGICAL AND OSMOREGULATORY EVALUATIONS OF THE SEMINOLE KILLIFISH, Fundulus seminolis, A CANDIDATE SPECIES FOR MARINE BAITFISH AQUACULTURE By Matthew A. DiMaggio August 2008 Chair: Cortney L. Ohs Cochair: B. Denise Petty Major: Fisheries a nd Aquatic Sciences U.S. baitfish production had a 2005 farm gate value of $38 million. Freshwater species currently comprise the majority of all cultured ba itfish. Aquaculture of mari ne baitfish species is still in its relative infancy and the increasing value of coastal property is forcing marine aquaculture inland. Fundulus seminolis a freshwater species ende mic to Florida, has shown economic potential for use as a marine baitfish, with a small number of commercial operations currently in production. The objectives of this study were a para sitological survey of wild F. seminolis broodfish, characterization of the salinity tolerance of th e species, evaluation of a point-of-care blood analyzer for use with F. seminolis, and elucidation of the physiology associated with their gradual seawater acclimation. In adherence with responsible aquaculture practices, a parasitic surv ey of the wild caught broodstock from Lake George, Florida was conducte d to identify potential health problems with the species. This is the first comprehensive parasitic survey of F. seminolis and the Lake George region. Thirteen distinct taxa were identified as parasites of F. seminolis Eight parasitic taxa were elucidated which had never before been recorded on F. seminolis 9

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Two separate acute acclimation experiments, na tural seawater and sodium chloride, were carried out to determine if survival was influenced by the salinity source. F. seminolis were able to tolerate acute transfer to 0, 8, and 16 g/L us ing both salinity sources but only specimens in natural seawater were able to survive in 24 g/L. No survival was observed in either salt source at 32 g/L. A gradual seawater acclimation was also investigated to examine survival at various acclimation rates. A survival rate of 100% was achieved when salinity was changed from 0 to 32 g/L over 24, 48, 72, and 96 h. The i-STAT point-of-care bl ood analyzer was evaluated ag ainst conventionally accepted instrumentation for determination of hemato crit, sodium, potassium, and chloride in F. seminolis. Whole blood and heparin diluted whole blood aliquot s were analyzed. Results analyzed by t-test, correlation coefficients, and the Bland-Altman method all indicated results obtained with the iSTAT unit were unreliable when compared with accepted conventional methodologies. A gradual seawater acclimation from 0 to 32 g/L over 24, 48, 72, and 96 h was conducted. Body weight, muscle water content, hematocrit, sodium, potassium, chloride, and plasma osmolality were analyzed. Generated data reveal ed physiological stress manifested in multiple variables analyzed after acclima tion times of 24, 48 and 72 h. Generally, data gathered from the 96 h acclimation suggest the initiation of physiologi cal acclimation as select analytes began to migrate back towards reference values derived from controls. Results of these experiments provide inform ation pertinent to the fields of physiology, ecology, and aquaculture regarding this rarely studied species. Additionally, experimental outcomes will help to diversify aquaculture within Florida and shape the marketing and distribution strategies for this ec onomically valuable killifish. 10

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CHAPTER 1 INTRODUCTION Floridas $7.5 billion annual economic impact for its recreational fisher y is the highest of any state in the U.S. according to the 2006 na tional survey of fishing, hunting, and wildlifeassociated recreation (Wattendorf and Sieber, 2008). The Florida Fi sh and Wildlife Conservation Commission reports that Floridas recreational saltwater fish ery had an economic impact of $5.2 billion in 2006 and was responsible for 51,500 jo bs (Wattendorf and Sieber, 2008). Despite these overwhelming statistics establishing Florida as a premier fishing locati on, of the 257 baitfish farms recorded in the 2005 USDA census of aqu aculture, only 2 were located in Florida (USDA, 2005). This disparity clearly illustrates the pote ntial for expansion and diversification of aquaculture within the state of Florida. Saltwater fishing practices and associated e quipment have made dramatic advances in technology and efficiency in recen t times. Interestingly, methods of marine baitfish procurement have experienced little change over the past 75 years. The use of nets and traps are commonly used to harvest these sometimes elusive organisms. Today the majority of marine baitfish sold in stores are wild caught. Availability of most sp ecies is seasonal yet demand remains relatively constant. Aquacultured marine ba itfish can potentially provide fishermen with a consistent supply of sought after species in desired sizes, regardless of season. Additionally, development of baitfish aquaculture w ithin Florida would help to divers ify the existing aquaculture industry and potentially alleviate collection pr essure on targeted wild populations. The seminole killifish, Fundulus seminolis is an endemic Florida fr eshwater killifish that has recently emerged as a potenti al candidate for marine baitfis h aquaculture. Its ability to complete its reproductive life cycle in freshwater is appealing for aquaculture in Florida as value of coastal property increases and access to se awater becomes more limited. Culture of this 11

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species in freshwater would allow production to occur away from co astal resources. Its large size and tolerance of poor water quali ty conditions make it a candidate worthy of investigation for baitfish aquaculture. Additionally, if this species is able to acclimate to full strength seawater, potential for disease transmission from the culture environment to the wild should be diminished because the seawater acclimation would ostensibly act as a prophyl actic treatment for freshwater ectoparasites and other salin ity sensitive pathogens. The objectives of this study were a parasitological survey of wild F. seminolis broodfish, characterization of the salinity tolerance of the sp ecies, evaluation of a point-of-care blood analyzer for use with F. seminolis, and elucidation of the physiology associated with its gradual seawater acclimation. Results from the parasitological investigation will provide data regarding the ecological diversity of parasites occu rring on a wild population of F. seminolis from Lake George, Florida. Additionally, parasite identific ation and enumeration are cruc ial for proper quarantine and biosecurity procedures within an aquaculture production setting. Salinity tolerance determinations will provide essential information influencing the marketing and distribution of this species as a ma rine baitfish. Information gathered from salinity tolerance experiments may also help to discern the role of salinity as a barrier to the species geographic dispersion. The use of point-of-care blood analyzers for physiological determinations in fish has recently become more prevalent. Validation of new technologies against standard techniques is essential for generation of reliable data. Evalua tion of the i-STAT point-of-care unit against conventionally accepted instrument ation will allow for the determination of agreement between 12

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the two methods and ultimately the validity of the i-STAT unit for use in fish physiology studies. Physiological studies should provide insight into underlying processes allowing the species to acclimate to seawater as well as establishing an osmoregulator y time frame within which it is capable of doing so. Experimental results may also contribute to the development of standardized acclimation protocols for use in commercial production settings. There is a noticeable paucity of published literature dealing with F. seminolis The consequent studies are intended to inform the scientific community and aquaculture industry regarding this emerging Fundulus species. Experimental results wi ll have immediate impact for Florida aquaculture and help to substantiate marine baitfish pr oduction as a viable aquaculture crop for the state and the region. 13

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CHAPTER 2 THE PARASITIC FAUNA OF THE SEMINOLE KILLIFISH, FUNDULUS SEMINOLIS, FROM LAKE GEORGE, FLORIDA Introduction The seminole killifish, Fundulus seminolis is an endemic Florida killifish with a geographic range within peninsular Florida from the St. Johns and New Rive r drainage basins to just south of Lake Okeechobee (Page and Burr, 1 991). Populations reaching as far south as NineMile Bend have been reported by Tabb and Ma nning (1961). This species, commonly referred to as a bullminnow or mudminnow, is one of the largest members of the genus, reaching total lengths of 20 cm (Hoyer and Canfield, 1994). It s popularity as a local baitfish for largemouth bass, Micropterus salmoides and other piscivorous game fish has generated interest in this species as a potential candidate for aquaculture. Relatively little is known regarding the life history of F. seminolis with only one public ation by DuRant et al. (1979) devoted entirely to the species. It has been referenced anecdotally or as a component in a larger study or survey in several publications (McLane, 1955; Phillip s and Springer, 1960; Tabb and Manning, 1961; Gunter and Hall, 1963; Gunter and Hall, 1965; Foster, 1967; Gr iffith, 1974A; Nordlie, 2006). To date there are no publications that have extensively examined the parasitic fauna of Fundulus seminolis Although Bangham (1940) included F. seminolis in his parasite survey, his sample size was only 14 individuals and most of the specimens were preserved in formalin prior to examination, likely altering the detectable para site burden. Dillons (1966) effort at compiling a list of parasites occurring on Fundulus spp. merely referenced Banghams work with no new additions. The most recent and extensiv e checklist of parasites occurring on Fundulus spp. compiled by Harris and Vogelbein (2006) excluded F. seminolis altogether. Therefore, the objective of this study is to eluc idate and enumerate the various pr otistan and metazoan parasites found within a population of seminole ki llifish from Lake George, Florida. 14

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Methods A total of 140 F. seminolis were collected from the eastern shore of Lake George (29 17 12 N 81 35 53 W) in Volusia County Florida. This broa d and shallow lake is part of the St. Johns River system and is the sec ond largest freshwater lake in the state of Florida. Fish were collected with a seine net ( 24.4 m X 1.2 m, 0.8 cm mesh) on thr ee separate occasions from March through May 2007. A sample size of 100 fish was determined to be suitable for the experiment based on previous work by Ossiande r and Wedemeyer (1973) and Simon and Schill (1984). This sample size would a llow detection with a 95% confiden ce level one carrier fish in a population greater than 1,000,000 with a 3% incidence of disease (Ossiander and Wedemeyer, 1973). Fish were captured with a seine net and transported to th e laboratory alive in water obtained from the collection site. A dissolved oxygen saturation of approximately 90% was maintained during transport. Water samples were collected prior to seining and were stored for later analysis. Water temperature was determined at collection sites. Dissolved oxygen (DO) and pH were both measured using Hachs HQ-20 meter whereas total ammonia nitrogen (TAN), nitrite, total hardness, total alkalinity, CO2, and free and total chlorine were measured using standard techniques (Hach Co., Loveland, Colorado). Salinity was determined using a refractometer. Upon arrival fish were individually weighed and measured and subsequently examined externally for gross signs of pa rasitism. If no gross signs of parasitism were evident, a skin biopsy was collected from the enti re length of the left lateral b ody wall of the fish, a gill biopsy (~3mm2) was collected from the specimens left second gill arch and a fin biopsy (~5mm2) was collected from the specimens caudal fin. Active le sions, erosions, erythemi c tissues, and visible parasites were given precedence and the area in question was biopsied instead. Wet mounts of all biopsied tissues were prepared for further anal ysis. Fish were subsequently euthanized in 15

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buffered tricaine methanesulfonate (MS-222, Ar gent Laboratories, Fi nquel, C-FINQ-UE-100G) and each specimens intestine was excised. Wet mounts of the complete intestine were prepared for further inspection. Skin, fin, gill, and intestin al biopsies were performed utilizing techniques described by Noga (1996). All wet mounts were prepared within 24 hours of capture and examined immediately thereafter. All parasitological terminology utilized adhere to the recommendations of Bush et al. (1997). Parasites were identifie d utilizing previously publis hed literature (Noga, 1996; Woo, 1995; Stoskopf, 1993) and with the help of B. De nise Petty, DVM, University of Florida. Slide preparations were examined using light micr oscopy at three different magnifications (40x, 100x, and 400x). Parasites were enumerated individually with the exceptions of Nematoda, Digenea, Myxobolus sp., and sessile ectocommensal ciliates of the order Sessilida (SECs). These four parasite groups were quantified utilizing a system similar to th e one implemented by Bravo et al. (2007). Three gradations, light, moderate, and hea vy, were determined for each parasite relative to numbers observed per field in five random fiel ds of view at a pred etermined magnification (Table 2-1). Prevalence, mean abundance, intensity range, and m ean intensity were calculated where appropriate. For categorical data, mean intensity was calculated by assigning numerical values to parasite descriptors. Results Thirteen distinct taxa were identified as parasites of F. seminolis six of which were identified down to genus and one to species. The most common parasitic group encountered in this survey was the subclass Digenea. Found in al l four of the tissues examined, digeneans were responsible for the highest prevalence recorded in this study, 95% in the intestine of sampled organisms (Table 2-2). Skin and gill biopsies yielded the greatest diversity of parasites with 8 taxa represented in each (Table 2-3 & Ta ble 2-4). Monogeneans accounted for the greatest 16

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prevalence on both the skin and gill biopsies, with 14% prevalence for Gyrodactylus sp. and 46% prevalence for Dactylogyrida respectively. Hirudi nea were the most common of all parasites found on the fin, with a prevalence of 39%, a mean abundance of 0.52, and a mean intensity of 1.3 (Table 2-5). The maximum mean intensity recorded was 5.00 for Ichthyobodo sp. on the fin and 5.00 for Piscinoodinium sp. on the gill. Trichodina sp. found on the gill biopsies demonstrated the broadest intensity range, 1-12 organisms per specimen analyzed. The largest calculable mean abundance of 0.73 was displaye d by Dactylogyrida on th e gill biopsies. The mean total length of F. seminolis collected was 106 2 mm with a range from 70 157 mm. The body weight of the study specimens ranged from 2.84 38.00 g, with a mean body weight of 13.02 0.71 g. Mean collection site wate r quality parameters were as follows; DO = 6.2 0.3 mg/L; pH = 7.6 0.1; temperature = 18.3 1.3 C; TAN = 0 mg/L; nitrite = 0 mg/L; salinity = 1 g/L; total har dness = 256.5 19.7 mg/L; total al kalinity = 96.9 5.7 mg/L; CO2 = 8.3 3.3 mg/L; free chlorine = 0 mg /L; total chlorine = 0 mg/L. Discussion This study provides the first comprehensive desc ription of the naturally occurring parasite fauna of F. seminolis. Additionally, to the authors knowledge this is the first published parasitological survey of fish collected from La ke George, Florida. As Floridas second largest water body and an important component of the St Johns River system, this survey provides valuable knowledge into the composition of the resident parasite community. Banghams 1940 survey of F. seminolis recorded six distinct ta xa and their corresponding quantities from which we were able to calculate the prevalence of each parasite. Interestingly, Bangham reports the digenean, Neascus vancleavei as the most common of the parasites observed (71% prevalence). This finding is consis tent with results from the present survey, as digeneans were found on all four of the tissues analyzed and exhibited the highest prevalence 17

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recorded of 95% in the intestinal biopsies. C onversely, Bangham failed to report the presence of Myxobolus sp., SECs, Hirudinea, Ichthyophthirius multifiliis Tetrahymena sp., Ichthyobodo sp., Piscinoodinium sp., and Trichodina sp.; the present study represen ts the first recorded accounts of these parasites infecting F. seminolis These results however cannot be accurately compared due to the small sample size used by Bangham as well as unclear diagno stic techniques and preservation of samples in formalin prior to parasite enumeration. The high prevalence of digenetic trematodes observed in the Lake George population of F. seminolis is possibly a direct result of the fishs diet. Upon intestinal excision it was noted that a predominant number of the specimens contained multiple gastropods in various stages of digestion. Similarities of parasite taxa found in the genus Fundulus to parasite taxa observed in the current study are evident in previous literature. Yoshino (1972) reported a wide array of digeneans in Fundulus parvipinnis, including N. vancleavei previously reported in F. seminolis (Bangham, 1940) Barse (1998) reported ten taxa found on the gills of F. heteroclitus in Chesapeake Bay, slightly more than the eight taxa we found on the gills of F. seminolis. Barse also examined the effect of seasonality, locali ty, and host sex and size. These factors were not investigated in this study, but c ould provide valuable data if anal yzed in future studies. Of note, Lake George had a salinity of 1 g/L during expe rimental collections. This salinity could have influenced the richness and abundance of paras ite species recorded during the survey. Adams (1985) reported six taxa infesting the gills of Fundulus kansae three of which were found on the gills of our study specimens. Trichodina spp. prevalence of 59% reported by Adams (1985) is considerably higher than the 5% prevalence on the gills of F. seminolis in the present study. Despite differences in Myxosporea genera, it is noteworthy that the para site was found in both F. 18

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kansae and F. seminolis. Additionally, 4 Myxobolus spp. have been reported in the banded killifish, Fundulus diaphanus, (Cone et al., 2006). Parasitological survey results of wild F. seminolis brood fish are integral in the establishment of effective quarantine and s ubsequent biosecurity procedures unique to aquaculture production facilities. Prevention of pathogen introductions both into and out of the culture environment must be ensured through implementation of res ponsible aquaculture practices. These findings will dictate treatment therapy options instrumental in the captive husbandry and culture of this emerging marine baitfish. To date, the most comprehensive checklist of parasite taxa infesting Fundulus spp. has been compiled by Harris and Vogelbein (2006). Ideally, future checklists w ill incorporate the data genera ted from this study and include F. seminolis with the other members of the genus. 19

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Table 2-1. Classification of parasite intensity per field of view at predetermined magnifications on the skin, fin, gill and intestine of Fundulus seminolis. # of fields of Light Moderate Heavy Parasite view (FOV) Magnification (Per FOV) (Per FOV) (Per FOV) Digenea 5 40x 1-10 11-25 26 Myxobolus sp. 5 400x 1 Xenoma or Individual 2-10 Myxosporea Xenomas 11 Xenomas Nematoda 5 40x 1-10 11-25 26 SECs 5 400x 1-10 11-25 26 Table 2-2. Parasite fauna observed on 100 intestin al biopsies of Fundulus seminolis Parasite Prevalence (%) Mean a bundance Intensity range Mean intensity Cestoda 2 0.03 1-2 1.50 Digenea 95 L-Ha La Myxobolus sp. 8 L-Ma La Nematoda 26 La La a Parasite descriptors per field of view (T able 2-1) L = Light; M = Moderate; H = Heavy Table 2-3. Parasite fauna observed on 100 skin biopsies of Fundulus seminolis. Parasite Prevalence (%) Mean abun dance Intensity range Mean intensity Dactylogyrida 1 0.01 1 1.00 Digenea 2 La La Gyrodactylus sp. 14 0.29 1-8 2.07 Hirudinea 5 0.06 1-2 1.20 Ichthyophthirius multifiliis 3 0.03 1 1.00 Myxobolus sp 2 La La SECs 2 L-Ha Ma Tetrahymena sp. 1 0.01 1 1.00 a Parasite descriptors per field of view (T able 2-1) L = Light; M = Moderate; H = Heavy 20

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Table 2-4. Parasite fauna observed on 100 gill biopsies of Fundulus seminolis. Parasite Prevalence (%) Mean abundance Intensity range Mean intensity Dactylogyrida 46 0.73 1-11 1.60 Digenea 12 La La Gyrodactylus sp. 1 0.01 1 1.00 Ichthyobodo sp. 1 0.01 1 1.00 Myxobolus sp. 1 La La Piscinoodinium sp. 1 0.05 5 5.00 SECs 1 La La Trichodina sp. 5 0.16 1-12 3.20 a Parasite descriptors per field of view (T able 2-1) L = Light; M = Moderate; H = Heavy Table 2-5. Parasite fauna observed on 100 fin biopsies of Fundulus seminolis. Parasite Prevalence (%) Mean abundance Intensity range Mean intensity Digenea 27 La La Gyrodactylus sp. 2 0.03 1-2 1.50 Hirudinea 39 0.52 1-3 1.30 Ichthyobodo sp. 4 0.20 2-8 5.00 Myxobolus sp. 4 L-Ma La a Parasite descriptors per field of view (T able 2-1) L = Light; M = Moderate; H = Heavy 21

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CHAPTER 3 EXPERIMENTAL SALINITY TOLERANC E DETERMINATIONS FOR THE SEMINOLE KILLIFISH, FUNDULUS SEMINOLIS Introduction The seminole killifish, Fundulus seminolis (Girard, 1859), is an e ndemic Florida killifish with a geographic range within peninsular Florida from the St. Johns and New River drainage basins to just south of Lake Okeechobee (Pag e and Burr, 1991). Relatively little is known regarding the life history of F. seminolis with only one publicati on by DuRant et al. (1979) devoted entirely to the species. It has been referenced anecdotally or as a component in a larger study or survey in several publ ications (McLane, 1955; Phillips and Springer, 1960; Tabb and Manning, 1961; Gunter and Hall, 1963; Gunter and Hall, 1965; Foster, 1967; Griffith, 1974A; Nordlie, 2006). This species, commonly referred to as a bullminnow or mudminnow, is one of the largest members of the genus, reaching to tal lengths of 20 cm (Hoyer and Canfield, 1994). Its popularity as a local freshwat er baitfish for largemouth bass, Micropterus salmoides and other piscivorous game fish has generated intere st in this species as a potential candidate for aquaculture. With previous data placing th e upper salinity tolerance of F. seminolis at 23.4 g/L (Griffith, 1974A), culture of this species for use as a saltwater baitfish warrants further investigation. If the species is able to acclimat e to full strength seawater, it could be produced exclusively in freshwater ponds or recirculation systems only needing to be acclimated to saline water prior to marketing and distribution. Add itionally, with coastal property values at a premium and limited access to seawater, baitfis h producers would be able to utilize inland resources for the culture of marine baitfish. Determination of salinity tolerance following gr adual and acute transfer is necessary to evaluate the species physiologi cal limitations which will influence culture and marketing 22

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practices. Similar studies have been conducted by Lotan (1971), Griffith (1974A), Stanley and Fleming (1977), Chervinski (1983) Nordlie (1987), Crego and Peterson (1997), Nordlie (2000) and Fuller (2008) on salinity tole rance of various members of th e order cyprinodontiformes. For the most recent review of cyprinidontoid salin ity tolerance consult Nord lie (2006). The previous studies examined multiple salinity ranges utilizi ng various salt sources (seawater and synthetic sea salts) and freshwater dilutions to achieve experimental salinitie s both hypo and hypersaline to natural seawater (32-35 g/L) Distinct salinity thresholds defined by survival and select hematological indices were then characterized for the species in question. Experimental salinity determinations such as these may be a more accurate representation of the organisms true salinity tolerance than maximum reported field sa linities (Kefford et al., 2004). This can be seen in Phillips and Springers (1960) record of F. seminolis inhabiting waters with a salinity of 13.5 g/L, the highest recorded salinity from published field observations Griffiths (1974A) subsequent experimental salinity determina tion for this species pl aced the upper salinity tolerance much higher, with an experimental sa linity range of 19.3 33.4 g/L. However, Griffith (1974A) reported high mortalities in maintaining this species in captivity and his sample size for salinity tolerance determinations was only four individuals. Alt hough the lower lethal temperature has yet to be established for this species, the experimental water temperature of 15C might have exacerbated the physiological stress caused by th e acclimation. Taken together, these circumstances call into question the accuracy of the salinity range reported by Griffith (1974A). Investigations into the salinity tolerance of freshwater species have shared a predominant ecological motivation. Investigatio ns by Bringolf et al. (2005) and Schofield et al. (2006) assessed salinity tolerances as barriers to invasion for the flathead catfish, Pylodictis olivaris and 23

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goldfish, Carassius auratus respectively. While the impetus for the current study is the evaluation of F. seminolis as an aquaculture candidate, invest igations into the salinity tolerance of F. seminolis should provide valuable informati on about the species ability to handle osmoregulatory stressors and may further be extrapolated for ecological applications. Salinity tolerance is an important considerati on in the culture of marine and freshwater organisms. It provides information about basic husbandry requirements necessary for the species to thrive in captivity as well as potential appl ications for the cultured organisms. Additionally, economic considerations associated with culture of marine or brackish water species make low saline or freshwater culture an a ttractive alternative. Research into low salinity aquaculture of marine species is common, but few studies ha ve been conducted on acclimation of freshwater species to seawater. Experiments examini ng abrupt transfer of black sea bass, Centropristis striata, to low salinities have helped to identify a salinity threshold for the successful culture of this species (Young et al., 2006). Si milarly, gradual acclimation expe riments with Nile tilapia, Oreochromis niloticus, and blackchin tilapia, Sarotherodon melanotheron, (Lemarie et al., 2004) as well as larval salinity toleran ce experiments with striped mullet, Mugil cephalus thick-lipped grey mullet, Chelon labrosus (Hotos and Vlahos, 1998), and cobia, Rachycentron canadum (Faulk and Holt, 2006), have provided valuable ev idence regarding the osmoregulatory ability of a species for use in conventional aquaculture conditions. The purpose of this study was to ch aracterize the salinity tolerance of F. seminolis a potential candidate for marine baitfish aquacult ure. Abrupt and gradua l salinity acclimations were evaluated as well as salinity sources (sodium chloride vs. seawater). This investigation represents the first comprehensive stu dy focused on the salinity tolerance of F. seminolis 24

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Methods F. seminolis were collected by seine net from the eas tern shore of Lake George, in Volusia County Florida and transported to the University of Florida Indian River Research and Education Center in Fort Pierce. Fish were assessed for pa thogens and treated accord ingly to ensure healthy research specimens for the subsequent salinity experiments. The acute salinity tolerance of F. seminolis to varying concentrations of sodium chloride (NaCl) and natural seawater (NSW) were investigated as well as survival following gradual NSW acclimation. Sodium Chloride Acute Salinity Tolerance Thirty five fish were transferred from a 6900 L recirculating system to 85 L glass aquaria with one fish per aquarium during the entire acclimation and experimental periods. Specimens weight and total length (TL) were recorded prio r to transfer. Length and weight ranges of 120 145 mm and 17.0 29.4 g were recorded with means of 131.2 6.3 mm and 23.2 3.6 g, respectively. Aquarium systems were maintained at < 1 g/L salinity well water and recirculated through biofilter media during the 96 h acclim ation period. Dissolved oxygen (DO), pH, temperature, salinity, total ammonia nitrogen (T AN), and nitrite were re corded daily during the acclimation and experimental periods with total alkalinity and total hardness recorded on days one and three of acclimation and daily during the course of the experiment. DO and temperature were measured using a YSI 550A meter (YSI Inc., Yellow Springs, Ohio). Salinity was determined using a handheld refractometer, pH was measured using a Hach sensION1 portable pH meter and total alkalinity and total hardness were determined using standardized titration techniques (Hach Co., Loveland, Colora do). TAN and nitrite were evaluated spectrophotometrically using a Hach DR 4800 spectrophotometer (Hach Co., Loveland, Colorado). Aquaria were held at ambient temp erature with a range of 19.9 26.5C and a mean 25

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temperature of 22.7C during the experimental period. Temperature diffe rences among aquaria never exceeded 2C. An ambient photoperiod of 11 L : 13 D was used during the experiment. Fish were fed once a day to satiation on days tw o and three of acclimation and food was withheld on days one and four of acclimation and dur ing the entire 96 h experimental period. Following the 96 h freshwater acclimation, aquaria were made sta tic and individual aquariums were randomly assigned to one of five treatments with seven replicates per treatment. Differences among treatment groups lengths and weights were not significant ( F 4, 30 = 0.68, p = 0.610; F 4, 30 = 0.20, p = 0.939, respectively). Treatment salinities examined were 0 (control), 8, 16, 24, and 32 g/L. Salinities were abruptly changed by removing the appropriate amount of fresh water and adding a predetermined volume of a concentrated brine solution made by dissolving 99.5% NaCl (Morton White Crystal So lar Salt, Morton Intern ational Inc., Chicago, IL) into well water. Control aquaria had a pr edetermined volume of fresh water removed and subsequently replaced to maintain similar trea tment of control and experimental groups. Tanks were aerated to thoroughly mix the water, then salinities were re measured to confirm the desired concentrations were attained. Individual biofilters which had been preconditioned to treatment salinities were placed in each tank to control nitrogenous wastes. Aquaria were examined for mortalities as follows: once every hour from 0 12 h, once every 6 h from 12 48 h, and once every 12 h from 48 96 h. A final weight was re corded upon discovery of a mortality or upon the termination of the 96 h exposure period. Mortality was defined as loss of opercular movement and no response to physical stimulus. Natural Seawater Acute Salinity Tolerance Methods for the NSW acute salinity toxicity tria l adhere to the previous methods listed for the NaCl acute salinity toxicity trial with excepti ons as noted. Treatment sa linities were abruptly changed by removing a predetermined volume of fresh water and adding a known volume of 26

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natural seawater collected from the Atlantic Ocean and filte red through a 1 micron cartridge filter. Control aquaria were treated as previ ously stated. Aquaria were held at ambient temperature with a range of 20.5 25.6C and a mean temperature of 23.4C during the experimental period. Temperature differences among aquaria never exceeded 2C. A ambient photoperiod of 11.5 L : 12.5 D was used during the experiment. Length and weight ranges of 122 146 mm and 16.6 31.2 g were recorded with means of 129.7 5.8 mm and 21.9 3.5 g, respectively. Differences among treatment groups lengths were not statistically significant ( F 4, 30 = 1.12, p = 0.368). Differences among treatment groups weights were statis tically significant ( p = 0.010) but were not considered to be biol ogically significant fo r the purpose of this experiment with a mean treatment weight range of 18.7 23.8 g. Differences among treatment weights did not violate any assumptions of subsequent survival analysis. Natural Seawater Gradual Acclimation Survival Sixty-four fish were transferred from a 6900 L recirculating system to 32, 85 L glass aquaria, divided in half by aquarium part itions (TDSU, Penn-Plax Inc., Happauge, NY USA) with one fish per aquarium subdivision during th e entire acclimation and experimental periods. Fish weight and total length (TL) were recorded prior to transfer. Lengt h and weight ranges of 124 152 mm and 21.7 38.3 g were recorded with means of 136.3 6.3 mm and 27.5 4.5 g, respectively. Aquarium systems were maintained at < 1 ppt salinity well water and recirculated through biofilter media during the 96 h acclim ation period. Dissolved oxygen (DO), pH, temperature, salinity, total ammonia nitrogen (T AN), and nitrite were re corded daily during the acclimation and experimental periods with total alkalinity and total hardness recorded on days one and three of acclimation and at the initia tion and cessation of experimental treatment periods. Methods for water quality analysis adhe re to previously stated techniques with the exception of salinity, which was measured using a YSI 30 salinity/conductivity meter (YSI Inc., 27

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Yellow Springs, OH USA). Aquaria were held at ambient temperature with a range of 17.5 27.2C and a mean temperature of 22.6C during the experimental period. Temperature differences among aquaria never exceeded 2C. A ambient photoperiod of 13 L : 11 D was used during the experiment. Fish were fed once a day to satiation on days two and three of acclimation and food was withheld on days one and four of acclimation and during the entire experimental period. Following the 96 h freshwater acclimation, aquari a were made static and individual tanks were randomly assigned to one of eight treatm ents with eight replicates per treatment. Differences among treatment groups lengths and weights were not significant ( p = 0.784; F 7, 56 = 0.38, p = 0.908, respectively). Among the treatment groups, four underwent a gradual salinity change from 0 to 32 g/L over predetermined time periods with the remaining four treatments serving as corresponding controls Acclimation times of 24, 48, 72, and 96 h were chosen with approximate salinity increases of 5.3, 2.7, 1.7, a nd 1.3 g/L, respectively, every 4 h. The final salinity of 32 g/L was achieved 4 h preceding th e termination of the tr eatment group in question. Salinities were gradually changed every 4 h vi a addition of NSW equa lly dispersed between aquaria subdivisions until the desired salinity was achieved. Excess water during salinity changes was allowed to flow out of the aquariums sta ndpipe. A single air stone within each subdivision provided adequate mixing within and betw een subdivisions ensuring a homogeneous environment within each aquarium. Control aq uaria had corresponding volumes of freshwater added in the same fashion as their saline counter parts. Aquaria were examined for mortalities as follows: once every hour from 0 12 h and on ce every 4 h from 12 96 h. A final weight was recorded upon discovery of a mortality or upon the termination of the acclimation exposure 28

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period. Mortality was defined as loss of opercular movement and no response to physical stimulus. Statistical Analysis Survival was estimated using a Kaplan-Meier product limit estimator (Kaplan and Meier, 1958). Log-rank tests were then performed to compare generated survivorship curves among treatments and within treatments between experi ments. Length, weight, and water quality data were analyzed using a one-way ANOVA with a Tukeys HSD means separation test. Nonparametric data were analyzed with a Kr uskal-Wallis test. Kaplan-Meier analysis and subsequent log rank tests were performed in S PSS version 12.0 (SPSS Inc. Chicago, IL USA). All other statistical analyses were performed in SAS version 8.02 (SAS Institute Inc., Cary, NC USA) All numerical data are represented as the mean SD unless otherwise stated. Statistical differences were consid ered significant if p 0.05. Results Sodium Chloride Acute Salinity Tolerance Survival was 100% at salinities of 0 (cont rol), 8, and 16 g/L throughout the entire 96 h experimental period (Table 3 1). Acute transf er to higher salinities yielded 100% mortality with mean survival times of 6 h (95% Confidence Interval [CI], 5 7 h) at 24 g/L and 2 h (95% CI, 2 2 h) at 32 g/L (Table 3 1). Despite the complete mortality observed in these two treatments, survival times were significantly different ( p 0.0001) from each other as well as from all other treatments examined ( p 0.0001) (Table 3 2). No biologically significant differences among treatments in the measured water quality parameters were observed throughout the course of this experiment. During the 96 h acclimation period the following ranges and means SD were recorded, respectively: DO, 6.38 7.57, 7.11 0.42 mg/L; pH, 8.33 8.52, 8.43 0.06; TAN, 0.00 0.02, 0.01 0.01 mg/L; nitrite, 0.0006 29

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0.0039, 0.0025 0.0008 mg/L; alkalinity, 136.80 153.90, 139.65 6.98 mg/L CaCO3; hardness, 188.10 239.40, 202.35 19.99 mg/L CaCO3. During the 96 h experimental period the following ranges and means were record ed, respectively: DO, 7.14 8.35, 7.73 0.33 mg/L; pH, 8.28 8.58, 8.43 0.08; TAN, 0.00 0.09, 0.03 0.03 mg/L; nitrite, 0.0125 0.3102, 0.0753 0.0565 mg/L; alkalinity, 119.70 153.90, 135.80 9.80 mg/L CaCO3; hardness, 171.00 256.50, 193.90 18.90 mg/L CaCO3. Natural Seawater Acute Salinity Tolerance Survival was 100% at salinities of 0 (contro l), 8, 16, and 24 g/L thr oughout the entire 96 h experimental period (Table 3 3). Acute transfer to 32 g/L yielded 100% mortality with a mean survival time of 13 h (95% CI, 11 16 h) (Table 3 3). This group was significantly different ( p = 0.0002) from all other treatments (Table 3 4). No biologically significant differences among treatments in the measured water quality parameters were detected throughout the course of this experiment with the exception of total hardness. Due to experimental dilutions of seaw ater, total hardness varied by treatment salinity. During the 96 h acclimation period the following ra nges and means were recorded, respectively: DO, 6.66 7.90, 7.13 0.41 mg/L; pH, 8.19 8.53, 8.37 0.11; TAN, 0.00 0.06, 0.02 0.02 mg/L; nitrite, 0.0000 0.0043, 0.0011 0.0017 mg/L; alkalinity, 153.90 205.20, 171.00 18.73 mg/L CaCO3; hardness, 153.90 239.40, 190.95 33.19 mg/L CaCO3. During the 96 h experimental period the following ranges and me ans were recorded, respectively: DO, 7.50 8.24, 7.87 0.19 mg/L; pH, 8.06 8.63, 8.36 0.13; TAN, 0.00 0.10, 0.03 0.02 mg/L; nitrite, 0.0018 0.0356, 0.0107 0.0074 mg/L; al kalinity, 119.70 171.00, 144.00 9.80 mg/L CaCO3. Hardness ranges and means by salinity are as follows: 0 g/L, 153.90 222.30, 182.60 21.40 mg/L CaCO3; 8 g/L, 1250.00 2570.00, 1557.10 300.00 mg/L CaCO3; 16 g/L, 2700.00 30

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3090.00, 2860.00 95.10 mg/L CaCO3; 24 g/L, 3830.00 5580.00, 4282.10 295.40 mg/L CaCO3; 32 g/L, 5700.00 6060.00, 5808.60 117.80 mg/L CaCO3. Sodium Chloride vs. Natural Seawater When survival was analyzed within treatmen t salinity between salt source, NSW and NaCl were significantly different ( p < 0.0001) in the 24 and 32 g/L salinity treatments. Natural Seawater Gradual Acclimation Survival For all treatment groups, survival was 100%. Therefore, no analysis of the data was required. With the exception of total hardness, no biologically signifi cant differences among treatments in the measured water quality parameters were observed throughout the course of the experiment. During the 96 h freshwater acclimat ion period the following ranges and means were recorded, respectively: DO, 7.23 7.98, 7.63 0.29 mg/L; pH, 8.03 8.41, 8.29 0.11; TAN, 0.00 0.02, 0.01 0.01 mg/L; nitrite, 0.0011 0.0036, 0.0025 0.0010 mg/L; alkalinity, 180 mg/L CaCO3; hardness, 188.10 205.20, 199.50 8.83 mg/L CaCO3. During the 96 h experimental period the following ranges and me ans were recorded, respectively: DO, 7.56 8.35, 7.86 0.19 mg/L; pH, 8.12 8.46, 8.31 0.07; TAN, 0.00 0.07, 0.03 0.02 mg/L; nitrite, 0.0014 0.0200, 0.0040 0.0022 mg/L; al kalinity, 160.00 180.00, 170.00 10.1 mg/L CaCO3. Due to experimental addition of seawater, total hardness varied between control and treatment groups. Hardness ranges and final mean s by salinity are as follows: 0 g/L, 205.20 mg/L CaCO3; 32 g/L, 205.20 5840.00, 5717.50 116.02 mg/L CaCO3. Discussion Survival analysis for NaCl and NSW acute salinity tolerance experiments revealed F. seminolis can tolerate direct transfer from freshwater to salinities of 16 and 24 g/L. Daily water quality analysis during the 96 h experimental periods helped to elimin ate potentially confounding variables in an effort to iden tify salinity as the driving fo rce behind observed mortality. Upon 31

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completion of the 96 h experimental exposure in acu te transfer experiments, surviving fish were left in their respective aquaria and monitored fo r 2 weeks. No mortalities were observed during this time period for any surviving treatments regardless of salinity source. This is evidence that results of 96 h survival experiments are useful indicators of a species ability to survive long term in selected treatment conditions. Additi onally, although 100% survival was noted at 16 g/L in both NaCl and NSW experiments, fish in th e NSW treatment generally appeared healthier. F. seminolis at 16 g/L in NaCl exhibited multiple areas of hyperemia, overall pallor, decreased activity, and increased mucus production. Excessive mucus has been previously reported in Fundulus kansae exposed to low calcium artificial seawater (Potts and Fleming, 1970). Differences between survival due to salin ity source were evident in 24 and 32 g/L treatments (Tables 3 1 and 3 3). Due to l ack of physiological data it is only possible to speculate as to the underlying cau ses for the recorded differen ces. The role of calcium in osmoregulation and ionic transport has been we ll studied (Potts and Fleming, 1970; Carrier and Evans, 1976; Isaia and Masoni, 1976; Pic a nd Maetz, 1981; Hunn, 1985). Although calcium was not measured directly, tota l hardness levels (mg/L CaCO3) were recorded throughout both experiments. Signifi cant differences ( p < 0.0001) in hardness were noted among NSW treatment salinities of 8, 16, 24, and 32 g/L. Additionally, hardness values from these treatment groups were also significantly different (p < 0.0001) from all NaCl treat ment groups and the NSW control. It can be inferred from this data that calcium and other divalent ions were in a greater abundance in the NSW treatment groups than thei r NaCl counterparts. Low external calcium in an environment hypersosmotic to fish has been shown to increase ion efflux in Lagodon rhomboides (Carrier and Evans, 1976) and alter ne t fluxes of both ions and water in Anguilla anguilla (Isaia and Masoni, 1976). Potts and Fleming (1970) reported a 35% reduction in the gill 32

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permeability of F. kansae in NSW when compared with fresh water, thereby decreasing ion efflux and drinking rate. Conve rsely they also reported F. kansae to have an increased drinking rate and water exchange rate when transferre d to low calcium synthe tic seawater. Although not directly tested, findings from the aforementi oned studies support th e hypothesis that the mortality observed in the NaCl experiment rela tive to the NSW experiment was a result of loss of hydro-mineral regulation due to lower environmental ion concen trations, particularly calcium. However, environmental deficienci es of other divalent ions su ch as magnesium, should not be dismissed because these ions also play a func tional role in salinity acclimation (Isaia and Masoni, 1976). Results of gradual NSW acclimation were encour aging for this marine baitfish candidate. F. seminolis was able to easily acclimate to 32 g/L in all 4 time intervals with no mortalities in any treatment. Modest lethargy in most replicates of the 24 h treatment group was the only outward sign of physiological stress observed among any of the treatments upon completion of the acclimation process. Salinity tolerance of the predominantly euryhaline Fundulus genus has been extensively investigated yet almost no experi mental evidence exists regarding F. seminolis until the present study. Griffiths (1974A) seminal investigation on salinity tolerance of the genus is the only study that has evaluated this emerging a quaculture candidate, but due to confounding experimental factors these data are potentially unreliable. An additional study by Griffith (1974B) attempted to elucidate the role of pituitary c ontrol in the freshwat er acclimation of the genus, but only one F. seminolis was used, again limiting the us efulness of the data. Salinity tolerance results from this experiment confirm F. seminolis can acclimate to the upper end of Griffiths (1974A) reported range. Survival by F. seminolis after acute transfer to 24 g/L 33

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indicates superior salinity tolerance when compared to Fundulus nottii s acute tolerance of 17 g/L (Crego and Peterson, 1997). However, no upper level salinity tolerance has been determined to date but it is unlikely F. seminolis will tolerate salinities of 95+ g/L commonly reported for F. grandis (Nordlie, 2000), F. heteroclitus (Griffith, 1974A) F. confluentus (Griffith, 1974A; Nordlie, 2000) F. similis (Nordlie, 2000) F. parvipinnis (Feldmeth and Waggoner, 1972), Aphanius dispar (Lotan, 1971), and Adenia xenica (Nordlie, 1987). F. seminolis is widely considered to be a freshw ater killifish, with upper field salinities reported at 2.4 (Gunter and Hall, 1963), 7.3 (G unter and Hall, 1965), and 13.5 g/L (Phillips and Springer, 1960). Results from our experiments clearly indicate F. seminolis is capable of tolerating brackish and full strength seawater for extended periods of time. Experimental results substantiate the potential of F. seminolis as a candidate for marine baitfish aquaculture following seawater acclimation. Ability to to lerate acute transfer from 0 24 g/L and gradual acclimation to 32 g/L NSW over a broad time period allows for flexibility in the development of a salinity acclimation protocol. Additionally, a mean survival time of 13 h after acute transfer to full strength seawater allows for marketing and use of freshwater acclimated fish in the marine environment if so desired. NSW acclimated fish provide an additional option for bait retailers utilizing saline holding facilities as well as anglers who wish to store their bait in saline livewells. Demands of bait retail ers and sportsman will ultimately dictate the acclimation regime for this new marine baitfish species. 34

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Table 3 1. Kaplan-Meier survival analysis for acute NaCl transfer. Statistic cannot be calculated for comparison of treatments with 100% survival for both. Treatment (g/L NaCl) Mean survival (h SE) Survival (%) (Control) 0 100 8 100 16 100 24 6 1 0 32 2 0 Table 3 2. Log-Rank analysis among tr eatments for NaCl acute transfer. Treatment 0 g/L 8 g/L 16 g/L 24 g/L 8 g/L 16 g/L 24 g/L p<0.0001 p<0.0001 p<0.0001 32 g/L p<0.0001 p<0.0001 p<0.0001 p<0.0001 Statistic cannot be calculated for comparison of treatments with 100% survival for both. Table 3 3. Kaplan-Meier survival analysis for acute NSW transfer. Treatment (g/L NSW) Mean survival (h SE) Survival (%) (Control) 0 100 8 100 16 100 24 100 32 13 1 0 Statistic cannot be calculated for comparison of treatments with 100% survival for both. Table 3 4. Log-Rank analysis among treatments for NSW acute transfer. Treatment 0 g/L 8 g/L 16 g/L 24 g/L 8 g/L 16 g/L 24 g/L 32 g/L p<0.0002 p<0.0002 p<0.0002 p<0.0002 Statistic cannot be calculated for comparison of treatments with 100% survival for both. 35

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CHAPTER 4 EVALUATION OF A POINT OF CARE BLOOD ANALYZER FOR USE IN DETERMINATION OF SELECT HE MATOLIGICAL INDICES IN FUNDULUS SEMINOLIS Introduction In fishes, determinations of hematocrit and plas ma electrolyte values are valuable tools for diagnostic and research purposes. The osmoregulatory abilities of fishes are commonly examined using these hematological indices. Fluctuations in plasma electrolyte and hematocrit levels also provide clinicians and research ers with valuable knowledge rega rding the physiological impacts of environmental and pathogenic stressors. Conventional methods of hematocrit and electrolyte determination, microhematocrit centrifugation, fl ame photometry, and chlo ride titration, are gradually being replaced by new technologies. Point-of-care (POC) blood analyzers are both efficient and user friendly. POC analyzers have undergone extensive te sting and validation by the U.S. Food and Drug Administration and inde pendent researchers for use in humans and similar testing has occurred for use in veterinary applications with canine, feline, equine and poultry models (Grosenbaugh et al., 1998; Loone y et al., 1998; Acierno and Mitchell, 2007; Steinmetz et al., 2007). As the use of such tech nologies becomes more pervasive in current literature, investigations into the accuracy a nd reliability of POC analyzers for evaluating hematological indices of fish is warranted. Th ere is currently little research validating POC analyzers against conventionally accepted instrumentation (CAI) for use in fish. The evaluation of a POC blood analyzer for use in rockfish, Sebastes spp., (Harrenstien et al., 2005) is the most comprehensive validation to date. The seminole killifish, Fundulus seminolis, is a freshwater killifish with a geographic range within peninsular Florid a. This species, commonly refe rred to as a bullminnow or mudminnow, is one of the largest members of the genus, reaching total lengths of 20 cm (Hoyer and Canfield, 1994). Its popu larity as a local baitfish fo r piscivorous game fish has 36

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generated interest in this species as a potential candidate for aquaculture Acclimation of this species to the marine environment for use as a baitfish will result in numerous physiological changes. Collection of limited blood quantities makes determinations of hematological indices problematic for this species. The blood volume requirement and rapid analysis time of the iSTAT unit makes it an attractive option for hema tological evaluations with this species. The purpose of this study was to evaluate a POC blood analyzer (i-STAT), and chosen cartridge (E3+) (Heska Corp., Fort Collins, CO, USA) against CAI for use in determination of hematocrit, sodium, potassium, and chloride values in F. seminolis Operating procedures following manufacturers specifications and alternative protocols using heparin diluted blood were analyzed. Methods F. seminolis were collected by seine net from the eas tern shore of Lake George in Volusia County Florida and transported to the University of Florida Indian River Research and Education Center in Fort Pierce, Florida. Fish were a ssessed for pathogens and treated accordingly to ensure healthy research specimens. Fish were held for a minimum of 6 months in an outdoor 6900 L recirculating system with biofilter media, a UV sterilizer and a 100 micron bag filter to maintain optimal water quality. Salinity was maintained at 2 g/L with an ambient temperature and photoperiod during the experimental period of February 2 March 20, 2008. On days of blood analyzation research specimens were co llected from the recirculating system and transported to the laboratory (~5min) where they were held in aerated 37.85 L aquaria filled with water from the recirculating system. All blood collection occurred within 24 h of transport. Experiments were performed to validate the accu racy of the i-STAT handheld clinical blood analyzer and the E3+ cartridge (Heska Corp., Fort Collins, CO, USA) against conventional hematological instrumentation. The E3+ cartridge is designed to measure sodium, potassium, 37

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chloride, hematocrit, and hemoglobin from w hole blood aliquots. Hemoglobin measurements were not examined in this study because hemogl obin is not directly measured by the i-STAT. Instead hemoglobin is automatically calculated by multiplying a human blood conversion factor (0.34) by the measured hematocrit percentage. The E3+ cartridge was chosen because it is one of the most basic cartridges produced by the manufacturer yet its analyzed parameters have application across numerous disciplines. Whol e blood was used to measure the remaining parameters in an initial experiment. A second experiment implemented a whole blood heparin dilution prior to analysis with the POC unit. No attempt was made to establish reference ranges for blood parameters in F. seminolis Whole Blood Analysis Twenty four F. seminolis were used in this experiment ranging in length from 126 154 mm with a mean length of 139.1 7.0 mm. Wei ghts ranged from 20.7 37.4 g with a mean weight of 27.0 4.7 g. None of the fish sampled exhibited any signs of gross parasitism or infection. Aquaria water did not contain anesthetic to prevent alteration of hematological indices of interest (Reinitz and Ri x, 1977). Fish were individually removed from the aquaria and weighed and measured. The specimens caudal peduncle was rinsed with nanopure water and blotted dry then promptly severed. A 100 l lithium heparinized capillary tube (7-000-1000, Drummond Scientific, Broomall, PA, USA) was filled first by placing the tube against the severed caudal vessel. Multiple ammonium hepari nized microhematocrit capillary tubes were then filled in the same fashion until blood flow ceased. Fish were then euthanized by pithing. All blood collection was completed within 5 minutes after removal from the holding aquaria. The 100 l lithium heparinized sample was immediatel y used to fill the E3+ cartridge to its prescribed level after which it was insert ed into the i-STAT unit and analyzed. The manufacturers operating pr ocedures for the i-STAT were adhered to throughout the 38

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experiment (i-STAT system manual, 1997). Twenty four E3+ cartridges were utilized in this experiment. Microhematocrit tubes were cappe d with sealant and centrifuged at 13,460 g (11,500 rpm) for 6 minutes, after which the hematocrit wa s determined. A mean hematocrit for each fish was calculated from the total number of micr ohematocrit tubes collected. Plasma was then separated from the red blood cells and frozen in mi crocentrifuge tubes at -20C for later analysis. Due to the small size of the fish sampled, blood volume and thus plasma volume was limited, allowing each sample to be run only once through the various analyzers. All samples were run within 30 days of freezing. Thawed plasma samples were analyzed for sodium and potassium concentrations utilizing a Jenway PFP7 flame photometer (Bibby Scientific Ltd., Essex, England) according to the manu facturers specifications. Plasma chloride concentration was determined utilizing a Labconco digital chlo ridometer (Labconco, Kansas City, MO, USA) according to the manufacturers directions. Whole Blood Heparin Dilution Analysis Methods for the validation of the i-STAT using a heparin diluted whole blood sample adhere to the methods previously listed for th e whole blood analysis except as noted below. Dilution of whole blood aliquots has not been validated by the manufacturer but was used by Suski et al. (2007). Fifteen F. seminolis were used in this experiment ranging in length from 129 155 mm with a mean length of 140.5 8.5 mm. Weights ranged from 18.4 37.7 g with a mean weight of 29.2 5.0 g. The specimens cauda l peduncle was rinsed with nanopure water and blotted dry after which it was promptly severed. A 90 l sample of whole blood was drawn up in a lithium heparinized capillary tube from the severed caudal vessel. The blood was then expelled into a sterile microc entrifuge tube into which 10 l of 150 usp/ml lithium heparin (H0878, Sigma-Aldrich, St. Louis, MO, USA) di ssolved in nanopure water was added. The sample was mixed by gently pipetting to avoi d hemolysis and then transferred to the E3+ 39

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cartridge and analyzed. The fina l heparin concentration of 15 usp/ml blood achieved was within the range of 14 16 usp/ml blood used by Harrens tien et al. (2005). Results obtained from the iSTAT were then corrected for the dilution factor Fifteen E3+ cartridges were utilized in this experiment. Remaining blood samples were coll ected and processed undiluted as previously stated for the whole blood experiment. Statistical Analysis Data generated by the i-STAT and E3+ cartr idge were compared with conventional instrumentation employed to analyze the indices under investigation. Numb ers of data points per analyte varied due primarily to cartridge error or results outside the reportable range of the POC analyzer. The Bland-Altman met hod for assessing agreement between two methods of clinical measurement was used (Bland and Altman, 1986) as well as calculations of correlation coefficients. A two tailed paired student t-test was also used to compare values generated by both methods of analysis. Bias was defined as the m ean percent difference between values generated by both methods. The limits of agreement (LA) we re defined as the bias 2 SD. Differences were considered significant when p 0.05. Results Whole Blood Analysis A 37.5% failure rate, defined as no recordab le results, was experienced with the POC analyzer. The error code unabl e to position sample was the mo st frequent code encountered. Of the remaining 15 cartridges not all yielded results deemed us eful for our analysis. Three cartridges reported hematocrit (< 10%) values outside the reportable range for this unit. Additionally, four cartrid ges reported chloride (> 140mmol/L) values outside the instruments reportable range as well. These va lues were not considered in s ubsequent comparisons. Analysis by CAI was 100% successful with no results above or below the instrumentations thresholds for 40

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all samples analyzed. Mean values by paramete r for each method of analysis are presented in Table 4 1. Significant differences ( p 0.05) were noted between methods for every parameter mean analyzed by t-test (Table 4 1). Correlation between methods were weak ( r < 0.9) and varied broadly with r values ranging from -0.52 0.79 (Table 4 1). Bland-Altman analysis revealed hematocrit to have a value lying outside the limits of agreement (Figure 4 1). LA values of 34.8, 14.6, 25.2, and 16.0% and bi as values of -44.5, -26.0, -10.3, and 12.8% were calculated for hematocrit, sodium, potassium, and chloride respectively. Whole Blood Heparin Dilution Analysis A 33.3% failure rate, defined as no recordab le results, was experienced with the POC analyzer. The error code unabl e to position sample was the mo st frequent code encountered. Of the remaining 10 cartridges, a ll yielded results deemed useful for analysis. Analysis by CAI was 100% successful with no results above or be low the instrumentations thresholds for all samples analyzed. Mean values by parameter for each method of analysis are presented in Table 4 2. Significant differences ( p 0.05) were noted between met hods for every parameter mean analyzed by t-test (Table 4 2) Correlation coefficients were ge nerally weak with the exception of hematocrit (r = 0.96). Coefficients again varied broadly with r values ranging from -0.02 0.96 (Table 4 2). Bland-Altman analysis revealed hematocrit and ch loride to have values lying outside the limits of agreement (Figure 4 2). LA values of 40.0, 17.1, 23.3, and 17.0% and bias values of -29.0, -16.0, 15.5, and 17.7% were calculated for hematocrit, sodium, potassium, and chloride respectively. Discussion Generally, mean values obtained from CAI were higher when compared with the POC analyzer using both whole and diluted blood samp les. Chloride values generated in both experiments as well as potassium values resulting from heparin diluted blood were exceptions. 41

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Significant differences observed between all mean parameter values analyzed by t-tests agree with previously reported result s by Harrenstien et al. (2005) in which all twelve parameters generated by the POC analyzer except potassium and glucose were found to be significantly different from their CAI counter part. Bias and LA values varied considerably throughout the experiments. Hematocrit bias (-44.5% and 29.0%) agreed with the trends observed by Harrenstien et al. (2005) in ro ckfish and the approximate -25% bias reported by Howard and Wack (2002) in birds. All LA values from both whole blood and heparin dilutions fell well outside Clinical Laboratory Improvement Act (CLIA) guidelines governing accepted discrepancies in testing methodologies for hematocr it ( 6%), sodium ( 4 mmol), potassium ( 0.5 mmol), and chloride ( 5%) (United States De partment of Health and Social Services, 1992). While these guidelines were developed to ensure accuracy and precision in a clinical setting, it should be a goal of individuals in the research field to adhere to such stringent scientific standards. Differences between the POC values and CAI values can not be easily explained. As the i-STAT is made to function with mammalia n blood and uses the Nernst equation to relate potential with concentration, possible temper ature effects of poikilothermic blood on the potentiometric determination of electrolytes could account for observed discrepancies. Numerous limitations of the POC unit and th e E3+ cartridge were identified through the course of this experiment. Successful sample analysis was hindered by the rapid clotting of collected blood despite immediate transfer. Simila r problems in sample analysis have been previously noted by Harrenstien et al. (2005), Olsvik et al. (2007) and Steinmetz et al. (2007) but not to the degree seen in this study. Dilution of samples with lithium heparin in subsequent experiments did not ameliorate this problem. Hematocrit and chloride values outside the instruments reportable range we re an additional difficulty enc ountered. Suski et al. (2007) and 42

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Olsvik et al. (2007) reported similar problems with hemoglobin and sodium respectively. Interestingly, Harrenstien et al. (2005) was not able to evaluate chloride in their experiment because all generated values were above the i-STATs reportable upper limit of 140 mmol/L. Brill et al. (2008) reported POC mean sodium values of 257 6 and 278 4 mmol/L and POC mean chloride values of 210 2 and 216 2 mmol/L for Carcharhinus plumbeus far exceeding the reportable range for either parameter. Su ski et al. (2007) reporte d diluting blood samples by 25% and it can be inferred from values reporte d by Brill et al. (2008) dilutions of the same magnitude or greater were used to circumvent out of range values. Heparin dilution of whole blood samples in our experiment brought values within the reportable range of the unit but overall, fared no better in regards to bias or LA values (Figure 4 2) than whole blood analysis. Despite only one validation study evaluating th e i-STAT against CAI for use in rockfish (Harrenstien et al., 2005), a myri ad of species and broad array of blood parameters have been subsequently analyzed and reported in scientific literature. bonefish, Albula vulpes (Suski et al., 2007), cod, Gadus morhua (Foss et al., 2006; Remen et al., 2008), Atlantic salmon, Salmo salar (Olsvik et al., 2007; Petri et al., 2008), spiny dogfish, Squalus acanthias (Mandelman and Farrington, 2007), Nile tilapia, Oreochromis niloticus (Choi et al., 2007), sandbar sharks, Carcharinus plumbeus (Brill et al., 2008), amur sturgeon, Acipenser shrenckii (Lu et al., 2005), and turbot, Scophthalmus maximus (Foss et al., 2007) comprise the increasing number of species whose blood has been analyzed by the unit. Recommendations by Harre nstien et al. (2005) against the use of the i-STAT for determination of chloride, potassium, glucose, total CO2, and HCO3 in fish as well as calls for additional studies on multiple species have been ignored. Foss et al. (2006), Choi et al. (2007), Foss et al. (2007), Ol svik et al. (2007), Brill et al. (2008), Petri et al. (2008), and Remen et al. (2008) used the iSTAT to analyze hematological parameters 43

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deemed unreliable and inaccurate with no effort to provide further methodological validation or correct the reported parameters for bias. Furthe rmore, the dilution of blood prior to analysis (Suski et al., 2007) is not recommended by the ma nufacturer and has yet to be evaluated against CAI. Choi et al. (2007) acknowledges the need for validation of the POC analyzer as its popularity increases, yet does not corroborate expe rimental POC values with CAI. However, Mandelman and Farrington (2007) made an effort to inform readers that pa rameter values are not absolute and should be evaluated with caution. Method validation studies (Grosenbaugh et al., 1998; Looney et al ., 1998; Acierno and Mitchell, 2007; Steinmetz et al., 2007) examining new technologies or new species are essential for the generation and dissemination of reliable da ta to the scientific community. Results from our analyses support the findings of Harrenstien et al. ( 2005) and we reiterate the need for further validation studies using multiple species. This PO C unit may be useful for elucidation of trends within analyzed parameters but results should be interpreted carefully as further testin g is still needed. None of the blood parameters analyzed by the i-STAT in this experiment could be considered reliable. Use of unvalidated instrumentation must be avoided to prevent publication and distribution of potentially erroneous data Methodological validation must be considered paramount for the introduction of new t echnologies in resear ch applications. 44

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Table 4 1. Mean SD values for analyzed hematological parameters using both the POC analyzer (i-STAT) and conventionally accepted instrumentation (CAI) generated from whole blood aliquots Significant results ( p values) from paired t-tests and calculated correlation coefficients ( r ) comparing both methods of analysis are reported. Parameter N POC analyzer CAI t-test ( p) r Hematocrit (%) 11 16 4 25 6 <0.0001 0.79 Sodium/Na+ (mmol/L) 15 140 2 182.3 12.1 <0.0001 -0.52 Potassium/K+ (mmol/L) 14 6.2 0.8 6.8 0.6 0.0118 0.36 Chloride/Cl(mmol/L) 11 137 2 121.0 10.4 0.0003 0.35 Table 4 2. Mean SD values for analyzed hematological parameters using both the POC analyzer (i-STAT) and conventionally accepted instrumentation (CAI) generated from heparin diluted whole blood aliquots. Significant results ( p values) from paired t-tests and calculated correlation coefficients ( r ) comparing both methods of analysis are reported. Parameter N POC analyzer CAI t-test ( p) r Hematocrit (%) 10 22 8 29 6 <0.0001 0.96 Sodium/Na+ (mmol/L) 10 142 5 166.5 12.7 0.0003 -0.03 Potassium/K+ (mmol/L) 10 6.6 0.9 5.7 0.8 0.0038 0.6 Chloride/Cl(mmol/L) 10 135 7 113.3 7.9 <0.0001 -0.02 45

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-90 -80 -70 -60 -50 -40 -30 -20 -10 0 10121416182022242628 Mean Hematocrit (%)% Difference (i-STAT CAI) -45 -40 -35 -30 -25 -20 -15 -10 -5 0 148 153 158 163 168 173 Mean Sodium (mmol/L)% Difference (i-STAT CAI) -40 -30 -20 -10 0 10 20 55 566 577 5 Mean Potassium (mmol/L)% Difference (i-STAT CAI) 8 -5 0 5 10 15 20 25 30 35 115 120 125 130 135 140 145 Mean Chloride (mmol/L)% Difference (i-STAT CAI) Figure 4-1. Bland-Altman plots of blood pa rameter percent difference (i-STAT conventionally accepted instrumentation [C AI]) versus overall mean blood parameter concentration ([i-STAT + CAI]/2) for hema tocrit, sodium, potassium, and chloride values generated from whole blood aliquots. Bias (mean % difference between the iSTAT and the CAI) is represented by the so lid line. Limits of agreement (bias 2SD) are represented by the dashed lines. Each point represents values generated from an individual F. seminolis 46

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-80 -70 -60 -50 -40 -30 -20 -10 0 10 20 12.0 17.0 22.0 27.0 32.0 37.0 Mean Hematocrit (%)% Difference (i-STAT CAI) -35 -30 -25 -20 -15 -10 -5 0 5 140.0 145.0 150.0 155.0 160.0 165.0 170.0 Mean Sodium (mmol/L)% Difference (i-STAT CAI) -20 -10 0 10 20 30 40 50 4.55.05.56.06.57.07.58.08. Mean Potassium (mmol/L)% Difference (i-STAT CAI) 5 0 5 10 15 20 25 30 35 40 113.0115.0117.0119.0121.0123.0125.0127.0129.0131.0 Mean Chloride (mmol/L)% Difference (i-STAT CAI) Figure 4-2. Bland-Altman plots of blood pa rameter percent difference (i-STAT conventionally accepted instrumentation [C AI]) versus overall mean blood parameter concentration ([i-STAT + CAI]/2) for hema tocrit, sodium, potassium, and chloride values generated from heparin diluted whol e blood aliquots. Bias (mean % difference between the i-STAT and the CAI) is repr esented by the solid line. Limits of agreement (bias 2SD) are represented by th e dashed lines. Each point represents values generated from an individual F. seminolis 47

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CHAPTER 5 PHYSIOLOGICAL EVALUATION OF FUNDULUS SEMINOLIS FOLLOWING FOUR SEAWATER ACCLIMATION PROTOCOLS Introduction Seawater acclimation in fishes is a culminati on of a myriad of physio logical processes; a current understanding of its va rious components is best summa rized by Evans et al. (2005). Research into ion secretion and uptake, wa ter balance, and morphological and enzymatic changes have elucidated a multitude of pro cesses allowing this acclimation. The strongly euryhaline killifish, Fundulus heteroclitus has been the focus of a majority of those examinations (Epstein et al., 1967; Karnaky et al., 1976; Jacob and Taylor, 1983; Wood and Marshall, 1994; Marshall et al., 1999; Marshall et al., 2000; Katoh et al., 2001; Mancera and McCormick, 2002). A natural euryhalinity has been a commonality of other experimentally utilized species. The gilthead sea bream, Sparus auratus (Laiz-Carrion et al., 2005), Mozambique tilapia, Oreochromis mossambicus (Foskett et al., 1981), and rainbow trout, Oncorhynchus mykiss (Madsen and Naamansen, 1989) are ju st several of the previously examined species. Few studies have assessed the osmoregulatory functions of relatively stenohaline fishes acclimated to atypical salini ties. Evaluation of low saline aquaculture of marine fishes have provided an impetus for these investigations (Faulk and Holt, 2006; Resley et al., 2006; Young et al., 2006; Fielder et al., 2007). The ability to culture marine fishes in a lo w saline or freshwater environment has many implicit advantages yet reproductive and physiologi cal limitations may impede success. As fish are transferred from a hypoosmotic to hyperosmotic environment, osmoregulatory complications may manifest in the form of increased plasma electrolyte concentrations and thus increased plasma osmolality. During seawater acclimation of F. heteroclitus, Marshall et al. (1999) reported increased plasma sodium concentrations from 1 24 h post tr ansfer with a peak 48

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osmolality occurring 24 h after transfer. Jacob and Taylor (1983) reported significant sodium and osmolality elevations up until 48 h post transfer. Investigations by Pickford et al. (1969) on F. heteroclitus blood serum showed an average decrease in sodium, potassium, and chloride of 9% in fish held in freshwater versus seawater Similar acclimation experiments conducted on the cyprinodontiform fishes F. kansae (Stanley and Fleming, 1976) and Aphanius dispar (Lotan, 1971) again described an increased elevation of plasma osmolality and ion influx. Loss of ionic homeostasis due to salinity acclimation is well es tablished and has been previously reported in commonly aquacultured species (Imsland et al., 2003; Resley et al., 2006; Young et al., 2006; Fielder et al., 2007). Elevated hema tocrit percentage is an indicat or of increased water efflux and hemoconcentration (Marshall et al., 2005). Wa ter efflux following hyperosmotic transfer may also be quantified in the calculated water conten t of a muscle tissue sample. Significant loss of muscle water content, such as that recorded by Altinok et al. (1998) in se awater acclimated Gulf of Mexico sturgeon, Acipenser oxyrinchus desotoi or seawater acclimated medaka, Oryzias latipes, (Sakamoto et al., 2001) can provide valuable osmoregulatory information and confirm observed trends in hematocrit values. Osmoregulatory limitations of candidate marine aquaculture species need to be evaluated to determine feasibility of production and marketing. The seminole killifish, Fundulus seminolis, is a naturally stenohaline freshw ater killifish (Phillips and Springer, 1960; Gunter and Hall, 1963; Gunter and Hall, 1965) which has recently emerged as a candidate for marine baitfish aquaculture. Griffiths (1974A) experimental salinity determination for this species placed the upper mean salinity tolerance at 23.4 g/L, with an experimental salinity range of 19.3 33.4 g/L. If the species is able to acclimate to full streng th seawater, it could be produced exclusively in freshwater ponds or recirculation systems, onl y requiring acclimation to saline water prior to 49

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marketing and distribution. Decreased reliance u pon saltwater and coastal access would allow for the utilization of inland resources for the culture of this species. Evans et al. (2005) recognized that molecular and biochemical events accompanying acute salinity transfers may be species specific. Osmotic evaluation of F. seminolis as a stenohaline freshwater analogue to F. heteroclitus could generate data which may elucidate physiological responses not previously observed in euryhaline Fundulus species. Therefore, the objective of this study was to measure the osmoregulatory effects (plasma osmolality, sodium, potassium, chloride, hematocrit, and muscle water content) of four different rates of seawater acclimation on F. seminolis Methods F. seminolis were collected by seine net from the eas tern shore of Lake George, in Volusia County, Florida and transported to the Universi ty of Florida Indian River Research and Education Center in Fort Pierce. Fish were a ssessed for pathogens and treated accordingly to ensure healthy research specimens fo r the subsequent salinity experiments Natural Seawater Acclimation Seventy two fish were transferred from a 6900 L recirculating system to 36, 85 L glass aquaria, divided in half by aquarium partitions (TDSU, Penn-Plax Inc., Happauge, N.Y.) with one fish per aquarium subdivision during the acclimation and e xperimental periods. Specimens weight and total length (TL) were recorded prio r to transfer. Length and weight ranges of 124 152 mm and 21.7 38.3 g were recorded with means of 136.1 6.4 mm and 27.5 4.5 g, respectively. Aquarium systems were main tained at < 1 g/L salinity well water (Na+ = 6.1, K+ = 0.3, Cl= 3.3 mmol/L) and recirculated through biofilter media during the 96 h acclimation period. Dissolved oxygen (DO), pH, temperature, salinity, total ammonia nitrogen (TAN), and nitrite were recorded daily during the acclimation and experimental periods with total alkalinity 50

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and total hardness recorded on days one and th ree of acclimation and at the initiation and cessation of experimental treatment periods. DO and temperature were measured using a YSI 550A meter and salinity was determined usi ng a YSI 30 salinity/conductivity meter (YSI Inc., Yellow Springs, Ohio). pH was measured using a Hach sensION1 portabl e pH meter and total alkalinity and total hardness were determined us ing standardized titrati on techniques (Hach Co., Loveland, Colorado). TAN and nitrite were eval uated spectrophotometrically using a Hach DR 4800 (Hach Co., Loveland, Colorado). Aquaria were he ld at ambient temperature with a range of 17.5 27.2C and a mean temperature of 22.6C dur ing the experimental period. Temperature differences among aquaria never exceeded 2C. A ambient photoperiod of 13 L : 11 D was used during the experiment. Fish were fed once a day to satiation on days two and three of acclimation and food was withheld on days one and four of acclimation and during the entire experimental period. Following the 96 h freshwater acclimation, a quaria were made static and randomized among nine treatments with eigh t replicates per treatment. Differences among treatment groups lengths and weights we re not significant ( F 8, 63 = 0.45, p = 0.885; F 8, 63 = 0.32, p = 0.954, respectively). Among the treatment groups, the precontrol (time 0) group was sacrificed immediately after the freshwater acclimation peri od; four treatments were subjected to a gradual salinity change from 0 to 32 g/L over predeter mined time periods with the remaining four treatments serving as controls for each corr esponding time period. Acclimation times of 24, 48, 72, and 96 h were chosen with approximate salinity increases of 5.3, 2.7, 1.7, and 1.3 g/L, respectively, every 4 h. The final salinity of 32 g/L was attained 4 h prior to the termination of each treatment group. Salinities were gradually ch anged every 4 h via addition of NSW equally dispersed between aquaria subdivisions until th e desired salinity was achieved. Excess water 51

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during salinity changes was allowed to flow out of the aquariums standpipe. A single air stone within each subdivision provide d adequate mixing within and between subdivisions ensuring a homogeneous environment within each aquari um. Control aquaria had similar volumes of freshwater added in the same manner as saline treatment tanks. Once the specified acclimation time had b een reached the treatment group and its corresponding control were sacrificed and bl ood and tissues were co llected. Fish were individually removed from thei r respective tanks and subsequently weighed. The fishs caudal peduncle was rinsed with nanopure water and blot ted dry after which it was promptly severed. Ammonium heparinized microhematocrit capillary tubes were then filled by placing the tube against the severed caudal vessels until blood flow ceased. This method was employed to prevent contamination of the blood sample by any e xogenous or endogenous fluids other than blood. Fish were then euthanized by pithing. Weights and blood collec tion were completed within 5 minutes after removal from tanks. Microhematocrit tubes were capped with clay and centrifuged at 13,460 g (11,500 rpm) for 6 minutes, after which the hematocrit determined. A mean hematocrit for each fish was calculated from the total number of microhematocrit tubes collected. Plasma was then separated from the red blood cells and frozen in microcentrifuge tubes at -20C for later analysis. Additionall y, a muscle tissue sample (mean weight = 0.804 0.227 g) was removed from each specimen to determine muscle water content (MWC). Samples were weighed in tared aluminum weigh boats be fore and after drying at 105C for 24 h (Altinok et al., 1998). Due to the small size of the fish sampled, blood volume and thus plasma volume was limited, allowing each sample to be run only on ce through the various an alyzers. All plasma samples were run within 30 days of freezing. Thawed samples were analyzed for sodium and potassium concentrations utilizing a Jenway PFP7 flame photometer (Bibby Scientific Ltd., 52

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Essex, England) according to the manufacturers specifications. Chloride concentration was determined utilizing a Labconco digital chlo ridometer (Labconco, Kansas City, MO, USA) according to the manufacturers acce pted protocols and osmolality was analyzed using a Wescor 5520 vapor pressure osmometer (W escor Inc., Logan, UT, USA). Statistical Analysis All data were analyzed with a randomized block ANOVA to elucidate any positional effects resulting from tank subdivisions followe d by a Tukeys HSD means separation test using SAS version 8.02 (SAS Institute Inc., USA). All percentage data was arcsine square root transformed prior to analysis All numerical data are represented as the mean SD. A p value 0.05 was considered statistically significant for all analyses. Results For all analyses, a randomized block ANOVA was used to elucidate any positional effects resulting from subdividing aquari a. No significant block effect was recorded for any of the quantified parameters in any of the subsequent analyses with the exception of osmolality when comparing the precontrol and control groups. This effect was marginally significant ( p = 0.0425) and not observed in any other analyses, thus ea ch subdivision was consid ered an independent experimental unit. Analyses of hematocrit, sodium, potassium, and chloride among the precontrol and control experimental groups showed no significant di fferences. The precontrol osmolality and MWC were significantly different ( p 0.0117, p 0.0008) when compared to all other control groups. Additionally, a significant difference ( p = 0.0425) in body weight change (BWC) between the precontrol and 96 h control was also noted. Plasma sodium (Figure 5 1), chloride (Figur e 5 2), and osmolality (Figure 5 3) values were all significantly ( p < 0.0001) elevated for all seawater acclimated fish when compared with 53

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their corresponding freshwater controls. BWC values (Figure 5 4) revealed variable weight loss across all control and saline acclimated groups ; however, NSW acclimated BWC values were significantly higher ( p < 0.0001) when compared with values obtained from control fish. MWC (Figure 5 5) percentages co rroborated the BWC data, show ing significantly less water (p < 0.0001) in the muscle samples of NSW acclimated fish compared with their freshwater counterparts. The hypertonic NS W exposure significantly ( p 0.0007) elevated plasma potassium (Figure 5 6) concentrations in all but the 48 h acclimation treatment ( p 0.1000). Conversely, hematocrit percentages (Figure 5 7) were variable and only the 96 h acclimation treatment differed significantly ( p = 0.0337) from the 96 h control. Table 5 1 summarizes all hematological values calculated from this experiment. MWC, BWC, and hematological parameters were compared among NSW acclimation rates of 24, 48, 72, and 96 h. Plasma potassium c oncentration, 11.5 0.9 mmol/L (Table 5 1), was highest following 24 h NSW acclima tion and was significantly different (p < 0.0001) from all other acclimation times (Figure 5 6). Mean osmolality ( p = 0.0106, p = 0.0414) and chloride ( p = 0.0446, p = 0.0038) values from the 24 and 72 h accl imation periods were significantly elevated when compared with values from those acclimated to NSW over 96 h (Figures 5 2 and 5 3). MWC percentages exhibited a similar trend, with the 96 h acclimation muscle samples containing a significantly ( p = 0.0081, p = 0.0083) larger proportion (1.9%) of water than both the 24 and 72 h treatments (Figure 5 5). Pl asma sodium concentrations from 72 and 96 h acclimation treatments were appreciably lower ( p 0.0438) than those recorded in 24 and 48 h acclimation periods. Analysis of hematocrit and BWC demonstrated no significant differences among any of the NSW acclimation periods. 54

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With the exception of total hardness, no biologically significant differences among treatments were detected in any of the measured water quality parameters throughout the course of the experiment. During the 96 h freshwater acclimation period th e following ranges and means were recorded, respectively: DO, 7.23 7.98, 7.63 0.29 mg/L; pH, 8.03 8.41, 8.29 0.11; TAN, 0.00 0.02, 0.01 0.01 mg/L; nitrite, 0.0011 0.0036, 0.0025 0.0010 mg/L; alkalinity, 180 mg/L CaCO3; hardness, 188.10 205.20, 199.50 8.83 mg/L CaCO3. During the 96 h experimental period the following ranges an d means were recorded, respectively: DO, 7.56 8.35, 7.86 0.19 mg/L; pH, 8.12 8.46, 8.31 0.07; TAN, 0.00 0.07, 0.03 0.02 mg/L; nitrite, 0.0014 0.0200, 0.0040 0.0022 mg/L; al kalinity, 160.00 180.00, 170.00 10.1 mg/L CaCO3. Due to experimental addition of seawater, total hardness varied between control and treatment groups. Hardness ranges and final me ans by salinity were: 0 g/L, 205.20 mg/L CaCO3; 32 g/L, 205.20 5840.00, 5717.50 116.02 mg/L CaCO3. Discussion Classic salinity acclimation experiments using F. heteroclitus (Jacob and Taylor, 1983; Marshall et al. 1999; Mancera and McCormick, 2 000) have focused on physiological effects resulting from abrupt transfer to NSW with an alyses usually occurri ng over a predetermined time course. Inability of F. seminolis to survive acute transfer to full strength NSW precludes replication of these studies. If effects of full strength NSW are to be elucidated, investigations into salinity acclimation of F. seminolis are thus restricted to physiological evaluations of abrupt salinity transfer into a maximum tolerated salinity or analysis of a gradual acclimation. Four gradual acclimation periods were investigated be cause full strength NSW is the terminal salinity of interest for marine baitfish. F. seminolis was able to tolerate NSW acclimation times of 24, 48, 72, and 96 h with varying physiological results. Hematocrit values in the 72 and 96 h acclimation groups were 55

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significantly lower than their co rresponding controls. Although every effort was made to treat fish equally, unknown stressors may account for the elevated control hematocrit values. Osmotic efflux of water would contribute to hemoconcentration and increased hematocrit values in more rapid acclimation periods. NSW acclimated fish lost significantly more weight than their controls throughout the course of seawater acclimation although no significant di fferences in BWC was seen among acclimation times. Similarly, the grea test decline in MWC was exhibited by NSW acclimated F. seminolis with a mean loss of 5.6 1.0% for all acclimation times when compared with freshwater controls, again suggesting in creased gill permeability and water efflux in response to the hyperosmotic e nvironment. Inve stigation of O. latipes by Sakamoto et al. (2001) revealed an 8% decrease in MWC apparent 2 h af ter transfer to NSW and requiring seven days to return to freshwater values. Comparable MWC va lues in response to salinity acclimation have also been reported in F. heteroclitus (Marshall et al., 2005) and A. oxyrinchus (Altinok et al. 1998). Katoh and Kaneko (2003) reported a decreas e in plasma sodium 12 h after freshwater transfer of saltw ater acclimated F. heteroclitus. Conversely, freshwater F. seminolis acclimated to NSW showed significantly higher plasma s odium concentrations than their controls, regardless of acclimation time. Plasma sodium concentrations were highest among 24 h NSW acclimated fish and gradually diminished with increased acclimation time suggesting superior ion extrusion capabilities in the slower acclim ation rates (Figure 5 1). In a time course experiment involving F. heteroclitus Marshall et al. (1999) repor ted a similar peak sodium concentration of 250 mmol/L 24 h after abrupt transfer, closely paralleling sodium concentrations of F. seminolis in the 24 and 48 h NSW acclimations (Table 5 1). Plasma potassium (Figure 5 6) peaked sharply in the 24 h NSW acclimation group while plasma chloride (Figure 5 2) exhibi ted its highest concentration in the 72 h acclimation but was not 56

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significantly different from 24 and 48 h NSW chlori de values. Ion fluctuations such as these reflect the ongoing physiol ogical restructuring of F. seminolis as it attempts to decrease gill permeability and actively excrete ions against a hyperosmotic gradient. Plasma osmolality is potentially the most useful of all quantified hematological parameters in osmoregulatory studies, delineating ionic and osmotic fluxes in a single value. Abrupt environmental salinity increases in F. kansae (Stanley and Fleming, 1976) elicited a p eak plasma osmolality occurring 20 h post transfer, similar to the 370 mm ol/kg peak osmolality recorded 24 h after the abrupt salinity transfer of F. heteroclitus (Marshall et al., 1999). Additionally, Pickford et al. (1969) reported an approximate decrease in osmolality of 8% in freshwater F. heteroclitus versus their saline counterparts. Mean osmolality values for NSW acclimated F. seminolis ranged from 490 40 505 23 mmol/kg for 24, 48, and 72 h acclimation periods and displayed a marked decrease, 447 27 mmol/kg, in fish acclimated to seawater over 96 hours. Few studies have examined NSW acclimation of fish that normally are found in freshwater stenohaline conditions. Experimental results clearl y demarcate the physiologi cal ramifications of seawater acclimation on F. seminolis and similar effects on other cyprinodontoids have been summarized by Nordlie (1987, 2000). Cumulativel y, BWC, MWC, and hematological data suggest an initiation of hydromineral regulation in the 96 h NSW acclimation treatment. Evans et al. (2005) proposed a potential deficiency in requ isite extrusion proteins in mitochondria rich cells or more subtle hormonal, renal, or intest inal deficiencies to e xplain the osmoregulatory difficulty experienced when freshwater fish ar e confronted with a hype rosmotic environment. Although no histological or enzymatic evaluations were carried out in this experiment, future studies incorporating these tech niques would provide more specific information regarding the physiology of gradual s eawater acclimation in F. seminolis. 57

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Although significant decreases among NSW acclimated F. seminolis were observed in BWC, MWC, plasma osmolality, and plasma ion concentration, none of these measured parameters returned to experimentally nor mal ranges as defined by their corresponding freshwater controls, regardless of acclimation rate (Table 5 1) Furthermore, Marshall et al. (1999) reported elevated plasma osmolality concentrations in F. heteroclitus 30 days after salinity transfer. Further salinity experiments with F. seminolis utilizing a time course approach and extending well beyond 96 h would help to elucidate an osmoregulatory time frame for this species in response to NSW acclimation. Despite the ability of F. seminolis to tolerate NSW acclimation over 24, 48, 72, and 96 h, results indicate that even duri ng the most gradual acc limation (96 h) the fish has not overcome osmotic stressors of the hypertonic environment. It is not yet clear whether further exogenous stressors, such as shipping or high density holding facilities, will exacerbate this transitional physiological status, potentially manifesting in immunocompromise or decreased survival. However, results from preliminary shipping a nd long term holding experiments show no such indications. Results of this investigation w ill contribute to the development of salinity acclimation protocols for the species use in commercial aquaculture as well as elucidate physiological responses to NSW acclima tion in this freshwater killifish. 58

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b, b, a, a, 0 50 100 150 200 250 300 02 44 87 29 6 Acclimation time (h)Sodium (mmol/L) Control (0 g/L) NSW (32 g/L) Figure 5 1. Mean plasma sodium by treatment (control, 0 g/L; NSW, 32 g/L) over experimental acclimation periods. Data are represented as means SD. denotes significant differences (Tukeys HSD, p 0.05) between individual NSW treatments and their corresponding controls. Different letters de note significant differences (Tukeys HSD, p 0.05) among all NSW treatments. Time 0 (p recontrol) sample was taken prior to initiation of the acclimation experiment. b, a, a, a, b, 0 50 100 150 200 250 300 02 44 87 29 6 Acclimation time (h)Chloride (mmol/L) Control (0 g/L) NSW (32 g/L) Figure 5 2. Mean plasma chloride by trea tment (control, 0 g/L; NSW, 32 g/L) over experimental acclimation periods. Data are represented as means SD. denotes significant differences (Tukeys HSD, p 0.05) between individual NSW treatments and their corresponding controls. Different letters denote significant differences (Tukeys HSD, p 0.05) among all NSW treatments. Time 0 (precontrol) sample was taken prior to initiation of the acclimation experiment. 59

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b, a, a, b, a, 0 100 200 300 400 500 600 02 44 87 29 6 Acclimation time (h)Osmolality (mmol/kg) Control (0 g/L) NSW (32 g/L) Figure 5 3. Mean plasma osmolality by tr eatment (control, 0 g/L; NSW, 32 g/L) over experimental acclimation periods. Data are represented as means SD. denotes significant differences (Tukeys HSD, p 0.05) between individual NSW treatments and their corresponding controls. Different letters denote significant differences (Tukeys HSD, p 0.05) among all NSW treatments. Time 0 (precontrol) sample was taken prior to initiation of the acclimation experiment. a, a, a, a, -20.0 -18.0 -16.0 -14.0 -12.0 -10.0 -8.0 -6.0 -4.0 -2.0 0.0 02 44 87 29 6 Acclimation time (h)Body weight (%) Control (0 g/L) NSW (32 g/L) Figure 5 4. Mean percent body weight change (BWC) by treatment (control, 0 g/L; NSW, 32 g/L) over experimental acclimation periods. Data are represented as means SD and untransformed for ease of interpretation. denotes significant differences (Tukeys HSD, p 0.05) between individual NSW treatments and their corresponding controls. Different letters denote significant differences (Tukeys HSD, p 0.05) among all NSW treatments. Time 0 (precontrol) sample was taken prior to initiation of the acclimation experiment. 60

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a, b, a, b, b, 66.0 68.0 70.0 72.0 74.0 76.0 78.0 80.0 02 44 87 29 6 Acclimation time (h)% Muscle water content Control (0 g/L) NSW (32 g/L) Figure 5 5. Mean percent muscle water conten t (MWC) by treatment (con trol, 0 g/L; NSW, 32 g/L) over experimental acclimation periods. Data are represented as means SD and untransformed for ease of interpretation. denotes significant differences (Tukeys HSD, p 0.05) between individual NSW treatments and their corresponding controls. Different letters denote significant differences (Tukeys HSD, p 0.05) among all NSW treatments. Time 0 (precontrol) sample was taken prior to initiation of the acclimation experiment. b, b, b a, 0 2 4 6 8 10 12 14 02 44 87 29 6 Acclimation time (h)Potassium (mmol/L) Control (0 g/L) NSW (32 g/L) Figure 5 6. Mean plasma potassium by treat ment (control, 0 g/L; NSW, 32 g/L) over experimental acclimation periods. Data are represented as means SD. denotes significant differences (Tukeys HSD, p 0.05) between individual NSW treatments and their corresponding controls. Different letters denote significant differences (Tukeys HSD, p 0.05) among all NSW treatments. Time 0 (precontrol) sample was taken prior to initiation of the acclimation experiment. 61

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a, a, a a 0 5 10 15 20 25 30 35 02 44 87 29 6 Acclimation time (h)Hematocrit (%) Control (0 g/L) NSW (32 g/L) Figure 5 7. Mean hematocrit by treatment (cont rol, 0 g/L; NSW, 32 g/L) over experimental acclimation periods. Data are represented as means SD. denotes significant differences (Tukeys HSD, p 0.05) between individual NSW treatments and their corresponding controls. Different letters de note significant differences (Tukeys HSD, p 0.05) among all NSW treatments. Time 0 (p recontrol) sample was taken prior to initiation of the acclimation experiment. Table 5 1. Mean ( SD) hematological parameters of F. seminolis acclimated to control (0 g/L) or NSW (32 g/L) over experimental time periods of 24, 48, 72, and 96 h. Treatment Hematocrit (%) Sodium (mmol/L) Potassium (mmol/L) Chloride (mmol/L) Osmolality (mmol/kg) Time 0 / 0 g/L 23 3 163.2 7.7 6.0 0.6 120.0 4.2 328 6 24 h / 0 g/L 21 3 163.9 5.4 5.6 0.6 119.6 3.3 316 6 48 h / 0 g/L 22 3 167.7 8.0 6.1 1.0 116.8 4.5 310 8 72 h / 0 g/L 25 3 166.4 4.5 5.8 0.6 115.9 8.6 307 9 96 h / 0 g/L 23 2 170.6 7.3 6.5 0.7 119.2 6.4 311 8 24 h / 32 g/L 25 5 263.0 11.3 11.5 0.9 207.9 14.1 505 23 48 h / 32 g/L 25 3 255.8 19.2 7.0 1.1 205.6 16.3 490 40 72 h / 32 g/L 22 5 233.2 16.2 8.1 1.0 216.5 24.1 495 48 96 h / 32 g/L 21 2 219.6 16.4 8.2 0.8 184.4 13.9 447 27 62

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CHAPTER 6 CONCLUSION Development of marine baitf ish aquaculture in Florida is predicated upon a strong consumer demand and identification of technol ogically and economica lly viable candidate species. With 2.7 million licensed anglers, identifi cation of a consistent consumer base within Florida seems intuitive. Preliminary evaluations of prospective culture species have identified F. seminolis as a marine baitfish candidate with economi c potential. Experiments carried out in this study evaluated potential barriers, both environmental and pathogenic, that may impede the culture and marketing of th is unique baitfish species. Parasitological survey results of wild F. seminolis brood fish are integral in the establishment of effective quarantine and s ubsequent biosecurity procedures unique to aquaculture production facilities. Additionally, pr evention of pathogen introductions both into and out of the culture environm ent must be ensured through implementation of responsible aquaculture practices. Thirteen distinct taxa were identified as parasites of F. seminolis Eight parasitic taxa never before recorded on F. seminolis were elucidated. This survey represents the first comprehensive examinati on of the parasitic fauna of F. seminolis These findings will dictate treatment therapy options instrumental in the captive husbandry and culture of this emerging marine baitfish. Analyses of select hematological indices are of great diagnostic value to clinicians as well as researchers. Recent technological advancements ha ve resulted in more efficient, portable, and operator friendly instrumentati on. The i-STAT is a point-of-car e (POC) blood analyzer whose use is becoming increasingly prevalent for hemato logical analysis of fi shes. Validation of new technologies against conventionally accepted inst rumentation (CAI) is crucial to prevent dissemination of erroneous data. Results from validation experiments in F. seminolis were highly 63

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variable and the accuracy of the unit was questiona ble when compared with CAI. Calculated bias was inconsistent thus precluding use of a correction factor. E xperiments using larger sample sizes and numerous species are needed to ascertain the reliability of this POC unit. Validation results were inaccurate and excluded the use of this analyzer in consequent experimental analyses. Determination of acute salinity tolerance and the physiologica l manifestations of natural seawater (NSW) acclimation were additionally investigated. Acclimation and survival of F. seminolis in full strength NSW is essential for feasib ility as a marine baitfish candidate. Two salt sources, NaCl and NSW, were used to assess acute salinity tolerance. F. seminolis was able to tolerate abrupt transfer into 16 g/L NaCl and 24 g/L NSW with 100% survival in both salinities. Even though no mortalities were observed in 16 g/L NaCl, poor physical appearance and atypical behavior suggested NSW to be far superior as an acclimation salinity source. Gradual NSW acclimation experiments contradicted previously published salinity thresholds for this species. F. seminolis exhibited 100% survival wh en acclimated to a salinity of 32 g/L over 24, 48, 72, and 96 h. Physiological analyses of NSW acclimati on rates yielded elevated plasma ion and osmolality concentrations accompanied by decreases in body weight and muscle water content. Although all of the NSW acclimated physiological endpoints measured remained significantly different from control values, a general tre nd signaling the initia tion of osmoregulatory compensation was noticed in 96 h values. Taken together, these results validate F. seminolis as a marine baitfish candidate and provide valuable da ta regarding the species salinity tolerance and underlying physiological processe s. Additionally, relatively few studies have examined the physiological adaptation of a freshwater ste nohaline fish to a marine environment. F. heteroclitus a euryhaline analogue of F. seminolis, has been the focus of extensive studies 64

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elucidating various physiological mechanisms of freshwater and saline acclimation in fishes. Future osmoregulatory studies involving F. seminolis a true freshwater killifish, may elucidate physiological mechanisms not employed by eury haline members of the genus. Furthermore, results from salinity experiments will have imme diate application for Florida baitfish producers and help to develop effective a nd efficient acclimation protocols. The culmination of this study provides a valuable assessment of an emerging Fundulus bait species with potential application in a marine environment. Diversification of Floridas aquaculture industry is vital to its continued longevity. Marine baitfish production is a logical and potentially lucrative endeavor for Florida aquaculturists. Through c ontinued research into candidate species and novel pr oduction methods, marine baitfish culture could soon establish itself as a viable aquaculture crop for the state and the region. 65

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REFERENCES Acierno, M.J., Mitchell, M.A. 2007. Evaluation of four point-of-care meters for rapid determination of blood lactate c oncentrations in dogs. Journal of the American Veterinary Medical Association 230(9), 1315-1318. Adams, A.M. 1985. Parasites on the gills of the Plains Killifish, Fundulus kansae in the South Platte River, Nebraska. Tran sactions of the American Microscopical Society 104, 278284. Altinok, I., Galli, S.M., Chapman, F.A. 1998. Ioni c and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon, Acipenser oxyrinchus de sotoi Comparative Biochemistry and Physiology 120, 609-616. Bangham, R.V. 1940. Parasites of fresh-water fish of southern Florida. Proceedings of the Florida Academy of Science 5, 289-307. Barse, A.M. 1998. Gill parasites of Mummichogs, Fundulus heteroclitus (Teleostei: Cyprinodontidae): effects of season, locality, and host sex and size. Journal of Parasitology 84, 236-244. Bland J.M., Altman D.G. 1986. Statistical me thods for assessing agreement between two methods of clinical measurement. Lancet 1, 307. Bravo, S., Almonacid, C., Oyarzo, C., S ilva, M.T. 2007. The parasite fauna of Galaxias maculatus in the estuary of Maullin River, Chile. Bulletin of the European Association of Fish Pathologists 27, 10-17. Brill, R., Bushnell, P., Schroff, S., Seifert, R ., Galvin, M. 2008. Effects of anaerobic exercise accompanying catch-and-release fishing on bl ood-oxygen affinity of the sandbar shark ( Carcharhinus plumbeus Nardo). Journal of Experiment al Marine Biology and Ecology 354, 132. Bringolf, R.B., Kwak, T.J., Cope, W.G., Larimore M.S. 2005. Salinity tolerance of the Flathead Catfish: implications for dispersal of in troduced populations. Tr ansactions of the American Fisheries Society 134, 927-936. Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. Journal of Parasitology 83, 575-583. Carrier, J.C., Evans, D.H. 1976. The role of envir onmental calcium in the freshwater survival of the marine teleost Lagodon rhomboids. Journal of Experime ntal Biology 65, 529-538. Chervinski, J. 1983. Salinity tole rance of the mosquito fish, Gambusia affinis (Baird and Girard). Journal of Fish Biology 22, 9-11. 66

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Choi, K., Lehmann, D.W., Harms, C.A., Law, J.M. 2007. Acute hypoxiareperfusion triggers immunocompromise in nile tilapia. Jour nal of Aquatic Animal Health 19, 128. Cone, D.K., Marcogliese, D.J., Barse, A.M., Burt, M.D.B. 2006. The myxozoan fauna of Fundulus diaphanus (Cyprinodontidae) from freshwater localities in eastern North America: prevalence, community structure, and geographic dist ribution. Journal of Parasitology 92, 52-57. Crego, G.J., Peterson, M S. 1997. Salinity toleranc e of four ecologically distinct species of Fundulus (Pisces: Fundulidae) from the northern Gulf of Mexico. Gulf of Mexico Science 1997, 45-49. Dillon, W. A. 1966. Provisional list of parasites occurring on Fundulus spp. Virginia Journal of Science 17, 21. DuRant, D.F., Shireman, J. V., Gasaway, R. D. 1979. Reproduction, growth and food habits of seminole killifish, Fundulus seminolis from two central Florida Lakes. American Midland Naturalist 102, 127-133. Epstein, F.H., Katz, A.I., Pickford, G.E. 1967. Sodiumand potassium-activated adenosine triphosphatase of gills: role in adaptation of teleosts to salt water. Science 156(3779), 1245-1247. Evans, D.H., Piermarini, P.M., Choe, K.P. 2005. Th e multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regu lation, and excretion of nitrogenous waste. Physiology Reviews 85, 97-177. Faulk, C.K., Holt, G.J. 2006. Responses of cobia Rachycentron canadum larvae to abrupt or gradual changes in salin ity. Aquaculture 254, 275-283. Feldmeth, C.R., Waggoner, J.P. III. 1972. Fiel d measurements of tolerance to extreme hypersalinity in the California killifish, Fundulus parvipinnis Copeia 1972 (3), 592-594. Fielder, D.S., Allan, G.L., Pepperall, D., Pankhurst, P.M. 2007. The effects of changes in salinity on osmoregulation and chloride cell mor phology of juvenile Australian snapper, Pagrus auratus Aquaculture 272, 656-666. Foskett, J.K., Logsdon, C., Turner, T., Machen, T., Bern, H.A. 1981. Differentiation of the chloride extrusion mechanism during seawater adaptation of a teleost fish, the cichlid Sarotherodon mossambicus, Journal of Experimental Biology 93, 209-224. Foss, A., Kristensen, T., Atland, A., Hustveit, H., Hovland, H., fsti, A., Imsland, A.K. 2006. Effects of water reuse and stocking density on water quality, blood physiology and growth rate of juvenile cod ( Gadus morhua). Aquaculture 256, 255. 67

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Foss, A., Imsland, A.K., Roth, B., Schram, E ., Stefansson, S.O. 2007. Interactive effects of oxygen saturation and ammonia on growth a nd blood physiology in juvenile turbot. Aquaculture 271, 244. Foster, N.R. 1967. Comparative studies on the biol ogy of killifishes (Pis ces, Cyprinodontidae). Ph.D. Dissertation, Cornell University 369 p. Fuller, R.C. 2008. A test for a trade-off in salin ity tolerance in early life-history stages in Lucania goodei and L. parva. Copeia 2008 (1), 154. Griffith, R.W. 1974A. Environment a nd salinity tolerance in the genus Fundulus. Copeia 1974, 319. Griffith, R.W. 1974B. Pituitary control of adap tation to fresh water in the teleost genus Fundulus. Biological Bulletin 146, 357-376. Grosenbaugh, D.A., Gadawski, J.E., Muir, W.W. 1998. Evaluation of a portable clinical analyzer in a veterinary hospital setting. Journal of the American Veterinary Medical Association. 213 (5), 691-694. Gunter, G., Hall, G.E. 1963. Biological investiga tions of the St. Lucie estuary (Florida) in connection with Lake Okeechobee discharges through the St. Lucie canal. Gulf Research Reports 1, 189. Gunter, G., Hall, G.E. 1965. A biologi cal investigation of the Caloos ahatchee estuary of Florida. Gulf Research Reports 2, 1. Harrenstien, L.A., Tornquist, S.J., Miller-Morgan, T.J., Fodness, B.G., Clifford, K.E. 2005. Evaluation of a point-of-care blood analyzer and determination of reference ranges for blood parameters in rockfish. Journal of th e American Veterinary Medical Association 226 (2), 255. Harris, C.E., Vogelbein, W.K. 2006. Parasites of Mummichogs, Fundulus heteroclitus from the York River, Virginia, U.S.A., with a Ch ecklist of Parasites of Atlantic Coast Fundulus spp. Comparative Parasitology 73, 72-110. Hotos, G.N., Vlahos, N. 1998. Salinity tolerance of Mugil cephalus and Chelon labrosus (Pisces: Mugilidae) fry in experimental conditions. Aquaculture 167, 329-338. Howard, L.L., Wack, R.F. 2002. Preliminary use and literature review of the i-STAT (a portable clinical analyzer) in birds, in Proceedings. Annual Meeting of the American Association of Zoo Veterinarians 96. Hoyer, M.V., Canfield, D.E. Jr. 1994. Handbook of co mmon freshwater fish in Florida lakes. University of Florida/Institute of Food and Agricultural Sciences. Gainesville, Florida. Spec. Pub. 160. 68

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Hunn, J.B. 1985. Role of calcium in gill function in freshwater fishes. Comparative Biochemistry and Physiology 82A (3), 543 -547. Imsland, A.K., Gunnarsson, S., Foss, A., Stefansson, S.O. 2003. Gill Na+, K+-ATPase activity, plasma chloride and osmolality in juvenile turbot (Scophthalmus maximus) reared at different temperatures and salinities.Aquaculture 218, 671-683. Isaia, J., Masoni, A. 1976. The effects of calcium and magnesium on water and ionic permeabilities in the sea water adapted eel, Anguilla anguilla L. Journal of Comparative Physiology 109, 221-233. I-STAT system manual. 1997. Fort Collins, Colorado: Heska Corp. Jacob, W.F., Taylor, M.H. 1983. The time course of seawater acclimation in Fundulus heteroclitus L. Journal of Experimental Zoology 228 (1), 33-39. Kaplan, E.L., Meier, P. 1958. Nonparametric estimation from incomplete observations. Journal of the American Statistical Association 53, 457-481. Karnaky, K.J. Jr, Kinter, L.B., Kinter, W.B., Stirling, C.E. 197 6. Teleost chloride cell. II. Autoradiographic localization of gill Na,KATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments. Journal of Cell Biology 70, 157-177. Katoh, F., Hasegawa, S., Kita, J., Takagi, Y., Ka neko, T. 2001. Distinct seaw ater and freshwater types of chloride cells in killifish, Fundulus heteroclitus Canadian Journal of Zoology, 79 (5), 822-829. Katoh, F., Kaneko, T. 2003. Short-term transformation and long-term replacement of branchial chloride cells in killifish transferred fr om seawater to freshwater, revealed by morphofunctional observations and a newl y established time-differential double fluorescent staining technique. Journal of Experimental Biology 206, 4113. Kefford, B.J., Papas, P.J., Metzeling, L., Nugegoda D. 2004. Do laboratory salinity tolerances of freshwater animals correspond with their field salinity? Environmental Pollution 129, 355-362. Laiz-Carrion, R., Guerreiro, P.M., Fuentes, J., Canario, A.V.M., Del Rio, M.P.M., Mancera, J.M. 2005. Branchial osmoregulatory response to salinity in the Gilthead Sea Bream, Sparus auratus. Journal of Experimental Zoology 303A, 563-576. Lemarie, G., Baroiller, J.F., Clota, F., Lazard, J., Dosdat, A. 2004. A simple test to estimate the salinity resistance of fish w ith specific application to O. niloticus and S. melanotheron. Aquaculture 240, 575-587. 69

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BIOGRAPHICAL SKETCH Matthew A. DiMaggio was born in Brooklyn, NY, moving to Staten Island, NY, shortly after. Summers were spent on Block Island, RI, where Matthew developed a passion for the ocean. He attended the State Univ ersity of New York at Gene seo, where he graduated in 2003 with a B.S. in biology and a minor in environmen tal sciences. The next three years were spent working in the urology research lab at the Univer sity of Rochester / St rong Memorial Hospital, investigating various urologica l cancers and concomitantly developing fundamental research skills necessary for his further educational aspirations Matthew moved to Florida in 2006 where he was accepted to a masters program in the De partment of Fisheries and Aquatic Sciences at the University of Florida. Matthew will complete his Master of Science in August 2008 and continue on with his graduate studies in pursuit of his Ph.D. 74