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1 DEVELOPMENT OF METHO DS FOR NON LETHAL HEALTH ASSESSMENT OF THE RED DRUM ( SCIAENOPS OCELLATUS CENTER NO TAKE FISHERIES RESERVE By CARLA M. GARREAU A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Carla M. Garreau
3 To my angel, Cookie
4 ACKNOWLEDGMENTS I would like to thank my major advisor Ruth Francis Floyd for her advice, constructive criticism and guidance throughout my graduate experience. I would like to thank Lou Guillette, Jr. for your belief in me that I could work full time and complete a graduate degree. I appreciate all of our f ield talks and your patience teaching me about endocrinology. I am extremely grateful for all the time Eric Reyier has spent teaching me fish scientific names in the field, quizzing me regularly, and his efforts helping me create the project design for th is research. I aspire to have his work ethic to always get the job done proficiently and professionally no matter what the task at hand. I am thankful for Roy Yanong who helped guide m e through my academic journey. David Westmark taught me how to bleed a red drum, and without him this project would not have been such a painless process for all the fish involved thank you! Conducting my field sampling would not have been possible without the help of the Eric Re yier, Karen Holloway Adkins, and Shanon Gann for their t ireless perseverance in the field. I would also like to express thank s to Carlton Hall, Donna Oddy and Tim Kozusko for assisting me in the field and laboratory. I greatly appreciated the statistics help from Eric Stolen and his patience in helping me grasp multivariate analysis. I w ish to express my gratitude to thank Angie Saul and Chad Young for allowing access to the S tock E nhancement R esearch F acility br oodsto ck as my initial patients for this protocol and also all the staff who help ed with the pilot study Josh Taylor, Josh Lunde, Mat Rhodes, Kerry Mesner, Micah Alo and Chris Young. I much appreciated the assistance of Patrick Thompson and Heather Maness from the University of Florida Aquatic Animal Health Program for their assistance with the Abbott i STAT analyzer I
5 am immensely grateful to Ashley Boggs from MUSC, and Heather Hamlin from University of Maine, for their help teaching me how to run radioimmun o assays in the laboratory and their patience with my learning curve f or understanding the results Finally I am eternally grateful to my entire family, especially my parents for encouraging me to pursue my graduate degree and cont inuing to reassure me to w ork hard and that it would all be worth it in the end. I wanted to thank my dad for teaching me how to fish, my brother for going fishing with his little sister when w e were kids, and to my mom for encouraging me to keep on fishing My pa rents taught me to be an independent and self reliant young adult and I credit them for doing a wonderful job raising me to work hard and play hard and to combine the two to discover my dream career as a marine biologist I want to thank Spiros for always believing in me and being a great support for me to lean on when times were difficult juggling work, school, family and friends, you helped me steer the course in the turbulent seas of a s thesis is dedicated to my angel, Cookie, who was the best friend a girl could have for 14 years. She provided me with unconditional love from end of her precious l ife. This research was funded in part by Inomedic Health Applications, Aquatic Animal Health Program of the University of Florida, the Medical University of South Carolina and Hollings Marine Lab. All research was performed under the authorization of the special activity license No. SAL 09 0512A0SR. All animal handling was in accordance with the Merritt Island National Wildlife Refuge Special Use Permit No. 2011SUP001 11 077.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 11 C HAPTER 1 RED DRUM ( SCIAENOPS OCELLATUS ) PHYSIOLOGY AND ENDOCRINOLOGY ................................ ................................ ................................ 13 Life History ................................ ................................ ................................ .............. 13 Red Drum Spawnin g ................................ ................................ ........................ 14 ................................ ......................... 16 Health Assessments ................................ ................................ ............................... 18 Wildlife Health Assessments ................................ ................................ ............ 18 Specimen Banking ................................ ................................ ........................... 19 American All igator Health Assessment ................................ ............................ 20 Marine Mammal Health Assessments ................................ .............................. 20 Fish Health Assessments ................................ ................................ ....................... 23 FWC Fish Assessment ................................ ................................ ..................... 25 SERF Hea lth Index ................................ ................................ ........................... 26 Collection Method ................................ ................................ ............................. 26 Handling Techniques ................................ ................................ ........................ 27 Sedation Techniques ................................ ................................ ........................ 28 Bleeding Techniques ................................ ................................ ........................ 28 Reproduc tive Health ................................ ................................ ......................... 30 Sex Hormones ................................ ................................ ................................ .. 30 Stress Response ................................ ................................ ................................ .... 32 Acute and Chronic Stress ................................ ................................ ................. 33 Cortisol ................................ ................................ ................................ ............. 34 Glucose ................................ ................................ ................................ ............ 36 Objectives and Hypotheses ................................ ................................ .............. 37 2 HEALTH ASSESSMENT OF ADULT RED DRUM ................................ ................. 41 Study Objectives ................................ ................................ ................................ ..... 42 Materials and Methods ................................ ................................ ............................ 44 Fish Capture Methods ................................ ................................ ...................... 44 Plasma Collection ................................ ................................ ............................. 44
7 External examination protocol ................................ ................................ .......... 45 Sex Identification ................................ ................................ .............................. 46 Tagging and Handling ................................ ................................ ...................... 46 Water Quality ................................ ................................ ................................ .... 46 Condition Factor ................................ ................................ ............................... 46 Steroid Assa ys ................................ ................................ ................................ 47 Glucose Analyses ................................ ................................ ............................. 48 i STAT Analyzer ................................ ................................ ............................... 49 Statistical Analyses ................................ ................................ .......................... 49 Results ................................ ................................ ................................ .................... 50 Morphometics & Condition Factor ................................ ................................ .... 50 Water Quality ................................ ................................ ................................ .... 51 Health Index ................................ ................................ ................................ ..... 51 Parasites ................................ ................................ ................................ .......... 51 Plasma Glucose Concentrations ................................ ................................ ...... 52 Plasma Cortisol Concentrations ................................ ................................ ....... 52 Plasma 11 KT Concentrations ................................ ................................ .......... 53 Plasma E 2 Concentrations ................................ ................................ ................ 53 Sex Identification ................................ ................................ .............................. 54 Recaptures ................................ ................................ ................................ ....... 54 Discussion ................................ ................................ ................................ .............. 55 Predicted Outcomes ................................ ................................ ......................... 55 Stress Res ponse ................................ ................................ .............................. 62 Water Quality ................................ ................................ ................................ .... 63 Further Research ................................ ................................ ............................. 64 LIST OF REFERENCES ................................ ................................ ............................... 74 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 82
8 LIST OF TABLES Table page 1 1 Health index scoring system developed for red drum by the Stock Enhancement Research Facility (Dukeman et al. 2006). ................................ ... 40 2 1 Morphometric data and condition factor for each reproductive period ................ 72 2 2 Plasma values for each reproductive period ................................ ....................... 72 2 3 Water quality in the KSC Reserve during each sampling period ........................ 73 2 4 Predicted sex for undetermined red drum caught in the KSC Reserve .............. 73
9 LIST OF FIGURES Figure page 1 1 Kennedy Space Center security zone de facto no take fisheries reserve is outlined in yellow. ................................ ................................ ............................... 39 2 1 Kennedy Space Center security zone de fa cto no take fisheries reserve. ........ 68 2 2 Collecting a 4ml blood sample from the branchial vessles in the gill of a wild caught red d rum. ................................ ................................ ............................... 69 2 3 Sex determination of wild caught red dru m in the field. ................................ ..... 69 2 4 Health index score of wild caught red drum ( S.D.) presented by sampling period ................................ ................................ ................................ ................ 70 2 5 Plasma cortisol concentrations of wild caught red drum ( 1 S.E.) presented by reproductive period and by sex. ................................ ................................ ..... 70 2 6 Plasma 11 KT concentrations of wild caught red drum ( S.D.) presented by reproductive per iod and sex. ................................ ................................ .............. 71 2 7 Plasma E 2 concentrations of wild caught red drum ( S.D.) presented by reproductive period and sex. ................................ ................................ .............. 71
10 LIST OF ABBREVIATION S 11 KT 11 ketotestosterone DFA discriminant function analysis E 2 estradiol ELISA enzyme immunoassay FIM fisheries independent monitoring FIT Florida Institute of Technology FL fork length FWC Florida Fish and Wildlife Conservation Commission GEA gross external abnormality GPS global positioning system HERA Health and Risk Assessment project IACUC Institutional Animal Care and Use Committee IRL Indian River Lagoon KSC Kennedy Space Center MINWR Merritt Island National Wildlife Refuge MPA marine protected area NASA National Aeronautics and Space Administration NIST National Institute of Standards and Technology PIT p assive integrated t ransponder RIA radioimmunoassay SERF Stock Enhancement Research Facility SL standard length TL total length
11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requir ements for the Degree of Master of Science DEVELOPMENT OF METHO DS FOR NON LETHAL HEALTH ASSESS MENT OF THE RED DRUM ( SCIAENOPS OCELLATUS EDY SPACE CENTER NO TAKE FISHERIES RESER VE By Carla M. Garreau December 2012 Chair: Ruth Francis Floyd Cochair: Louis J. Guillette, Jr. Major: Fisheries and Aquatic Sciences D espite significant value of the Florida r ed drum (S ciaenops ocellatus) fishery a lack of sex and stress hormone data are available C urrent non lethal health ass essment programs do not collect this information. This was the first study to assess sex and stress hormones for adult red drum in Florida, with the goal of develop ing protocols defin baseline data This project incorporated the Stock Enhancement Research Facility (SERF) external health index with blood chemistry analysis of glucose, cortisol, 11 ketotestosterone (11 estradiol (E 2 ) Red drum (n=126) were collected from the oldest fully protected no take fisheries reserve in the United States during t hree different reproductive periods to evaluate seasonal variation and effect of reproductive activity on stress response. Fish in a ll periods scored near or above the for the SERF health index The lower scores were at tributed to wild fish hav ing more parasites than cultured fish. On average c ondition factor ranked all fish as excellent exceptional. Glucose
12 cortisol, and E 2 levels were significantly different among reproductive periods Cortisol values ranged between 0.93 1.25 ng/ml, well below typical 10 ng/ml found in teleosts. 11 KT was s ignificantly elevated during the reproductive period for both sexes Blood collection occurred in less than three minutes a nd may have minimized the glucose and cortisol response associated with handling. Results from this study illustrate the potential value of future comparisons of red drum near the study area the range of the species and as a model for other sciaenids
13 CHAPTER 1 RED DRUM ( SCIAENOPS OCELLATUS ) PHYSIOLOGY AND ENDOCRINOLOGY Life History The r ed drum, Sciaenops ocellatus ( family Sciaenidae ) are part of an important inshore recreational fishery in Florida ( Adams and Tremain 2000 ) Florida is recognized Hunting and Wildlife Associated Recreation t hat shows Florida is the number one recreational fishing state. Florida attracts about 2.8 million anglers annually In 2007, the Indian River Lagoon (IRL) was calculated to have a commercial fishing value of $3.8 million dollars and $1.5 billion dollars for recreational fishing ( Hazen and Sawyer 2008 ) Red drum are a long lived fish so me exceeding 30 years of age in Florida Atlantic waters ( Murphy and Taylor 1990 ) The species inhabit s nearshore and estuarine waters from Massachusetts to the Gulf of Mexi co coa st. Annual commercial landings of ( Winner et al. 1999 ) Declines in adult red drum resulted in closure of the commercial fishery in Florida in 1986 ( Murphy 2009 ) At that time, t he recreational fishery became regulated by a slot size of 18 27 inches (457 685 mm) and a daily bag limit of one fish ( Johnson and Funicelli 1991 ) These measures were put in place by state and federal managers to safeguard the spawning stock biomass and help e nsure stable annual recruitment ( Murphy 2009 ) The recreational fishing effort has more than doubled o n both the Atlantic and Gulf coasts since 1989 ( Winner et al. 1999 ) Large juvenile size classes (200 600 mm) comprise the majority of all red drum fishery landings in Florida waters ( Adams and Tremain 2000 ) It is important for fisheries managers to have a n understanding of the
14 use of essential fish habitat area of red drum to protect this species Recreational landings combined from the Gulf and Atlantic in Florida were estimated at 2,499,324 fish in 2007 ( Murphy 2009 ) T he latest red drum stock assessment conducted in 2008 indicated that overfishing was not occurring on either coast of Florida ( Murphy 2009 ) In February 2012 the Florida Fish and Wildlife Conservation Commission divid ed the state into three fishery management a reas for red drum : Northwest, Northeast, and the South The bag limit was increased to two fish per day in the Northwest and Northeast regions, which indicates success in the management of the species. It is di fficult to assess exploited fish populations and to evaluate the risk involved in fishery management decision s and difficult to determine when management actions are truly working to sustain stocks ( Murray et al. 1999 ) Red Drum Spawning Red drum have demonstrated a pattern in spawning site selection by returning year after year to spawn in similar locations, since red drum eggs and planktonic larvae have been collected repeatedly in parts of its range including Texas, Alabama, Mississippi, and Florida ( Johnson and Funicelli 1991 ) Starting in late August throu gh October, the majority of red drum produce numerous planktonic larvae in nearshore waters near estuarine inlets ( Perez Dominguez and Holt 2002 ) L arval red drum however, have been found commonly in July and early August and occasionall y in June in the IRL Florida ( Reyier et al. 2008 ) After a few weeks in the plankton, the larvae settle in seagrass beds within estuaries that serve as primary nursery habitat for juvenile red drum. Sexual ly mature red drum have been reported in bays and estuaries in Texas, North Carolina and Florida despite its usual offshore spawning. Recent d ata has shown that adult red drum reside and spawn within certain estuaries including
15 Pamlico Sound, North Carol ina ( Ross et al. 1995 ) Savannah River and Atlamaha River Estuaries, Georgia ( Lowerre Barbieri et al. 2008 ) Tampa B ay and IRL System, Florida ( Murphy and Taylor 1990 ; Reyier et al. 2011 ) and Chan deleur Sound Louisiana ( Comyns et al. 1991 ) Reyier et al. ( 2011 ) stated that estuarine reproduction is an important life strategy for the species in east central Florida. M anagement of estuarine spawning r ed drum may depend in part on factors such as identification, preservation, and management of spawning and nursery areas and preferred estuarine habitats for the various life history stages ( Johnson and Funicelli 1991 ) The proportion of the population involved in this inshore spawning activity is currently unknown; however these fish may be at greater risk from intensive inshore recreational fishing harvest. Red drum spawning in the south ern portion of the species range tends to occur later in the year and is more protracted beginning as early as August and extending into December in the northern Gulf of Mexico, Texas and Tampa Bay Spawning behavior has been observed between mid September and mid February in the Evergla des National Park ( Jannke 1971 ) Perez Domi nguez and Holt ( 2002 ) found that the benefit of evolving an estuarine depende nt lifestyle increases the growth potential of fry and juveniles in fluctuating environments Red drum larvae are very tolerant of tempera ture fluctuations from 22 27C ( Perez Dominguez and Holt 2002 ) Juveniles have strong site affinity to their inshore estuarine habitat, but growing evidence demonstrates that adult red drum are also utilizing estuarine habitats throughout their life and can complete their life cycle there without the need for an offshore residency period. Reyier et al. ( 2011 ) found that acoustically tagged and monitored red drum in the IRL Florida, exhibit strong site fidelity from winter thr ough
16 early summer with movement increasing within its range during the fall spawning months; however the majority of tagged fish remained within the lagoon year round. Th e non migratory behavior of this population seems to be unique to the northern IRL re gion as most mature adults from other areas emigrate to nearshore waters with maturity ( Reyier et al. 2011 ) Johnson and Funicelli ( 1991 ) believe that red drum may di splay some degree of homing or imprinting instincts which has been observed in other fish species facilitating their ability to return to spawn at their place of origin. Red drum eggs were collected in Mosquito Lagoon during October and early November i n clumps suggesting they were from spawning aggregations ( Johnson and Funicelli 1991 ) Adams and Tremain ( 2000 ) observed that estuarine creeks are critical nursery habitats used by juveniles and suggested that the use of warmer creek waters during winter months was crucial for survival when lagoon temperatures decline d Murphy and Taylor ( 1990 ) studied red drum in Florida and found that males matured at smaller sizes and younger ages than did females on both the west and east coasts They estimate d the lengths at which 50% of fish were mature on the Atlantic coast for males at 511 mm and females at 900 mm. The average observed sizes for mal es were significantly larger at ages 1 and 2 on the Atlantic coast than the Gulf Coast Red drum in Florida appear to grow more rapidly than red drum in Mississippi, South Carolina and Texas ( Murphy and Taylor 1990 ) Kennedy Space Center Reserve Mature resident red drum are known to spawn throughout the fall period within the Reserve ( Stevens and Sulak 2001 ; Reyier et al. 2008 ) The study area includes 33 km 2 of estuarine waters within the KSC boundary and a de facto marine fisheries reserve
17 that was established in 1962 to safeguard rocket launch operations (Figure 1 1). This is the oldest fully protected no take fi sheries reserve in the United States ( Roberts et al. 2001 ) and is managed by Merritt Island National Wildlife Refuge (MINWR). A no take reserve is an area where extractive activities are banned excep t for limited scientific and educational purposes ( Bartholomew and Bohnsack 2005 ) Few no take reserves exist in the Un ited States ( Murray et al. 1999 ) The KSC reserve area is a shallow estuary, with a mean depth of 1.5 m eters T here are a few dredged areas with depths up to 15 m that were created during construction of rocket launch pads and roadways, as well as a navigational channel that is 3 4 m in depth. This estuary is isolated fr om the Atlantic Ocean by barrier islands which have only five widely spaced inlets, the closest being Sebastian Inlet, approximately 95 km south of the study area ( Reyier et al. 2011 ) Many marine fishes utilize estuaries for a crucial life history component due to highly productive waters that serve as feeding and nursery grounds for the species that comprise 75 percent of U.S. commer cial landings ( Chambers 1992 ) The estuary has extensive seagrass beds of shoal grass ( Halodule wrightii ), manatee grass ( Syringodium filiforme ) and salt marsh that provide refuge from predators and foraging opportunities for red drum throughout their life cycle. The KSC Reserve minimizes many of the challenges to growth and survival experienced by red drum elsewhere in the southeastern U.S. These may include intense angling pressure, reduced habitat quality caused by coastal development, and storm water r unoff contaminants from anthropogenic sources. Previous research has demonstrated that sportfish within the KSC Reserve achieve a larger mean size and exist in higher densities than those in adjacent public areas ( Johnson and Funicelli
18 1991 ) Red drum have strong site fidelity ( Reyi er et al. 2011 ) and those from KSC Reserve waters spend a large portion of their adult lifespan in the reserve Red d rum spawning in the KSC Reserve may have the positive benefit of being protected from adjacent public waters, protecting a portion of the spawning stock from exploitation ( Bohnsack 1993 ) Stevens and Sulak ( 2001 ) did a mark recapture study with red drum in public waters surrounding the KSC Reserve and found that large adult sportfish were being protected in the reserve area. It was functioning as a replenishment zone for the reproduct ively active population much like a marine protected area (MPA ). The KSC Reserve is off limits to public anglers and fits the most stringent guideline of an MPA which is a no take reserve for all animals residing in its area. Health Assessments Wildlife H ealth A ssessments Marine animals are faced with health threats including infectious agents ( viruses parasites, bacteria, and fungi ), entanglements, catch and release stress and exposure to, or accumulation of toxic pollutants. Health assessments on mari ne animals h ave been developed to improve our understanding of the biology and overall health and vigor of wildlife populations Tracking and analyzing health trends in populations of marine animals suggest they can function as sentinel species and provid e a useful tool for evaluation of the well being of aquatic ecosystems ( Bossart 2011 ) Marine mammals and reptiles have been extensively studied by using specific health assessment protocols that are proving valuable in comparing populations from different geographic regions. Data may include presence or absence of disease, contaminants or physiologic al metrics. The protocol for wild animal sampling is trending t oward non lethal assessments, and incorporation of methods that facilitate the return of animals to
19 the population being studied This in turn allows for long term sampling of the same animal if tagged with a unique identification number facilitating as sessment of changes over time status can be extrapolated from assessments conducted on individual animals. Ranges hematocrit, packed cell volume, and glucose are used to assess the overall health of the sentinel species using the animals as bioindicators. If a range of these known data are available it adds to the decision in classifying a health status. S everal ef forts currently underway, are intended to cor relate select health metrics with exposure to pollution, biotoxins, and disease by use of live capture techniques and releas e techniques thus decreasing the need for lethal sampling ( Kucklick et al. 2010a ) Specimen Banking A very important aspect of health assessment protocols is specimen banking, which ensure s that geographic and temporal trends can be examined retrospectively w hen questions arise or conditions change. Traditional specimen banking has relied heavily on lethal sampling, but recent studies have proven the value of non lethally collected marine mammal tissue samples for assessment of pollutant exposure ( Kucklick 2010b ) The National Institute of Standards and Technology (NIST) developed uniform collection protocols for marine mammal tissues which included blood, blubber, and organ tissues. Marine mammal assessment data includes: body condition, notation of lesions/wou nds, body weight, eye exam, analysis of blood, skin, and reproductive parameters. Also implantation of a passive integrated transponder (PIT) tag before the animal is released will facilitate access to data from the same animal in the future. NIST also s upports monitoring of sea turtle health and developed a
20 uniform protocol to collect blood and scute samples from live capture and release ( Kucklick 2010b ) Aguirre and Lutz ( 2004 ) state that marine turtles can serve as sentinels of ecosystem health for benthic environments in Florida. However to determine the health status of an individual or a population, normal functional physiology and disturbed or pathological physiology must be distinguished ( Aguirre and Lutz 2004 ) American Alligator Health Assessment Hamlin et al. ( 2010 ) w ere able to determine the seasonal and environmental influence on androgen cycles in the adult male American alligator, Alligator missi ssippiensis in Florida, at Merritt Island National Wildlife Refuge (MINWR), a site with known heavy metal contamination N on lethal methods were used, including blood sampl ing These techniques facilitate mark recapture studies that may help identify se asonal changes in health parameters. Boggs et al. ( 2011 ) also studied the American alliga tor using non lethal techniques and were able to ascertain seasonal variation in plasma thyroid hormone concentrations from two locations in Florida. Wildlife sentinels may more accurately reflect the real world exposure conditions of contaminants or sele ct pathogens ( Hamlin and Guillette 2011 ) Marine Mammal Health Assessments Bossart ( 2011 ) explains that marine mammals are excellent sentinel species due to their long life spans, long term coastal residen cy high t rophic level diet and fat stores that contain anthropogenic toxins. Goldstein et al. ( 2006 ) noted that it is important to include physical examination, history, and diagnostic testing as part of the health a ssessment of marine mammals. Atlantic bottlenose dolphins, Tursiops truncatus have been studied in Sarasota Bay, Florida since 1970 and annual health
21 assessments continue there and in other locations in the southeastern United States ( Wells et al. 2004 ) A large data set of blood parameters has been collected and integrated to create an objective, quantitative and comparable health assessment sco ring system F our different designations have been developed for assessment of wild dolphin population health status. P arameters from baseline blood values were used to assign a health status score. These range from apparently good health to a serious m edical condition that requires treatment ( Wells et al. 2004 ) Some concerns with this type of scoring system include variations in analytical tec hniques used by different laboratories and missing values can bias health scores down Further, the scoring system does not include body condition ( Wells et al. 2004 ) Bottlenose dolphins have also been studied in a collaborative effort in Charleston, South Carolina, and the IRL Florida as part of a Health and Risk Assessment (HERA) project from 2003 2005. Bossart et al. ( 2008 ) explained that the goals of the HERA project were to develop standardized tools for health and risk assessment. These included physical exam, sampling of teeth for age determination, skin/blubber, ultrasound to detect pregnancy, hematology, serum chemistr y, gastric, urinalysis, blowhole and fecal cytology. Hematology was used to evaluate relative leukocyte counts, hemoglobin, red blood cell count s mean corpuscular platelet volume, mean corpuscular hemoglobin, white blood cell count s and total platelets all of which were determined by an automated analyzer. Serum chemistry included analyses for sodium, potassium, chloride, bicarbonate, anion gap, blood urea nitrogen, creatinine, uric acid, calcium, phosphorus, magnesium, glucose, total direct and indirec t bilirubin, cholesterol, triglycerides, iron and fibrinogen. Specific i mmunological tests and antibody titers were
22 also run from collected blood sample s. Data from these dolphins can be compared to previously collected data through nine different collab orative projects that are involved with HERA. Conclusions include insights on sexually transmitted tumors that are occurring in these dolphin populations M ultiple abnormalities in serum chemistries, protein electrophoresis, and immunological parameters were found in dolphins with tumors when compared to healthy dolphins ( Bossart et al. 2008 ) Dolphins were considered healthy when they were categorized to be free of disease by Bossart ( 2008 ) An example of establishing normal parameters i ncludes the serum range of glucose for wild bottlenose dolphins which is 62 139 mg/dL ( Varela et al. 2006 ) This becomes a reference range for v eterinarian s to use in assessment of wild and captive dolphins. The H ERA project researchers caution that interpretation of the immune system data may not be possible yet. D ata on wild dolphins has still not been well defined and further research is needed to defin e the complex immune function and overall health of dolphins ( Bossart et al. 2008 ) The Florida manatee, Trichechus manatus latirostris is a federally endangered marine mammal that has been extensively studied in Florida for over 25 years. Subsequ ently a large health database has been assembled from non lethal health assessments conducted on this species in Florida ( Bonde e t al. 2004 ) Long term recapture data allows researchers to notate differences in captive and wild populations over time. Harvey et al. ( 2007 ) noted differences in blood plasma values measured in wild and captive manatees during routine diagnostic health assessments. Differences in blood plasma values for these popula tions were attributed to many varia bles including diet, time from last feeding, water c hemistry environmental stressors, presence of
23 bacterial contaminants, body condition, capture stress and seasonal variations. Differences between captive and wild popu lations are expected, but poorly defined for many species. W hen conducting health assessment s on a wild population since many protocol without deviation to attempt to eliminate sampling errors in the field. Fish Health Assessments The use of fish as biological indicators o f estuarine conditions has many advantages. Useful health indicators are capable of detecting responses to synergistic, sub lethal stress which can affect the fitness of an organism with population level consequence such as condition index, organ function liver glycogen content or liver somatic index ( Leamon et al. 2000 ) Fish health assessment is playing an increasing role in both fishery management and env ironmental monitoring policy. Hematologic and plasma chemistry data can provide insight into organ function, general meta bolic homeostasis and immune function, once baseline information has been established. Fish can provide a more sensitive assessment of the link between human activities and their ecological consequences ( Schlacher et al. 2007 ) One concern with biological indicators such as blood parameters is that normal ranges for a particular fish species must be determined prior to being use d in a monitoring pro gram ( Leamon et al. 2000 ) A comparison of the range and relations hip of these indicators between proximate but separate non impacted locations would present the natural range of values for the species. Baseline blood parameter data from unfished stocks can vastly improve estimates of population parameters for harvested species ( Murray et al. 1999 ) Leamon et al. ( 2000 ) stated that data obtained from a non impac ted location study site, such as a reserve with no fishing allowed, could function as a standard for evaluating other sites.
24 All of the above reasons make the KSC reserve an excellent study location for determining baseline blood values of wild red drum Red drum are suitable study animals for immune responses of natural populations due to their life history as sedentary juvenile s through adults that remain in the same estuarine habitat for four to five years ( Evans et al. 1997 ) This would be an important component of a health index valuable for future use and comparis ons with other red drum stocks. Fish health can be assessed using morphological, hematological and immunological examination as well as by using experimental disease challenges ( Maita 2007 ) The extent to which natural environmental variables and anthropogenic stressors influence the immune response of marine teleosts to antigenic challenges in their natura l environment remains unknown ( Evans et al. 1997 ) However non lethal health assessments currently h ave limited adapt ations for work in fishery science. Fish health as sessments conducted in the field are commonly conducted by post mortem necropsy potentially removing healthy animals from wild stock s Red drum throughout the state of Florida including the IRL have been tested for total mercury content since it is known to bioaccumulate in fish tissue Human fish consumption has been correlated with mercury content in fish ( Adams and Onorato 2005 ) Data from this study w ere utilized by the Florida Department of Health to issue a health advisory suggesting limit s to human consumption of red drum in the Florida Keys Florida Bay area. Currently the legal recreational slot size for red drum is an effective filter to prevent human consumption of fish with potentially higher concentr ations of mercury caused by bioaccumulation
25 FWC Fish A ssessment The Florida Fish and Wildlife Conservation Commission (FWC) has conducted health evaluations on wild fish populations during their Fisheries Independent Monitoring (FIM) program since 1990. Fish are usually captured using a trammel net or bag seine. These methods result in fish being in the net for at least 10 25 minutes b efore they are moved to a live well for further process ing While fish are struggling in the confined space of the net or live well, their primary and secondary stress responses become elevated over the duration of the cap ture. Currently evaluations conducted through the FIM program are focused on gross external abnormalities (GEA) such as lesions, wounds, infections or other obvious signs of injury. If no external GEA are observed the fish is counted and released. If a fish does exhibits a GEA, then it is culled and sent to the FWC Fish and Wildlife Research Institute for necropsy and further analysis Length and weight measuremen ts are condition factor is calculated later in the laboratory. Condition factor has been accepted as a gross health index o f general fish condition and is thought to provid e information on energy reserves and the potential for an animal to tolerate toxicant challenges or other environmental stressors ( Grund et al. 2010 ) Tissues collected include: otoliths and dorsal spines used for age determination, fins for genetic sampl ing reproductive organs for gonad histology, muscle sample for determination of mercury concentrations, and stomach contents for dietary determinatio n. After the fish is evaluated, a potential cause of death is assigned to determine if anthropogenic causes are involved and to see if any mitigation can be performed to p revent future fish kills or GEA s. FWC does
26 not have any non lethal field methods to examine internal health of wild fish populations or GEAs. SERF H ealth I ndex The Stock Enhancement Research Facility (SERF), operated by FWC in Port Manatee Florida, has developed a health index for juvenile red drum which they use to assess the health status of their fingerlings. The health index relates all quantitative and qualitative data associated with external evaluations. H ealth challenges of cultur ed fish are often related to water quality and stocking densities ( Dukeman et al. 2006 ) Fish are netted from their tanks and euthanized with a lethal dose of MS 222 to be processed for the assessment. Th is met ric creates an assessment using the external condition of the fish (i.e., physical abnormalities, parasite load, microbial infections, and condition factor). The health index provides a score in the range of 0 50 for each fish examined (Table 1 2). When a red drum scores a value below 45, it is determined to have had compromised health whereas a score of 45 and above is considered in the If fish score below 45 water quality, stocking density and feeding regime are examined to determine the cause of the compromised health of the fish. Collection M ethod Many stress hormones have a short latency period making a baseline determination difficult or impossible. Handling of any kind will result in changes of stress hormones occurring within a few minutes. Collecting fish from a wild setting involves many variables that are not a concern in a laboratory setting. These may include how long it takes to collect the fish, variable water quality, water temperature, unknown age and sex of the fish being targeted. Techniques used to collect w ild fish include hook and line, trammel netting, seining, and long lining.
27 The ideal capture method for non lethal testing would be the quickest way to get fish on board with minimal stress followed by rapid rel ease Many species of fish can be captured by hook and line fairly easily; however a disadvantage of this technique is the risk o f gut hook ing the fish or causing structural damage to the mouth or jaw. Trammel nets, seining and long lining all require th e fish to be entangled in a net or on a hook for at least 10 20 minutes before the animal would be retrieved for assessment. Using long lining as a method for fish collection, Pankhurst and Sharples ( 1992 ) reported that snapper, Pagus auratus that were in the set for one and half hours had cortisol va lues of 22 ng/ ml and that these increased to 58 ng/ml within sixty minutes of landing. A study by Lowerre Barbieri et al. ( 2003 ) of hook and line catch and release fishing on a wild spawning aggregation of common sno ok was conducted to determine effect on reproductive output. Findings from this study conducted on the Atlantic coast of Florida found that the stress of capture and release did not appear to cause the common snook to cease spawning based upon recapture d ata that evaluated ovarian development. The study also concluded that females implanted with sonic tags continued to spawn even after encountering considerable handling and stress involved with the surgery to implant the tags Handling T echniques Once a f ish is captured, a few options are available in the field for handling the animal Restraint options include plac ement in a cooler with water and aeration, restrain t in a sling, or use of an inverted v tray without water. The option of p lacing the fish i n water would be ideal, however contamination needs to be considered especially in a remote field setting where only the surrounding ambient wa ter is available for rinsing, and parasite s could be moved between fish.
28 Another consideration is ambient air and water temperature S ince field sampling in Florida can mean hot weather and even hotter water temperatures, this parameter needs to be monitored and controlled to be kept within ambient water ranges. If fish are confined in a small container and wate r temperature is high, the dissolved oxygen being. Sedation T echniques Once the fish is in hand, some researchers employ the use of anesthesia, trica i ne methane sulfonate (MS 222), which current ly is the only US Food and Drug Administration approved anesthes tic for fish; however it can have effects on blood chemistry values. Thomas and Robertson ( 1991 ) found that in red drum when MS 222 is used at a dosage of 80 mg/l for two and half to six minutes, it can block the plasma cortisol biochemical response to handling stressors Further, MS 222 cannot be used on legally edible wild fish t hat c ould be caught by the public unless they are held for a 21 day withdrawal period before being released ( US Food and Drug Administration 1997 ) Since field sampling often is a catch and release practice, use of anesthetics is not feasible given current legal constraints and concerns for anesthetic residue in edible tissue. A few main reason s to use anesthetics are to sedate and immobilize the animal and to eliminate pain or discomfort to the animal. Manual r estraint needed for venipuncture and evaluati on of external morphometric measurements do es not typically require the use of an anesthetic. Bleeding T echniques Maita ( 2007 ) e xplained that measurement of blood parameters, such as hematocrit value, hemoglobin concentration, red blood cell counts, and plasma components are tools that can be used to monitor fish health Plasma components
29 commonly assessed are total protein, urea nitrogen, glucose, total cholesterol, triglyceride, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase. These components are used to infer health by comparing it to data gathered previously and deciding if the current values are within a healthy range. If values obtained are outside of the ranges previously detected it could mean that the current health status of the animal is in decline. These plasma components measure the fish response t o stressors and everyday life. An ad vantage of using blood samples for health assessments is that these parameters are routinely measure d using commercially available kits and equipment and several analyses can be measured from the same animal using a small amount of blood. A n advantage of using blood collection techniques as a measure of fish health is that they usually can be carried out without killing the fish (Maita 2007) A disadvantage is that many factors can confound hematological data such as differences in species, age, sex, wate r quality, water temperature, and handling methods. All these variations can make comparisons between studies difficult and make setting normal ran ges, or reference ranges, challenging ( Maita 2007 ) Ways to mitigate these variations are to create a static protocol to collect samples that is repeatable with boundaries on fish length, weight, time of year to sample, time of day, and a standard collection method. Blood is usually collected from one of four locations in the fish, caudal vein, heart dorsal aorta, or the branchial vessels There are advantages and disa dvantages associated with each site Collecting a blood sample from either the caudal vein, by cardiac puncture or from the dorsal aorta leaves a chance for other constituent body fluids to enter the sample. Also cardiac puncture is typically only performed on sedated
30 fish that are going to be euthanized. Using the branchial vessels in some species of larger fish is quick and e fficient The choice of blood collection techniques will depend on the fish species size, body morphology, protected status, available restraint methods, and lethal or non lethal outcome. Reproductive H ealth The health and success of a species ultimately relies on successful reproduction. Understanding baseline values for sex hormones is necessary to ascertain wh at sex hormone ratios could potentially impact reproductive success of red drum ( Roberts et al. 1999 ) E ffective management practices reproductive biology. When a species of fish is under threat from excessive fishing pressure during its spawning period this can lead to detrimental effects on larval output ( Coleman et al. 1996 ) An approach to measuring fishing pressure is to examine fish after undergoing capture noting the duration of capture, and evaluating their stress hormones. Stres s can un favorably affect reproductive physiology in fish by causing changes in reproductive hormone concentrations, fecundity, egg size, and survival of eggs and larvae ( Billard et al. 1981 ; Ca mpbell et al. 1994 ; McCormick 1998 ) Sex Hormones Seasonal changes in gonadal steroid hormone concentrations have been well documented in many freshwater teleost species. L ess information is available on gonadal steroid hormone concentrations for marine species ( Prat et al. 1990 ) Wild white sturgeon h ave been studied by Webb et al. ( 2002 ) for potential classification of sex and stage of gonadal maturity using plasma hormones 11 ketotestosterone (11 KT) and estradiol ( E 2 ). Th e y reported that fish between 96 152 cm fork length with values of 5 ng/m l or higher of 11 KT were classified as male and those with values of
31 2mg/m l or higher of E 2 were classified as females. Their conclusion was that the use of plasma sex steroids provide d an accurate and less invasive method for calibrating current population models They were able to use plasma hormone analysis to replace the ne ed for surgical gonadal biopsies on these sexually mono morphic fish. Red drum are also sexually mono morphic Mature males may be recognized if a drumming sound is heard, or if milt is seen extruding from the vent during spawning season Traditionally f is heries research has require d the sacrific e of individuals for accurate sex identification which is necessary for population dynamics studies and stock assessments A study on cultured and captive red drum by Kucherka et al. ( 2006 ) evaluated circulating sex steroid concentrations of (11 KT) and (E 2 ) The key results were that a minimum concentration of 11 KT 1.0 ng/ml is the threshold to distinguish male sex of fish during summer or early fall, of cultured adult red drum. It is imperative to be able to correctly identify the sex of red drum to delineate seasonal differences in blood plasma parameters These parameters may vary based on their sex, since plasma steroid profiles vary during different stages of the gonadal cycle in both males and females ( Kucherka et al. 2006 ) Males had values of 0.7 ng/ml of 11 KT in early fall and 1.0 ng / ml during late fall while females had values of 0.5ng/ml in early fall and late fall respectively According to Hamlin et al. ( 2007 ) few studies have been conducted using fish to define the relationship between stress and possible changes i n sex steroid concentrations. By contrast, general s tress responses to numerous environmental conditions have been studied in a range of fish species, especially long lived species of high economic importance ( Hamlin et al. 2007 )
32 Stress Response Fish are exposed to stressors on a daily basis both in the ir natural habitat and under aquaculture conditions ( Bayunova et al. 2002 ) Bonga ( 1997 ) defines stress as a response evoked in conditions that cause discomfort, fright, pain, or a sensory perception of an adverse stimulus or its effects. It is a common misconception among fishery biologists that stress in itself is detrimental to the fish Stress is a necessary adaptive mechanism that allows the fish to cope with stressor s and maintain homeostasis ( Barton 2002 ) The stress response of fish is comparable to th at of mammals and conforms to a general vertebrate pattern ( Bonga 1997 ) The effects of stressors can disturb the h omeosta sis of an animal and cause a coordinated set of behavioral an d physiological responses that attempt to enable the animal to overcome the threat ( Bonga 1997 ) Stress responses in fish are part of an integrated process which can be broken down into four components The primary stress respo nse, the immediate reaction of the response s are the immediate actions and effects of hormones at the blood and tissue level These may include increases in cardiac outp ut, oxygen uptake, mobilization of energy and changes in osmoregulation Some tertiary responses, such as changes in blood physiology, are seen over longer periods of time and may inhibit growth, reproduction, and the immune response ( Bonga 1997 ) The quaternary responses are seen long term at the population level as reproductive outputs, die offs and changes in birth rates.
33 Acute and Chronic Stress According to Pankhurst ( 2011 ) under most circumstances, recovery from acute stress will occur in most fish species over a period of six hours or less. In some species elevated plasma cortisol concentrations can persist for days if the stressor is chronic or severe ( Hamlin et al. 2007 ) Magnitude of change in plasma cortisol concentration is affected by sampling techniques, size, season, age and rearing temperature in white sturgeon ( Hamlin et al. 2007 ) The typical cortisol and glucose response following exposure to a stressor would be a rapid increase reach a peak then a slow decrease to resting concentrations after t he stressor has been eliminated Chronic s tress in cultured fish causes an increase in cortisol and may cause negative physiological conditions such as impaired immune function ( Tort et al. 1996 ) increased oxygen radical production ( Ruane et al. 2002 ) and reproductive impairment ( Pankhurst and Van Der Kraak 1997 ) S tressors may negatively affect reproduction; however there is a growing amount of evidence that states otherwise Cortisol is a normal endocrine comp onent of the reproductive system but it can suppress reproduction in some cases ( Hamlin et al. 2007 ) Th is relationship has been studied i n Siberian sturgeon ( Acipenser baer i i ), and the findings were that plasma concentrations of E 2 testosterone and 11 KT did not decrease with elevated plasma cortisol concentrations following acute handling stress This is consistent with observations in many other species of fish. Wingfield ( 1994 ) explained that studies on wild populations show that severe environmental conditions and the demands of reproduction are not necessarily stressful if they are predictable. Fish hav e predictable reproductive periods and most usually have site fidelity year after year. Some harsh environmental conditions could include adverse water quality such
34 as extreme temperature s or salinit ies but if seasonal patterns occur annually within the tolerance range of a fish then these seemingly extreme conditions may be well tolerated. Consequently, stress caused by a situation that is reoccurring on a n annual basis may be minimal or non existent for wild populations. Cortisol Stress physiology stud ies on fish have focused on the aquaculture industry with the goal of maximizing production ( Barton 2002 ) Stressors initiate a c omprehensive endocrine response in fish characterized by hypersecretion of catecholamines and cortisol which then induces a variety of secondary effects previously mentioned. Stress responses of red drum have been studied in cultured fishes ( Robertson et al. 1987 ) with an emphasis on use of plasma cortisol and glucose as reliable indices ( Wedemeyer and Yasutake 1977 ) Cortisol is the predominant corticosteroid in an acute stress response in red drum. Cortisol combines mineralocorticoid and glucocorticoid functions in fish which reflect the relationship between energy metabolism and hydromineral control. The mineralocorticoid functions provided by cortisol include the promotion of differentiation of the chloride cells in the gills, intestin e and kidneys. The glucocorticoid functions affect carbohydrate, protein and lipid metabolism ( Bonga 1997 ) Stress can be acute or chronic each state having different effects on the endocrine system of the animal. In a chronic stress response, cortisol concentrations may remain elevated but below peak concentrations for prolonged periods of time Basal concentrations were reported as low as 5 ng/ml in red drum ( Barton and Iwama 1991 ) Chronic stress responses in cultured fish have been assessed with handling disturbance, heavy metals, organic pollutants, rapid temperature changes acidic water, and confrontations
35 with predators ( Bonga 1997 ) Barton and Iwama ( 1991 ) reviewed corticosteroid values in sixteen fish families with varying sample sizes, including data on pre stress and post stress values which ranged from <1 ng/ml to 2000 ng/ml depending on the various species, stressor and conditions. A laboratory study on Atlantic cod by Morgan et al. ( 1999 ) reported that fish stressed for half an hour three times a week for ten weeks by capture/confinement had higher plasma cortisol concentrations than undisturbed control fish Fish in both groups were however, able to be spawn ed and there was little difference in the production of eggs, fertilization rates, and hatching success of larvae. Due to sampling complications, less information on stress and its physiological and endocrine effects in natural settings is available for wi ld stocks of fish ( Pankhurst 2011 ) Thomas and Robertson ( 1991 ) stated that common aquacultu re procedures such as netting, handling, disease treatments and transportation are stressful events for fish and maybe associated with increased susceptibility to disease and a reduced capacity to maintain homeostasis Pankhurst and Sharples ( 1992 ) found that to date the lowest values measured for corti sol in wild fish were 1.7 ng/ml in snapper, Pagrus auratus which were captured by net and sampled underwater by scuba divers in less than ten minutes The length of time from capture to blood sample collection is also an important factor to consider for sample design. An important factor to remember when sampling wild fish is that there is a short time lag that precedes the increase in corticosteroids. Changes caused by the acute stress response can be minimized if sampling occurs rapidly ( Pankhurst 2011 ) Rapid hook line capture is a suitable field protocol to combat the issue of increasing corticosteroids soon after the perturbation. Basal cortisol values in teleost fishes are
36 typically <10 ng/ ml ; however a number of species have much higher values in unstressed fish ( Pankhurst 2011 ) F or example cortisol values in Salmonidae have been reported at 544 ng/ml Oncorhynchus mykiss and 122 ng/m l Coregonus lavaretus Reported cortisol values for Cyprinidae have been Carassius auratus 66 ng/ml, and for Percichthyidae, Morone saxatillis 250 ng/ml were recorded values ( Barton and Iwama 1991 ) Variability in corticosteroid concentrations can be due to a wide range of factors besides stress The sex of the fish and maturity, time of day, time since last feedi ng, and seasonal variations all influence corticosteroid concentrations ( Pankhurst 2011 ) E stablishing causal relationships between environmental stressors and observed effects in fish from natural systems is difficult due to many intrinsic environmental variables Presently there are no widely accepted and proven approaches for determining their relationships ( Adams 2003 ) Adams ( 2003 ) found that field studies have advantages, in that they represent the natural environmental conditions, but they also have the limitations that causal factors co occur resulting in high variability Glucose G lucose can be used as an indicator in assessments of fish health ( Robertson et al. 1987 ) The standard methodology for measurement of glucose has been to collect blood and then run a laboratory assay to analyze the sample. Recent technolo gy created for diabetic patients uses handheld glucometers that provide an instantaneous reading. These handheld glucometers may not capture the full range of glucose values for fish as they were created to detect mammalian ranges. A point of care system that has been utilized by veterinarians for many years on a wide range of animals including marine mammals, and reptiles is an Abbott i STAT analyzer. An Abbott i STAT
37 analyzer can analyze a suite of blood parameters. The Abbott i STAT is sensitive to t emperature and humidity levels and therefore can be difficult to use under field conditions. If the temperature of the unit is too hot it will give an error reading requiring the use of more than one cartridge per fish which becomes expensive. There have been a few studies that have e valuat ed the use of handheld meters in fish ( Iwama et al. 1995 ; Wells and Pankhurst 1999 ) The ir ease of use, portability and quick sample analysis make handheld meters promising alternatives to traditional laboratory methodologies ( Venn Beecham et al. 2006 ) B oth an i STAT meter and Contour glucometer will be used to test glucose concentrations according to the methods of Venn Beecham et al. ( 2006 ) and Brown et al. ( 2008 ) The Abbott i STAT and Contour glucometer are not validated for the red drum species and values obtained will need to be validated for ac curacy. A l aboratory glucose absorbance assay validation will evaluate the accuracy sensitivity, and reliability of these two field glucometers Objectives and Hypotheses To date no studies exist that have incorporate d a non lethal health assessment on a wild population of red drum and that evaluate the sex and stress hormones of the fish temporally T he goal of this study (Chapter 2) is to evaluate non lethal techniques to assess the exter population Kennedy Space Center (KSC) Reserve. We hypothesize that 1) The red drum population inside the KSC Reserve will score in the healthy range, ( above 45 ) of the SERF health index, 2) t here will be a significant seasonal dif ference in glucose va lues of red drum due to foraging pattern changes that correspond to the reproductive period such as a hyperglycemic response during pre and post spawning and a hypoglycemic response during spawning 3) t here will be seasonal variation in the sex steroid p rofiles
38 of 11 ketotestosterone and 17 estradiol that correspond to the reproductive period and 4) t here will be seasonal variation in the concentration of cortisol that corresponds with the reproductive period.
39 Figure 1 1. Kennedy Space Center s ecurity z one d e f acto no t ake f isheries r eserve is outlined in yellow.
40 Table 1 1 Health index scoring system developed for red drum by the S tock E nhancement R esearch F acility ( Dukeman et al. 2006 ) Max imum possible scores are shown below A score of 45 or greater is considered consistent standard condition factor equation was used for the calculation. Fish ID Example C ondition factor x 5 10 Eyes 1 Mouth 1 Mucous 3 Microbial infections 2 Gill parasite pathogen 2 Gill parasite # 4 Gill parasite load 4 Gill a bnormal 5 Skin/fin/ parasite pathogen 2 Skin/ fin parasite # 4 Skin/ fin/ parasite load 4 Scale loss 2 Wounds 2 Other a bnormality 4 Health i ndex s core 50
41 CHAPTER 2 HEALTH ASSESSMENT OF ADULT RED DRUM Understanding s easonal variation in plasma concentrations of hormones a nd other parameters is important for the assessment of health, and physiological activities including reproduction, growth and metabolism. Although significant work in this area has been done with captive populations of various fish species, mostly those associated with fish stocking programs and aquaculture, an improved understanding of wild fish in their natural environmen t is critical if we are to understand the impact of human activities and environmental perturbations. This is especially critical for economically important sport fish including red drum ( Sciaenops ocellatus ) N on lethal health assessments have not been carried out on red drum in Florida despite the significant recreational catch and release fishery present. The impact of this fishery on the health of individual fish is poorly understood. F ew studies have resulted in baseline values for blood parameters. Thus, there has been little data for comparative studies among wild populations. Previous studies on the reproductive biology of th is species are also limited primarily to age and growth studies complemented by histo logical assess ments of maturation rates ( Murphy and Taylor 1990 ; Murphy 2009 ) Stock assessments are currently conducted by Florida Fis h and Wildlife Conservation Commission (FWC) These consist of m onthly sampl ing of red drum from both coasts of Florida using a variety of fishing gear, targeting different size classes of fish. N on lethal assessment s are conducted on fish that do not ha ve obvious gross lesions. These fish are measured and released ; however minimal data is collected to assess the internal health of th e fish in hand. The se non lethal samples provide insight into catch rates over time T he most
42 recent stock assessment s were conducted in 2008 and indicated that overfishing was not occurring on either coast of Florida ( Murphy 2009 ) Lethal exams are conducted so that gonad s and otoliths can be harvested for reproduction status, age and growth analysis. I t is not ideal that lethal sampling must be conducted to verify that fish are indeed healthy from a n economic perspective of the fishery R ed drum stock assessments can be improved so they provide more information while decreasing the use of lethal harvest techniques Some limits to non lethal sampling are that age and growth determination from otoliths is not possible, along with internal organ assessments for condition i ndexes and sex determination. New methods such as fin clips for age determination and blood hormone analysis for sex determination are currently being investigated as alternative means of collecting this data. The Stock Enhancement Research Facility (SERF ) developed a health index for juvenile red drum which is use d to assess the health status of their fingerlings. The health index can be p erformed without lethal harvest SERF currently utilizes this as a lethal method since information on internal organ health is needed by the hatchery The health index metric assess es the external condition of the fish utilizing quantitative and qualitative data based on the evaluation created by Dukeman et al. ( 2006 ) This health index range s in a possible score of 0 50 based upon external condition When a red drum scores a value below 45, its health is determined to be compromised, whereas a Study Objectives The goal of th is study wa s to develop non lethal techniques for assessment of population Reserve The objectives of this project were to evaluate the potential use of the SERF
43 health in dex on a wild population of red drum inside the KSC Reserve t o determine if there are seasonal changes in blood glucose concentrations in wild red drum that change with reproductive period t o evaluate plasma concentrations of sex steroid hormones 11 keto testosterone and 17 estradiol in three different reproductive periods and t o evaluate plasma cortisol concentrations in three different reproductive periods We hypothesize that 1) t he red drum population inside the KSC Reserve will average their score in the healthy range, ( above 45 ) of the SERF health index, 2) t here will be significant seasonal differences in glucose values of red drum due to foraging pattern changes that correspond to a higher appetite and body demand during the reproductive p eriod 3) t here will be seasonal variation in the sex steroid profiles of 11 ketotestosterone and 17 estradiol that correspond to the reproductive period and 4) t here will be seasonal variation in the concentration of cortisol that corresponds with the reproductive period. Health can be loosely defined as the level of functional or metabolic efficiency of a living organism. Many blood parameters are utilized to assess health in marine mammals and reptiles as well as fish Given the lack of baseline blo od parameters for red drum, this project concentrated on four blood parameters which were most commonly assessed in the literature. We propose that data such as these will enable researchers and state or federal agencies interested in the well being of f ish for management or sport able to refer to reference values for sex and stress steroids in a wild population of red drum.
44 Materials and Methods Fish Capture M ethods All fish were handled according to procedures approved by the Institutional Animal Care and Use Committee (IACUC) NASA, protocol # GRD 11 077. Fish were sampled according to the guidelines of the special activity license # SAL 09 0512A SR, issued from the Florida Fish and Wildlife Conservation Commission (FWC) and a special use permit # 2011SUP001, issued from the Merritt Island National Wildlife Refuge (MINWR). All fish were caught using a hook and line approach designed to decrease fight time. Barbs we re shaved off of circle hooks to lessen any potential injury to the fish or staff and to minimize handling time Large surf rods and 30 lb. test line w ere used to land fish in the boat quickly and reduce handling time. Only fish over 650 mm standard lengt h (SL) were used for this project All fish were caught in the KSC Banana River reserve area and all fishing sites were visited during each of the three reproductive periods (Figure 2 1 ). The three reproductive periods were defined as pre spawning ( May ) spawning ( September and October ) and post spawning ( December ) Angling time and handling time were recorded for each fish using a handheld timer. The timer was started when the angler felt the fish bite the hook. Fight time was recorded and kept under five minutes. Fish were netted when close to the boat, immediate ly de hooked, and then placed in lateral recumbency for the blood draw procedure. Plasma Collection Fish were manually restrained on an inverted v tray with a biologist holding the operculum away from the body wall to allow access to the gills. A 4 ml b lo od sample w as extracted from the branchial vessel s
45 20 gauge needle, and drawn into a lithium heparin V acutainer (BD, Franklin, NJ) tube labeled with an individual identification number (Fig ure 2 2 ) Th e time interval from the fish being hooked to blood sample collection was recorded, and was kept under seven minutes. Several drops of blood were immediately extracted from the vacutainer for glucose analyses using a Contour (Bayer Healthcare Tarrytown, NY) glucose strip for every fish. A subset of ten fish from pre spawning and seven from spawning were tested for glucose on an i STAT (Abbot, Princeton, NJ) analyzer B lood was placed on ice until it was returned to the laboratory and centrifuge d for a minimum of 10 minutes at 3000 g to separate the plasma and cell fractions P lasma w as then aliquoted into 1.8 ml cryovials and frozen at 7 0 C. External examination protocol The external exam included traditional morphometric measurements outlined by Lagler ( 1962 ) These included standard length (SL: tip of snout to the posterior end of the last vertebra), total length (TL: tip of snout to tip of the caudal fin ) and weight which were recorded to the nearest centimeter and gram, respectively. A g ill biopsy and skin mucus were collected using plastic microscope slide covers Wet mounts of these tissues were examined immediately at 10 0 x for the presence of external parasite fauna and the number of organisms per sample was recorde d. The external exam also evaluated the eye s, mouth, external mucous layer, scales, and the fins of the fish. Physical deformities were noted, along with the presence of lesions that could suggest bacterial, fungal or other infections (e.g. presence of u lcers). External parameters were recorded and scaled according to severity using the SERF health index ( Dukeman et al. 2006 ) ( Table 1 2 )
46 Sex I dentification Field attempts to identify sex of collected fish i drumming soun d and applying pressure to the abdomen to determine if milt was exuded during the fall spawning period, both indicative of a male ( Fig ure 2 3 A ) Possible females were probed in the vent area with a pipette to attempt to determine sex by locating the urinary orifice, anus, and ovipositor (Fig ure 2 3 B ) If sex was not clear ly distinguished sheet. Tagging and H andling A s a means to permanently identify fish, a PIT tag (Biomark 12 mm 125 kHz) w as inserted using a sterile12 gauge needle placed subcutaneously near the cheek on the left side Also the dorsal portion of the caudal fin was clipped as an external mechanical tag Capture positions w ere recorded using global positioning system (GPS) equipment The fish were released immediately after examination and sample collection was completed, typically < 15 minutes from hooking time Water Quality Water chemistry parameters (e.g., temperature, salinity, dissolve d oxygen and pH) were recorded using a Yellow Springs International water quality sonde, model 6920, at all fish capture locations during each sampling period. The temperature data collected was compared to NOAA buoy, station 41113, the Cape Canaveral Nea rshore station using a t test. Condition Factor Condition factor was calculated for all fish using the equation of fish weight (g) x 100 /standard length 3 ( Fulton 1904 )
47 Steroid Assays A solid phase radioimmunoassay using a 96 well plate format (Perkin Elmer, Boston, MA, Protein A Flash Plate Plus) was used to determine concentrations for estradiol 17 (E 2 ) as previously described by Hamlin et al. ( 2011 ) Plasma samples were thawed on ice and extracted twice using 5 ml diethyl ether by vortexing the plasma samples vigorously with ether. The aqueous phase was removed by snap freezing in liquid nitrogen and the organic pha se was decanted into a new clean tube. Th e diethyl ether extract was evaporated over filtered air and stored overnight at 20C. Prior to running the assay, each sample was reconstituted with phosphate buffered saline with gelatin (1%). The samples were run in duplicate and each of the four plates contained duplicate wells for interassay variance and a blank. Sample plates were analyzed using a microplate reader (Perkin Elmer MicroBeta 2 2450 Microplate counter, Waltham, MA). E 2 was validated in triplicate for red drum by verifying that serial dilutions of pooled plasma were parallel to the standard curve. The interassay variance samples were verified by measuring the steroid in extracts obtained from a sample of plasma pooled from t hree males and three females. The assay validation response was parallel to the respective RIA standard curve for E 2 Spikes were also performed and were validated. The interassay variances were less than 5 % among plates Unknown concentrations were e xtrapolated from standard curves plotted as the % bound versus the log 10 concentration. Plasma E 2 concentrations in some fish were higher than the standard curve, so the max value approach was utilized. The highest value validated on the standard curve w as substituted for the values that were too high off the standard curve.
48 Plasma 11 ketotestosterone ( 11 KT ) and cortisol were assayed using commercially available enzyme immunoassay kits (ELISA) (Cayman Chemical, Ann Arbor, MI, USA; cat. 582751 and cat. 500 360 respectively). Plasma samples for both 11 2 Desiccated samples were reconstituted with EIA buffer provided with the kit. Both assays were validated for red drum by verifying that serial dilut ions of plasma and spiked standards were parallel to the standard curve. The samples were run in duplicate and each of the six plates contained duplicate wells for interassay variance and a blank. Both kits f ollow ed the recommended incubation time of eig hteen h ours at 4 C Sample plates were analyzed at a wavelength of 412 nm using a microplate reader (Molecular Devices Versa Max tunable, Sunnyvale, CA). The interassay variances were less than 7 % for all plates of the assays Unknown concentrations wer e calculated from standard curves plotted as the % bound versus the log 10 concentration. Glucose Analyses Glucose was analyzed using several methodologies, including hand held devices potentially useful in the field and a standard laboratory based techniqu e. First, a drop of blood was immediately obtained following the collection of the blood sample in the field and applied to a Bayer brand Contour glucose strip. The strip then was inserted into the Contour glucometer and the glucose value recorded. Pla sma glucose also was measured by an absorbance assay, using an Invitrogen, Amplex Red Glucose/ Glucose Oxidase Assay Kit (A22189). Plasma samples were thawed on ice and evaluated according to the instructions provided. The samples were diluted 20X with buffer to keep the concentrations within the limit of the kit. The four
49 sample plates were analyzed using a microplate reader (BioTek Synergy HT, Winooski, VT). i STAT Analyzer A sub sample of fish during the pre spawning (n= 10) and spawning period s (n = 7) also were tested in the field with an Abbott i STAT analyzer using one drop of blood per cartridge. The CG4+ and CG8+ cartridges were used. The CG4+ cartridge provided values for lactate, PCO 2 PO 2 TCO 2 HCO 3 sO 2 The CG8+ cartridge provide d values for glucose, hematocrit and hemoglobin. The Abbott i STAT analyzer was not available for use during the post spawning reproductive period, so only glucose values for those periods were utilized to compare results with the laboratory assay and fie ld glucometer. Statistical Analys e s Individual red drum served as sampling unit replications Treatments consisted of the three sampling periods ( pre spawning, spawning, and post spawning ) representative of three reproductive periods. To compare water qu ality data, cortisol data, 11 KT data and E 2 data ANOVA and t tests were used. Health index d ata did not pass the normality test, Shapiro Wilk ( p = 0.03, 0.02, 0.02) data w ere log transformed, and a one way Analysis of Variance was used to test for diffe rences among the three periods of reproduction, F (2, 123) = 36.58, p = 0.14. To compare glucose data, Kruskal Wallis non A p value was considered significant if lower than 0.05. Statistical a nalyses were performed with Sigma Plot (11.0, San Jose, CA). Discriminant function analysis (DFA) was used to predict the sex of fish for which sex could not be determined in the field ( Venables 2002 ) Known values for the
50 paramet ers c ortisol, 11 KT, E 2 and lab glucose were used as explanatory variables. Predictor variables were log transformed to meet the assumption of multivariate normality. Values of 11 KT and cortisol below the detection threshold were replaced with one half of the minimum detection value. DFA, with all explanatory variables, was used to predict the sex of undetermined fish. A step wise DFA was used to explore reduced models that could be useful in understanding relationships between variables for use in sex determination in future field studies. Graphical methods and statistical tests were used to access how well the data met the two key assumptions of DFA multivariate normality and equality of the variance covariance matrices ( McGarigal et al. 2000 ) Quadratic discriminant function analysis was used to verify results of the DFA for indications that failure to meet statistical assumptions influenced the results. All statistics were performed in R Deve lopment Core Team ( 2012 ) DFA was conducted using the MASS package ( Venables 2002 ) and stepwise DFA was performed using the klaR package ( Weihs et al. 2005 ) Results Morphometics & Condition Factor A total of 12 6 fish were caught during the three sampling periods T otal length of pre spawning period fish (n=38 ) averaged 774 96.2 S.D. mm with average weights 5.39 2.72 S.D. kg and an average condition factor of 1.6 0.10 S.D. in May 2011. Spawning period fish (n=45) had a n average total length of 797 79 S.D. mm with average weight of 6.12 2.79 S.D. kg and an average condition factor of 1.72 0.18 S.D. in September and October 2011 The post spawning period fish (n=42) had an average length of 847 79 S.D. mm, average weight of 7.64 2.71 S.D. kg and an
51 average condition factor of 1.77 0.17 S.D. in December 2011 M orphometric data and condition factor are presented in Table 2 1. Water Quality Temperature, dissolved oxygen, salinity and pH data for each sampling site per capture day exhibited little variation among sampling periods (Table 2 2). An independent samples t test was conducted to compare the field temperature to the NOAA buoy station 41113. There was no significant difference between the measurements for field temperature ( M = 25.53, SD =4.38) and NOAA buoy temperature ( M = 24.63, SD =3 .33) t (4) = 0.28, p = 0.79. Health Index Using the SERF health index, t he fish obtained during this study exhibited average scores above 45 during the pre spawning and spawning periods, which is considered the threshold for healthy at the SERF hatchery. The average score decreased slightly to 42 in post spawning fish (Fig ure 2 4 ). A post hoc Tukey tes t showed reproductive periods, s pawning vs. p ost spawning differed significantly at p <0.05, and p re spawning vs. p ost spawning differed significantly at p < 0.05 The s pawning vs. p re spawning periods were not significantly different p = 0.25. Parasites The majority of red drum (90%) sampled in this study had grossly visible external parasites. Three types of parasites were commonly identified : Argulus s p. Caligus sp. and Ergasilus sp. One Argulus sp. per fish was observed, on average, usually on the ventral surface. In contrast, Caligus sp. was more common, averaging 15 per fish, again, found on the ventral surface. A mean of one Ergasilus sp. per fi sh was found inside the operculum of each fish.
52 Plasma Glucose Concentrations Three different methods were used to determine glucose concentrations for the Pre spawning period and Spawning period T he Abbott i STAT was not used in the Post spawning period S ignificant difference s in glucose values were found between the glucometer and the lab assay ( Table 2 2 ) Since only a sub sample of glucose values were obtained with the Abbott i STAT there were not enough data values to make statistical comparisons between the lab g lucose data or the glucometer. Glucose data for the lab absorbance assay and the glucometer for all three reproductive periods did not pass the normality test Shapiro Wilk ( p <0.05). A Kruskal Wallis Analysis of Variance on Ranks was used to test for differences among the three periods of reproduction between the absorbance assay and the glucometer, H (2) = 22, p = <0.001. A pairwise multiple comparison procedure post hoc test, was performed, s pawning period vs. p re spawning differed significantly Q (4.01), p<0.05, s pawning period vs. p ost s pawning differed significantly Q (4.09), p<0.05. Pre spawning period and p ost spawning were not significantly different Q (0. 03) p >0.05. Linear regression between fish fight time and lab glucose concentration had an R 2 value of 0.03 S i milarly a linear regression between total fish handling time from hook up and lab glucose concentration also had an extremely low R 2 value of 0. 007. Plasma Cortisol Concentrations On average, plasma cortisol concentrations ranged from 0.93 ng/ml to 1.25 ng/ml for the three reproductive periods examined (Table 2 2). The EIA for cortisol reported nine fish concentrations above the m axi mum value on the standard curve, so a m ax imum concentration approach was employed. The highest value validated on the standard curve was substituted for the values that were originally higher than the
53 highest value validated on the standard curve A t test was co n ducted to compare cortisol among the reproductive periods T here was a significant difference between the periods ( M = 1.11, SD = 2.02); t (111) =5. 81, p <0.001 (Fig ure 2 5 ). Plasma 11 KT Concentrations On average, plasma 11 KT concentrations ranged from 0 04 0 0 .058 ng/ml during the three reproductive periods examined (Table 2 2). The EIA for 11 KT reported values that were below the minimum value on the standard curve so a minimum concentration approach was employed. This approach substituted half of the lowest value from the validated standard curve for those values that were below the minimum value of the standard curve. A t test was co nducted to compare 11 KT among the reproductive periods T here was a significant difference among the periods ( M = 0.23, SD = 0.55); t (121) =4.73, P <0.001 (Figure 2 6 ). Plasma E 2 C oncentrations On average, plasma E 2 concentrations ranged from 0.41 ng / ml to 2.28 ng / ml during the three reproductive periods (Table 2 2). E 2 had some values that were higher than the standard curve, so the max imum value approach was engaged. The highest value validated on the standard curve was substituted for the values that were originally higher than the highest value validated on the standard curve. Only four fish had values that were too high and they were positively field identified as females in the field during spawning period. A t test was co nducted to compare E 2 among the reproductive periods T here was a significant difference between the periods ( M = 1.1, SD = 2.71); t (124) =4.51, P <0.001. Plasma concentrations of E 2 were significantly different among reproductive periods according to the t test (M = 1.1, SD = 2.71); t (124) =4.51,
54 P<0.001 (Figure 2 6). However, plasma E 2 concentrations were significantly elevated in females during the spawning period compared to males. Sex Identification Cortisol and lab glucose met the assumptions of the DFA multivariate normality and equality when comparing the means and plots. E 2 and 11 KT resulted in differences of group means an d plots indicating some departure from normality. The classification function derived was: 1.110972 0.1028649log (cortisol) + 0.6881698 log (11 KT) 1.0840710 log (E 2 ) +1.5684448log (lab glucose). The group mean for females was 1.123013 and for males w as 1.314225 Among the fish of known sex used to derive the discriminant function, 22 of 25 females and 41 of 48 males were correctly classified. The quadratic DFA resulted in 24 of 25 females and 43 of 48 males correctly classified. Quadratic DFA does not assume multivariate normality, so the DFA could perform better when this assumption is not met ( Venables 2002 ) Using this approach, we predicted that twenty four of the unknowns were female and that eleven were likely to be male (Table 2 5). Recaptures The majority of fish (n=124, 98 % ) used for this study were only captured once, but two individuals were captured twice The within study recapture rate ( [ # of fish recaptured/ # of fish captured ] mult iplied by 100 ) was 1.6 % One male initially caught during the pre spawning (May 9, 2011) period was recaptured during the post spawning period December 15, 2011 period. The measurements for this fish display rapid growth in length and weight gain, from 710 mm TL to 761 mm TL and 3.75 kg to 4.90 kg. The second recaptured fish, a female that was initially caught during the spawning period, October 10, 2011, at 784 mm TL and 5.00 kg and was recaptured during the post
55 spawning period, December 13, 2011 Sh e was caught at the same location and measured 784 mm and weighed 5.75 kg. Discussion This study defined parameters that can be used to assess wild red drum in the field and further defined baselines for plasma concentrations of cortisol, glucose, estr adiol (E 2 ) and 11 ketotestoterone (11 KT) for adult red drum in the KSC Reserve during three phases of the reproductive cycle Further, we evaluat ed wild caught fish using external parameters outlined in the SERF health index This is the first study in red drum to examine the relationship between a measure of stress (e.g., plasma cortisol concentrations) and potential reproductive function status, as indicated by the plasma concentrations of various sex steroids in a de facto no take fisheries reserve. This is also the first study to obtain blood samples from the branchial vessels in non anesthetized wild red drum. Predicted Outcomes I predicted that a no take fishery reserve, such as the KSC Reserve, would have fish that s core in the healthy range of the SERF health index We observed such a pattern for fish caught during the pre spawning and spawning period s Fish caught in the post spawning period scored slightly below this range A factor that contributed to the depressed score during t he post spawning period was parasite load. The external parasites identified in this study were those that were visible to the naked eye These were primarily found on the ventral surface of the fish and under the operculum. Microscopic examination of g ill tissue and skin mucus did not reveal protozoans or other parasites beyond those macroscopically visible. None of the gill biopsy samples showed any abnormal signs of hyperplasia or hypertrophy.
56 The SERF health index was developed to assess cultured ju venile red drum These fish are not expected to have parasites, which wa s the main difference in the health scores between hatchery reared fish and wild fish sampled in this study Over 90% of wild fish sampled had parasites This is not surprising when evaluating wild fish, and Landsberg et al. ( 1998 ) suggested that p arasites o n wild fish may be indicative of healthy ecosystems However excessive parasite loads on a fish suggest a compromised immune system and may be indicative of comprised health Evans et al. ( 1997 ) considered wild red dru m from estuaries in South Carolina with fewer than three determination was used in this study, only 10% of the study animals would have been trophy sized fish according to their condition factor. A ll fish with three types of parasites evaluated in this study were still robust in length and weight and had excellent condition factor scores. These fish had no ulcers or other external lesions. Even while using barbless circle hooks, we were able to catch trophy size red drum with ease suggesting that barbed hooks may be unnecessary for catching these fish Barbed hooks may cause more harm if swallowed during catch and release procedures Fish collected during the post spawning sampling period actually had the highest average condition factor suggesting that there was minimal spawning condition deterioration in this population. The reason for the slightly lower health index score i n this group was due to higher parasite numbers on these fish. Since fish collected in all impression that the sampled population of red drum in the reserve were thriving. One
57 cave at is the hook and line technique employed for this research would not catch fish that did not bite the hook. Fish that were c aught during this study were hungry or interested in feeding since they attempted to feed on our bait / tackle. Using the techni que of netting/ encircling fish would have eliminate d that concern and could have resulted in a different outcome. Previous research has demonstrated that sportfish within the KSC Reserve achieve a larger mean size and exist in higher densities than those in adjacent public areas ( Johnson and Funicelli 1991 ) My second hypothesis stated that there would be a significant difference in seasonal blood glucose concentrations potentially indicative of metabolic disturbance caused by changes in fish feeding habits or envi ronmental conditions The detectable range for the Contour glucometer for this study was 10 600 mg / dL. Glucose values obtained using the Contour glucometer had a broad range of values during each reproductive period There were no seasonal trends in glucose values. A population of f ish having large ranges for glucose is not uncommon H owever to pinpoint a reason for this is more difficult without stomach analysis to determine what the diet consisted of or when the fish last ate T he capture stress and handling also can affect glucose values, typically inciting a hyperglycemic response. Since the fish captured in this study were all brought to the boat in less than seven minutes and a blood sample was collected in under eight and a half minutes, the time necessary for the glucose to reflect any The values collected may then reflect actual baseline values of the animal. Handheld glucometers may not capture the f ull range of glucose values for fish since they were created to detect mammalian ranges. A study by Iwama et al. ( 1 995 )
58 investigat ed the use of glucometers for field use on cultured Atlantic salmon by holding the fish in a dip net out of the water for thirty seconds and then collecting a blood sample at 2, 4, 6 8 and 24 hours afterwards from the caudal vein They found that glucose values obtained by the glucometer were two times higher than the laboratory assay and the reason for the difference was unknown. Venn Beecham et al. ( 2006 ) compar ed a glucose meter to a laboratory assay for c hannel catfish The study was performed with MS 222 and then some container with MS 222 B oth groups were sampled from the caudal vasculature for a blood sample. They found that the Accu Chek Advantage meter was consistently lower than the values obtained from the laboratory reference method for both groups of fish although there was low variability between replicates for the meter. Glucose meters can be useful tools for measuring plasma glucose values in th e field; however they should only be utilized when relative measurements are appropriate. The laboratory glucose absorbance assay had average values of 36 mg/dL during the pre spawning period, 38 ml/dL for spawning and 29 ml/dL for post spawning. These va lues are similar to the basal values reported for Siberian sturgeon. Hamlin ( 2007 ) reported basal values for plasma glucose for captive Siberian sturgeon of 36.3 mg/dL wi th a peak during capture and handling stress of 84 mg/dL using the same commercial glucose oxidase kit Some higher glucose values were obtained for red drum that were similar to those values for Siberian sturgeon that endured handling stress, however the re were only weak correlations between the times from fish hook up to landing, and from hook up to the blood sampling. It is possible that the element of time on the hook
59 or time to blood sample collection was not highly correlated with glucose concentrat ion but rather the individual response of the red drum is highly variable. There was a significant difference between the absorbance assay and glucometer for the s pawning period vs. p re spawning period and s pawning vs. p ost spawning period. These differen ces are difficult to isolate to a specific cause, however it is noted that there were no significant differences between p re spawning and p ost spawning glucose concentrations, suggesting that the s pawning period may be affecting glucose concentrations. Re productively active periods are overall taxing on fish as much of their energy reserves are utilized for production of eggs and sperm, and their feeding behavior may be altered to put more effort into spawning, which may be a factor for these observed diff erences. A lessened appetite may cause a reduction in glucose values if food was not eaten recently in conjunction to the sampling time. The third hypothesis examined seasonal variation in the sex steroid profiles of 11 KT and E 2 during the three reprodu ctive periods examined. T h e sex of individual fish had to be determined during field collections. Unfortunately, red drum have no external characteristics that allow easy identification of the sex of an individual unless drumming or exuding milt during spawning w ere observed which would confirm that the fish was male Thirty five fish were not identified to sex during field collection. A discriminant function analysis was employed using the hormone data from the fish of known sex from this study A pr edicted sex was assigned to the fish based on this data from this study I was able to establish baseline values for the sex steroid hormones 11 KT and E 2 which is the first time sex steroid profiles for wild red drum have been reported Both 11 KT and E 2 had significant differences among reproductive periods.
60 Both males and females had significantly elevated 11 KT during the spawning period. Higher 11 KT concentrations during spawning for males are likely due to final maturation of the sperm. Webb et al. ( 2002 ) found that plasma concentrat ions of 11 KT in mature males were higher than that found in mature females for wild white sturgeon. Kucherka et al. ( 2006 ) measured plasma 11 KT concentrations that were significantly higher in males, 0.8 ng/ml, than females, 0.5 ng/ml, during early stages of gonadal growth in both cu ltured and captive red drum This study found no significant difference between sex es or reproductive period when measuring E 2 concentrations. This data precludes E 2 as a n indicator for sex identification; however future research should be performed to co mpare these values to other populations of red drum One reason for these low values could be that some fish sampled were not adults since some of the males sampled were below the 50% sexually mature length cut off The E 2 concentrations of red drum sam pled by Kucherka et al. ( 2006 ) were low er in captive males and females before gonadal recrude scence and then increased significantly with the progressi on of vitellogenesis in females M ales used in this study were all over the 50% maturity length of 511 mm (FL) since the sampling cutoff was set at 650 mm (SL), however Atlantic coast females have a 50% maturity length of 900 mm (FL), and only 11% of the female sampled in this study were over 900 (TL) The E 2 values for all females on average for spawning and post spawning reproductive periods were higher than males. Only four fish had values that were higher than the standard curve and they were positively field identified as females during the spawning period. W ebb et al. ( 2002 ) measured levels of E 2 for mature wild white sturgeon females to be higher than those of mature males
61 Kucherka et al. ( 2006 ) was able to establish a 2 ng /ml threshold of E 2 for females wh en attempting to determine unknown sex ; however that threshold is not reflected in this study. All the values obtained for red drum in this study are an order of magnitude lower than previously published studies. This may be a factor of newer RIA test ki ts which have decreased the cross reactivity between the hormones in the blood sample from 15% to less than 0.01% in this study. Cultured red drum are typically kept between 23.7 28.4 C and kept at 26C when maintaining spawning condition ( Adams et al. 2010 ) The temperatures recorded in the field during the spawning period averaged the same as the NOAA buoy temperature data which was 28C This temperature falls within the spawning temperature range that is utilized at hatcheries. This information may be relevant to understand ing why some red drum in east central Florida are spawn ing in the estuary and not inlets or the ocean during a spawning period. Reyier ( 2011 ) found that acoustically tagged and monitored red drum in th e IRL Florida, exhibit ed strong site fidelity from winter through early summer Movement increased within its range during the fall spawning months ; however the majority of tagged fish remained within the lagoon year round. Lastly the fourth hypothesis was that there wou ld be variation in cortisol concentrations during different reproductive periods. This hypothesis was accepted since there was a significant differen ce in cortisol concentration among period s. The recorded levels of cortisol for red dru m in this study, some below detectable limits of the assay and up to 1.88 ng/ml were low compared to other fishes in the literature. I believe that these low values were partly due to improvements in analytical techniques and that the collection technique s used for this study were very fast.
62 Fish were not anesthetized during our study. Many studies have used MS 222 anesthesia for cortisol studies in fish. MS 222 is recognized as a powerful anesthe tic for fish and its use may affect cortisol data especi ally when compar ing fish sampled without anesthesia This difference is because unbuffered MS 222 is a powerful stressing agent for fish and previous studies resulted in higher cortisol values ( Blaxhall 1972 ) Anesthetics may lessen the eff ects of stress and reduce the cortisol response in fish ( Crosby et al. 2006 ) Another factor that may affect cortisol val ues is the time of day the sample was collected. Cortisol concentrations in green sturgeon, white sturgeon and Florida gar have been shown to be highly sensitive to diurnal variation and it is necessary to specify the sampling time period to reduce the po ssibility of time as a confounding variable ( Hamlin et al. 2007 ) All fish in this study were collected between 9:00 AM and 2:00 PM to minimize effects of potential daily f luctuations in hormone concentrations. However many other parameters may affect cortisol values in fish such as handling treatments, handling time, and salinity. Barton and Iwama ( 1991 ) reported prestress cortisol values for Sciaenops ocellatus as <5 ng/ml and a post stress range fr om 14 250 ng/ml based on studies that had differing procedures for handling treatments, handling times and salinites Stress Response When drawing general conclusions from available literature on stress responses in fish, only a very small fraction of the teleost species have been investigated ( Bonga 1997 ) The basal levels of cortisol reported for Siberian sturgeon were 5 ng/ml with peak levels during stress studies reaching 75 ng/ml ( Hamlin et al. 2007 ) In a previous study cultured juvenile red drum were challenged with a 2 minute transfer stressor This consisted of transferring fish by dip net and exposing them to air for two minutes
63 after which a blood s ample was collected by cardiac puncture. The results from this study by Thomas and Robertson ( 1991 ) noted rapid elevations in plasma cortisol and glucose concentrations after fifteen minutes Concentrations reached > 100 ng/ml for cortisol and hyperglycemia >100mg/100ml for glucose at fifteen minutes post stressor and persist ed for 120 minutes. However when red drum were challenged with a five second exposure to air during transfer, there was no plasma cortisol stress response, and only a slight hyperglycemic response ( Thomas and Robertson 1991 ) Bonga ( 1997 ) stated that for most stress studies fish from domesticated stocks were used and such fish may have a blunted stress response when compared with wild type strains of the same species. The adult red drum used in this study were challenged with an air exposure time ranging from between two to seven minutes, and all blood samples were collected between two and a half minutes and eight and half minutes. This rapid collection of the blood sample is a main point to consider when comparing the low cortisol values of this study to others in the literature. C ortisol values were below 2ng/ml which is well below the basal levels reported for sturgeon and equal to the basal levels reported for cultured red drum ( Kucherka et al. 2006 ; Hamlin et al. 2007 ) Perhaps the intensity of the hook and line rapid capture was below the threshold required to induce corticosteroid responses in adult red drum. Water Quality During sampling the water quality parameters did not change markedly among each of the three period s. Adams and Tremain ( 2000 ) noted that seasonal movements by juvenile red drum in similar estuarine systems are likely related to changes in water temperature, salinity or prey availability. Gonadal steroidogenesis is regulated by gonadotrophins, and gametogensis in red drum can be initiated by environmental cues
64 such as water temperature and day length, similarly to common snook ( Roberts et al. 1999 ) Also Johnson and Funicelli ( 1991 ) suggested that stable estuarine salinity provides suitable conditions for egg and larval survival mitigating the need for ontogenetically cued estuarine shelf migrations. Salinity measurements during the post spawning period averaged 30 ppt which is within the optimal range for red drum egg survival ( Holt et al. 1981 ) Reyier et al. ( 2011 ) stated that such a high incidence of red drum estuarine spawning may mostly be facilitated by mild winter water temperatures in East central Florida wh ich is recognized as a climactic transition zone that has temperate winters compared to other estuaries of the south U. S. Atlantic and Gulf of Mexico. Further Research One effective way to have an understanding of the impacts of human activities on natural systems is to have reference areas with minimal human impact ( Bohnsack 1993 ) Reference areas can help resource managers detect changes and attempt to distinguish if these changes are natural or caused by human actions. T he KSC Reserve is a great example of a reference area with minimal impacts due to its protected status. The next step toward a better understanding of red drum physiology and building baseline stress and sex hormone data base would be to compare the KSC R eserve red drum population to other nearby heavily fished waters such as Mosquito Lagoon which is part of the IRL system but open fishing to the public year round. Thomas and Robertson ( 1991 ) noted that fish that had been repeatedly exposed to similar shallow water stressors during routine aquaria maintenance had reduced subsequent biochemical stress responses to the adverse stimulus. Barton ( 2002 ) also
65 found that repeated exposures to mild stressors can desensitize fish and attenuate the neuroendocrine and meta bolic responses to sub sequent exposure to stressors. R esults obtained from fish used in this study may be compared to other sub populations of red drum in adjacent areas that experience intense angling pressure, and more urbanization of habitat. Collabora tion with FWC or fishing tournaments for red drum could aid in providing fish to be sampled according to the health assessment outlined by this project. F ield logistics, sample volumes, personnel training and availability of sample storage are issues to b e considered when developing collaborati ve studies ( Kucklick 2010b ) Current assumptions within the sportfish industry are th at catch and release fishing returns fish back to their environment and they survive the process without detrimental effects. However underestimating the discard mortality rate of a fishery was associated with the collapse of an entire red snapper fishery ( Diamond and Campbell 2009 ) Sportfish in general and red drum specifically, are known to tolerate repeated catch and release events from recreation al fish ing but not much is known about how these repeated events may affect the overall health and reproductive potential of the animal. Adult red drum in Mosquito Lagoon, which is adjacent to this study site were part of a n acoustic telemetry study and had a conservative 41% estimated angler recapture rate which far exceeds most other mark recapture studies ( Reyier et al. 2011 ) Anglers have d iscovered that good fishing and the largest fish are likely to be caught near reserves ( Bohnsack 1993 ) Bohnsack ( 2011 ) repo rted on the coastal protected areas in Florida and their effects on recreational world records and found that the highest concentration of r ecords was near the KSC Reserve. Red drum specifically had 55% of records near
66 recreational anglers to achieve more records than if the area had been regulated only by statew ide regulations ( Bohnsack 2011 ) High fishing pressure on an estua rine dependent spawning red drum population may have significant negative impacts on the breeding population. Further research is needed to determine if detrimental effects from angling pressure including catch and release can be det ermined by utilizing a similar health assessment protocol. Since the area of east central Florida seems to contain a population of estuarine dependent spawning adults within the confines of a reserve and relatively small scale management actions could res ult in rapid enhancement to local populations adjacent to areas managed by MINWR and Canaveral National Seashore. R eyier et al. ( 2011 ) suggested s easonal closures, limited access areas, or catch and release only zones. Growing scientific evidence indicates that marine reserves are effective and benefit both fishery and non fishery activities ( Bohnsack 1993 ) Before more management actions are employed additional quantitative health assessment data would benef i t the decision making process. This study provides much needed baseline data for stress and sex hormones on adult red drum in a reserve area safe from angling pressure and many other anthropogenic stresses However for reserves to be successful, public education and awareness about their functions and importance is needed. Also as resources within reserves increase, adequate surveillance and enforcemen t is necessary ( Bohnsack 1993 ) The continental shelf on the east coast of Florida has had relatively minimal research on the abundance and distribution of adult red drum so it remains unclear i f
67 the estuarine spawning fish constitute a large portion of the spawning biomass ( Reyier et al. 2011 ) Data from this study support that year round residency as well as spawning is occurring in the KSC Reserve area of the IRL complex. The red drum data may also be comparable to other physiologically related estuarine sportfish including such as the black drum, ( Pogonias cromis ) in this region. The new insights of this non lethal health assessment approach for wild red drum could positively impact fishery management by decreasing the need to cull fish for collection of data needed to manage the fishery. There is already a large database in Florida on length weight relationships relative to age of red drum as well as gonadosomatic index data. Blood plasma data could fill in sex data and age could be extrapolated based on fish lengths. Also fin clips co uld be utilized for age determination and compared to test the accuracy of the methodology. There is an increasing concern about how stable the red drum stock status is in Florida, although the bag limit was increased in 2012 for certain areas S tate man agers are still considering a red drum necessary before starting a stocking project. The health parameters presented here provide the baseline data necessary to monitor the health o f red drum in the future. More plasma parameters such as hematocrit, hemoglobin, erythrocyte counts, leukocyte counts, and comparing the structure of the blood cells may need to be evaluated in the future to attempt to determine health status differently if endocrine disrupting patterns or other deteriorations in health are observed. An important concept to move forward is to implement more non lethal methods for red drum health assessments which would be a win win for fisheries management and recreationa l anglers.
68 Figure 2 1. Kennedy Space Center s ecurity z one de f acto no t ake f isheries r eserve Study area of the KSC Reserve is outlined in yellow.
69 Figure 2 2 Collecting a 4ml blood sample from the branchial vessles in the gill of a wild caught red drum A 1 inch needle and lithium heparin vacutainer (BD, Franklin, NJ) were used Photo courtesy of Carla Garreau. Figure 2 3 Sex determination of wild caught red drum in the field. A ) Soft pressure applied to the abdomen of a red drum during spawning period induced free flowing milt indicating it was a male B ) Examination of a red drum that was not heard drumming was considered to be a possible female. The fish was probed in the vent area with a pipette t o determine sex and if the urinary orifice, anus, and ovipositor were found it was a female. Photos courtesy of Carla Garreau. A B
70 Figure 2 4 Health index score of wild caught red drum ( S.D.) presented by sampling period Pre Spawning, May n=38, Spawning September October n=46 and Post Spawning December 2011 n=42. Figure 2 5 Plasma cortisol concentrations of wild caught red drum ( 1 S.E.) presented by reproductive period and by sex. 36 38 40 42 44 46 48 50 Pre-Spawning Spawning PostSpawning Health Index Score Reproductive Period 0.00 0.50 1.00 1.50 2.00 2.50 Pre-Spawning Spawning Post-Spawning Cortisol (ng/ml) Reproductive Period male female
71 Figure 2 6 Plasma 11 KT concentrations of wild caught red drum ( S.D.) presented by reproductive period and sex. Figure 2 7 Plasma E 2 concentrations of wild caught red drum ( S.D.) presented by reproductive period and sex. 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 Pre-Spawning Spawning Post-Spawning 11 KT (ng/ml) Reproductive Period male female 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 Pre-Spawning Spawning Post-Spawning E2 (ng/ml) Reproductive Period male female
72 Table 2 1 Morphometric data and condition factor for each reproductive period Sampling p eriod n C ondition f actor ( k) m ean S.D. Weight (kg) m ean S.D. Total l ength (mm) mean S.D. Pre s pawning 38 1.60 0.10 5.39 2.72 774 96.2 Spawning 45 1.72 0.18 6.12 2.79 797 79 Post s pawning 42 1.77 0.17 7.64 2.71 847 79 Table 2 2 Plasma values for each reproductive period Pre s pawning Spawning Post s pawning Plasma parameter n Mean S.D. n Mean S.D. n Mean S.D. Glucometer g lucose (mg/dL) 38 19 .0 5.00 46 26 7.92 42 19 4.23 Abbott i STAT g lucose (mg/dL) 10 34.2 4.24 7 44.3 9.81 0 n/a Lab a ssay g lucose (mg/dL) 38 36 .0 6.83 46 38 9.97 42 29 6.08 Cortisol (ng/ml) 33 0.93 1.53 41 1.13 1.88 39 1.25 2.50 11 KT (ng /ml) 37 0.05 0.04 46 0.58 0.80 40 0.04 0.04 E 2 (ng/ml) 3 7 0.40 0.35 4 6 2.28 4.21 42 0.41 0.35
73 Table 2 3 Water q uality in the KSC Reserve during each sampling period Pre s pawning Spawning Post s pawning Mean S.D. N Mean S.D. N Mean S.D. N T emperature (C) 2 7.61 1.64 10 28.57 2.22 21 20.57 1.07 9 Dissolved oxygen (mg/L) 7.9 1.42 10 6.78 2.57 21 7.41 1.58 9 Salinity (ppt) 35.63 1.35 10 37.78 1.89 21 30.57 2.37 9 pH 8.39 0.15 10 8.24 0.50 21 7.79 0.15 9 Table 2 4 Predicted s ex for undetermined red drum caught in the KSC Reserve Mean f emale p robability S.D. Mean m ale p robability S.D. Predicted s ex 0.9 0.11 0.10 0.11 female 0.32 0.12 0.68 0.12 male
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82 BIOGRAPHICAL SKETCH Carla Marie Garreau was born in Winfield, Illinois. She grew up in the suburbs of Chicago, Illinois graduating from Trinity High School in 2002. During that time she spent many summers in northern Wisconsin at her family summer home on a lake which encouraged her love of fishing as well as going on family vacations to Mexico and the Caribbean. She earned dual B achelor of S cience degrees in m arine b iology and a quaculture from Florida Institute of Technology (FIT) in 2006. She also holds her Dive Master certification from the Professional Association of Diving Instructors, and has completed over 6 00 dives around the world. Before she graduated from FIT, she worked as an Environmental Sciences intern many threatened and endangered speci es at KSC, which is managed by Merritt Island National Wildlife Refuge, such as sea turtles, gopher tortoises, scrub jays and the Southeastern beach mouse. She currently works for Inomedic Health Applications at KSC under the medical and environmental ser vices contract. Upon completion of her m aster s d egree program Carla will continue her thirst for knowledge by looking to become a part time teacher at Brevard Community College in addition to her career at NASA Carla has become an avid sprint triathlon at hlete and has recently completed her first ma rathon and hopes to continue working and playing outdoors in Florida.