1 DETERMINATION OF TEMPERATURE THRESHOLDS FOR THE NORTHERN HARD CLAM, AND EVALUATION OF BACKCROSSED F1 HYBRIDS ( MERCENARIA MERCENARIA X MERCENARIA CAMPECHIENSIS ) By MELISSA ANN BRODERICK A THESIS PRESENTED TO THE GRADUATE SCHOO L OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA
2 2012 Melissa Ann Broderick
3 To my wonderful friends and family for their unwaverin g encouragement and faith in me
4 ACKNOWLEDGMENTS I thank Florida Sea Grant and United States Department of Agriculture for their support. Dr. Shirley Baker, thank you so much for giving me an opportunity to pursue my dreams. Your guidance has hel ped me to grow as a scientist. I thank Dr. John Scarpa and Ms. Leslie Sturmer for their knowledge and advice which was crucial to this project. Thanks to Dr. Charles Cichra for his wisdom as a supervisory committee member and graduate coordinator. I thank Mr. William White, Mr. Reggie Markham and Mr. Barry Clayton in the clam shack for their hard work and willingness to teach me. I would not have been able to do this without my many friends at the University of Florida and scattered across the United States and all their love and support, especially: Drew Garnett, Ashley Jean Hoffman, Alexander Broderick, Virginia Hays, Landon Proctor, Anastasia Yakaboski, Alisha Huettig, Ethan Holscher, Larry Lawson, Chelsey Campbell, Nick Cole, and Mike Dickson. Special t hanks to my mom and dad, Susan and Timothy Broderick, for their unwavering faith in me. I did it!
5 TABLE OF CONTENTS page CHAPTER 1 Aquaculture of Mercenaria mercenaria 3 2 Laboratory Challenge of Backcrossed F1 Hybrids ( M. mercenaria X M. campechiensis 3 Water Quality Six Month O ld Bac Twelve Month O ld Backcross Hybrids.. 4 APPENDIX : WATER QUALI TY DATA...
6 LIST OF TABLES Table P age 2 1 The group number, parental cross, and families used in the backcross experiments;M= M. mercenaria C= M. campechiensis A 1 Mean water quality values in each replicate of treatment 15 ppt normoxia for 12 month A 2 Mean water quality values in each replicate of treat ment 25 ppt normoxia for 12 month A 3 Mean water quality values in each replicate of treatment 25 ppt hypoxia for 12 month A 4 Mean water quality values in each replicate of treatment 35 ppt normoxia for 12 month A 5 Mean water quality values in each replicate of treatment 15 p pt normoxia for six month A 6 Mean water quality values in each replicate of treatment 25 ppt normoxia for six month A 7 Mean water quality values in each replicate of treatment 25 ppt hypoxia for six month A 8 Mean water quality values in each replicate of treatment 35 ppt normoxia for 12 month
7 LIST OF FIGURES Figure P age 2 1 2 2 2 3 Backcross experiment daily checks 3 1 The daily dissolved oxygen concentration (mg/L) averaged for each treatment in the six month old backcross hybrid challenge 3 2 The daily dis solved oxygen concentration (mg/L) averaged for each treatment in the 12 month old backcross hybrid challenge 3 3 Upper acute temperature limit of M .mercenaria 3 4 Mean days of survival across all groups by treatm ...34 3 5 Box and whisker plot of mean days of survival by group with quantiles to display variability in data..
8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DETERMINATION OF TEMPERATURE THRESHOLDS FOR THE NORTHERN HARD CLAM AND EVALUATION OF BACKCROSSED F1 HYBRIDS ( M ercenaria mercena ria X Mercenaria campechiensis ) By Melissa Ann Broderick August 2012 Chair: Shirley Baker Major: Fisheries and Aquatic Sciences The northern hard clam, Mercenaria mercenaria (Linnaeus1758 ), is an important aquaculture species in Florida. Florida water temperatures are at the upper temperature range for M. mercenaria and high mortalities in the summer months may be a result of the combined stress of high temperatures, extreme salinities, an d low dissolved oxygen. Basic breeding techniques, such as hybridization and polyploidy, may be useful to develop a more stress tolerant hard clam suited for Florida waters. Basic aspects of thermal biology are not known for the northern hard clam grown b y Florida shellfish farmers, nor have experimental hard clam lines, such as backcrossed hybrids, been evaluated in laboratory challenges. Therefore, the objectives of my research were to determine the upper acute temperature limit of cultured M. mercenari a and to M. mercenaria M. mercenaria x M. campechiensis ) and reciprocal crosses) in laboratory challenges. The upper acute temperature limit was determined by exposing cultured M. mercenari a (n=40/trt) to four target temperatures (32, 34, 36, 38C) and recording number of clam mortalities in four hour intervals. All hard clams died within 28 hours of exposure in the 38C treatment, whereas no clams died at any other treatment temperatures, i ndicating that the upper acute temperature is
9 near 38C for Florida cultured M. mercenaria Six month old and 12 month old backcrossed hybrid hard clams and controls were challenged in mimicked summer stressor conditions in Florida: oxygen stress (<3 ppm), high temperature (32C), and various salinities (15, 25, 35 ppt). Unfortunately, backcrossed hybrid hard clams exhibited no significant improvement in survival. Understanding stress limits in Florida strains of M. mercenaria and hybrids will contribute to potential management of summer mortality events and further development of hardier clam strains for Florida.
10 CHAPTER 1 INTRODUCTION Background Mercenaria mercenaria the northern hard clam, is an important aquaculture species in Florida The $19 million dollar industry (USDA 2007) grew rapidly over the last three decades and shows potential for further growth. Mercenaria mercenaria inhabit coastal waters ranging from Canada to the Gulf of Mexico with a difference in mean annual temperature of 25C betwee n its northern and southern distributions ( Abbot 1974, Pickard and Emery 1982). This large geographic range indicates potential to survive in a wide array of climates. The water s in which the aquaculture industry of Florida operates, are at the upper tempe rature range of the northern hard clam High mortalities of this species occur during the summer in Florida probably as a result of physiological stress of high temperature s combined with extreme salinities, low phytoplankton abundance, and low dissolved oxygen concentration (Scarpa et al. 2005) Efforts are being made to mitigat e the e ffect of summer conditions on this valuable crop. One means of reducing summer mortalities is to develop a c lam stock that is more tolerant of high temperatures. Selective b reeding and hybridization for improved cultured stocks is common practice in fin fish aquaculture and throughout agriculture. The Sunshine bass is a hybrid cross of the striped bass, Morone saxatilis and the white bass, Morone chrysops and is commonly aq uacultured because of the improved growth rate and culture characteristics compared to either parent species (Bartley 2001) Rainbow trout and char ( Oncorhynchus mykiss x Salvelinus sp .) are hybridized for improved disease resistance along with many othe r salmonid species ( Dorson et al., 1991 Bartley 2001 ) Many other freshwater fishes including carps, tilapias, loaches, catfishes, and drums are all hybridized to improve their performance as aquaculture species (Bartley 2001). The potential of breeding p rograms for bivalves is relatively
11 unexplored however, with only five well known efforts world wide ( Haskin Shellfish Research Laboratory New Jersey; Hatfield Marine Science Center Oregon; Virginia Institute for Marine Science Virginia; The French Resea rch Institute for Exploration of the Sea ( IFREMER ) France; Commonwealth Scientific and Industrial Research Organization, Australia ). O yster breeding programs for disease resistance are successful at the Haskin Shellfish Research Laboratory Hatfield Marin e Science Center and Virginia Institute for Marine Science. Hybri dization of the northern hard clam and southern quahog appears to produce a clam with improved thermal stress tolerance (Baker et al. 2011) The hybrids of M. mercenaria and M. campechiensi s (produced from single parent crosses with parents randomly selected from existing broodstock) have produce d some families that consistently outperform pure M. mercenaria in survival when reared under commercial conditions ( Sturmer et al. 2012 ). However, the hybrid clams gape excessively in refrigeration making them unacceptable for commercialization (Sturmer et al. 2012) Therefore, the next step is to examine the effectiveness of backcrossing hybrids with parental species to withstand summer conditions a nd tolerate refrigeration. Aquaculture of Mercenaria mercenaria in Florida From 2005 to 2008, the annual value of exported hard clams from the aquaculture industry in the United States grew from $34 million to $58 million, but between 2008 and 2010 decline coast (Bergquist et al. 200 9 ). The United States Department of Agriculture census of aquaculture in 2005 reported the hard clam industry in Florida had $18 million in sale s in 2001 and $9.8 million in sales in 2005 (USDA 2007) ; the decrease in 2005 was due to the hurricane season of
12 2004 Most of the growth of the hard clam aquaculture industry in Florida can be attributed to the increasing expansion of areas cultivated, ra ther than improved stocks or methods. Culture methods for hard clams in Florida begin in the hatchery. Most growers purchase seed from hatcheries, where clams are spawned and raised until they are 1 mm or larger in shell length (Whetstone et al. 2005). Nu rsery systems serve as an intermediate holding place before clams are ready to be planted. Nurseries are usually weller or raceway systems, and hold clams unti l they are 5 to 6 mm in shell length, which is the usual minimum size to be field planted (Whets tone et al. 2005). Grow out takes place on lease areas, which support about 1 ,0 00,000 seed/acre (Whetstone et al. 2005). Most Florida growers use soft polyester mesh bags staked to the substrate to grow clams (Whetstone et al. 2005). These bags provide pro tection from predators and biofouling organisms, and aid the grower in organizing their crop, much like crop rows in traditional agriculture. The mesh size is increased after about 6 months of growth, when clams are sorted into their new bags to finish gro wing (Whetstone et al. 2005). The last stage of grow out takes 12 to 24 months, depending on environmental conditions and food availability (Whetstone et al. 2005) After the bags are collected for harvesting, the clams are brought to a wholesaler to be p rocessed. Processing includes cleaning, size grading, counting, and packaging (Whetstone et al. 2005). The climate in Florida allows for almost year round growth and continual harvesting (Bergquist et al 2008). Survival in summer months ha s recently bee n below the average of 50 to 70% (FAO) with 0% survival in the Big Bend (Dixie, Levy, and Citrus counties) area in 1998 ( S. Maddox, USDA, Farm Service Agency, pers. comm). Summer water temperatures in Florida can exceed 30C which is above the upper tempe rature threshold (27 to 30C) of many phytoplankton species, limiting the amount of available food (Bergquist et al 200 9 Hoff and Snell 1987).
13 Elevated temperature also increases clam metabolism and energy demands at a time when food availability is low (Weber et al. 200 7 ). Additional stress factors stem from lower dissolved oxygen and potentially lower salinites from the increased number of tropical storms in summer months. Salinity was almost 0 ppt in April 2003 and can be highly variable during summer months ranging between 10 and 30 ppt (Bergquist e t al 200 9 ). Determination of the acute upper temperature limit may contribute to better managing the summer losses, which prevent Florida from achieving its full commercial clam aquaculture potential by in forming a selective breeding program. Hard Clam Physiology and E nvironment Geographically, M. mercenaria is native to the Gulf of St. Lawrence Canada, to Indian River Lagoon, Florida re cently introduced by clam aquaculture (Arnold e t al 2004, Baker et al 2008). This range encompasses huge extremes in temperatures, indicating the ability of the hard clam to acclimate tropical climat e allows for a long growing season. I n recent years however, temperatures in the summer have exceed ed the upper range of optimal temperatures for hard clam growth (Baker et al 2011, Sturmer et al. 2012). An estuarine species, M. mercenaria is usually fou nd in habitats with salinities between 20 to 30 ppt and optimum temperatures between 15 to 25C (Grizzle et al. 2001). The effect of salinity on behavior and physiology is well documented for M. mercenaria The limits for survival are between 12 and 46 pp t for adults and pumping rates are inhibited below 15 ppt and above 36 ppt (Castagna and Chanley 1973, Hamwi 1969). Behaviors indicating stress, including gaping and reduced burial activity, occur when salinities dip below 15 ppt clams will close their
14 val ves to keep water of 12 ppt or less out of their internal tissues (Chanley 1957, Castagna and Chanley 1973). Outside the optimal temperature range of 15 to 25C clams become stressed and growth can be adversely affected (Grizzle et al. 2001). Under stres s, shell growth of the clam will become lateral or thicker, and produce dark growth bands when growth is slow or suboptimal; these bands are laid down in both summer and fall in Florida (Arnold 1991). Clams investing energy in lateral shell growth may be d iverting energy from possible tissue or meat growth. Growth ceases when temperatures exceed 31C (Ansell 1968) as they did in June 2004, August 2005, August 2007, June 2008, July 2009, and July 2010 ( Gulf Jackson Lease Area, Levy County, water quality data FDACS data, 2004, 2007 2010) Water temperatures in Florida are also highly variable and can fluct uate by as much as 11C in a 24 hour period, preventing time required for acclimation (Weber et al. 2007). Temperature affects metabolism and water pumping rates. Because clams are ectotherms, i ncreased metabolism from high water temperatures results in high energy demand. Pumping rates are maximally effic ient at temperatures between 20 to 25C, but cease to undetectable levels around 32C (Hamwi 1969). Clam s respond to high temperatures by closing their valves and resorting to anaerobic metabolism (Hamwi 1969). Other signs of metabolic stress include rising to the surface of the sediment and gaping, or opening the valves At some unknown critical high t emper ature even anaerobic metabolism ceases, stopping growth, reduci ng immune response, and causing death (Weber et al 2007). H igh water temperatures are also correlated with low phytoplankton densities causing further physiological stress from low food ava ilability at a time when energy needs are increased (Bergquist et al. 2009)
15 Dissolved oxygen level is a compounding environmental factor a ffecting hard clam behavior and survival While low dissolved oxygen, or hypoxic condition s ( dissolved oxygen le ss than 2 mg/L ) alone may not be stressful to hard clams, reduced survival is noted when combined with high temperatures (Baker et al. 2002). Under hypoxic conditions, oxygen uptake is maintained by increasing the efficiency by which oxygen is pulled fro m the water column, but under extreme environmental stress, the valves close leaving the clam to metabolize anaerobically (Hamwi 1969), which can be sustained for up to 18 days, but only at low temperatures (1 to 6C) (Loosanoff 1939). Hard clam physiolo gy with regard to environmental limits is a relevant contemporary topic of research, because of the implications for the speci es in regard to climate change This paradigm links an increased cellular demand for oxygen with increasing temperature to the env ironmental availability of dissolved oxygen. Warmer water is typically lower in dissolved oxygen. When oxygen availability is limited, an animal switches to anaerobic metabolism and protects its cellular functions with emergency mec hanisms. An animal susta ined in this state is not a healthy and actively growing animal. Given these recent development s in understanding the effects of exceeding optimum temperature ranges, it is prudent to pursue the development of a hard clam strain for the Florida aquaculture industry with high thermal tolerance. Determining the acute upper temperature limit in M. mercenaria will contribute to understanding and potentially managing summer mortality events, which are problematic for clam growers in Florida. When water temperatu res are expected to be in the upper critical range, a grower could for example harvest larger clams, rather than risk losing them. It is clear that, for Florida to remain competitive in the clam aquaculture industry, a hardier strain of Mercenaria mercenar ia is needed.
16 Bivalve Breeding Methods Stock improvement is common practice in aquaculture through hybridization and selective breeding for various traits including disease resistance, high growth rates, improved shelf life, improved flesh quality, sterili ty and improved environmental tolerance (Bartley 1997). Quantitative trait loci (QTL) are phenotypic traits that vary in degree, like skin or hair color, and contribute to polygenetic effects. Multiple genes contribute to an animal s taste, growth, envir onmental tolerance, or other commercially valuable characteristics (Newkirk 1979). Heat tolerance, or degree of heat tolerance is likely a QTL (Newkirk 1979). Improving environmental tolerance in a cultured population helps to keep it healthy in possibly u npredictable conditions (Bartley 2001). This would be especially true of clam and other bivalve culture which is mostly subject to the natural conditions of the body of water and stochastic events versus a tightly controlled fin fish recirculating facilit y for example. Research has been done on selective breeding of oysters for disease resistance and improved flesh quality in a handful of programs world wide. The Haskin Shellfish Research Laboratory of Rutgers University has breeding programs in place to produce fast growing Eastern oysters, Crassostrea virginica resistant to Juvenile Oyster Disease, MSX, and Dermo, for mid Atlantic restoration projects ( Allen et al. 1993 ). The Coastal Oregon Marine Experiment Station at Oregon State University has a Moll uscan Broodstock Program that aims to improve Pacific oyster Crassostrea gigas and Asian oyster Crassostrea sikamea broodstock to enhance commercial yields, create a broodstock management program for industry for sustainable commercial production, and main tain a repository for selected top performing oyster families ( Langdon et al. 2003, Brake et al. 2003, Evans et al. 2003 ). College of William and Mary Virginia Institute of Marine Science details their extensive selective breeding program for
17 disease resis tance, meat yield, and growth in depth in the 2009 report of The Aquaculture VIMS 2009 ). Australia national scientific organization, Commonwealth Scientific and Industrial Research Organisation, has breeding programs for the Pacific oyster and hybridization programs for abalone. The French Research Institute for Exploration of the Sea ( IFREMER ) program is devoted to selective breeding of commercially important bivalves including oysters and blue mussels Although these programs have been successful at producing genetically superior shellfish for aquaculture their limited number demonstrates the untapped potential for growth in using breeding methods for improving shellfish aquaculture, particular ly that of hard clams. Hard clams are diploid, having two sets of chromosomes in each somatic cell. During reproduction, haploid (one set of chromosomes) gametes combine to create diploid (two sets of chromosomes) offspring with a set of chromosomes from each parent. Triploid animals have three sets of chromosomes and usually cannot reproduce. Triploids are often favorable in aquaculture, because they are, for the most part, sterile and often larger (Beaumont and Fairbrother 1991, Guo and Allen 1994, Evers ole et al. 1996). The increase of genetic material in each cell is theorized to create an additive effect of a larger animal, known as polyploidy gigantism (Guo and Allen 1994). It has also been suggested that energy diverted from reproduction is allocated into tissue or meat growth (Beaumont and Fairbrother 1991, Eversole et al. 1996). The hypothesis of increased heterozygosity states fitness of an animal is increased with genetic diversity, therefore the third set of chromosomes improves fitness and gener al growth (Beaumont and Fairbrother 1991). Triploidy is commonly used in Pacific oyster aquaculture; several methods have been developed for inducing triploidy. The most reliable method is to breed a tetraploid (four sets of
18 chromosomes) with a diploid, wh ich results in 100% triploidy (Guo et al. 1996). Other methods include interfering with polar body development in meiosis I and II by introduction of chemical, high pressure, or temperature stress (Beaumont and Fairbrother 1991). Of these methods suppressi on of the second polar body with cytochalasin B produces the highest percent of triploid larvae, but is dangerous for the technician because of toxicity (Guo et al. 1996). Triploid hard clams have been tested for stress resistance. Triploid hard clams were challenged in a laboratory under salinity (10, 25, 40 ppt) and oxygen stress (D.O. < 2 mg/L). Their survival and burial was compared to diploid hard clam siblings. Triploids had better survival in the 25 ppt hypoxic treatment, but overall were not found t o survive significantly better than diploid hard clams (Hoover 2007). Hybridization is the breeding of two individuals within or between species, and aims to specifically highlight an advantageous trait or generally improve the h ard iness of the stock for culture conditions (Bartley et al. 2001) Research has been conducted on hybridizing M. mercenaria with the southern hard clam, M. campechiensis which both occur and hybridize (Dillion and Manzi 1989) M. campechiensis is not favorable for culture because of its limited shelf life, gaping only days after harvest, but tolerates high summer water temperatures more readily than M. mercenaria (Menzel 1989) M. campechiensis has a natural range extendi ng as far north as southern New Jersey, but is most common from North Carolina to the Gulf of Mexico, and has been reported in the Caribbean ( Baker et al. 2008 ). Although these two species will readily hybridize in the wild, they usually remain geographica lly separate preferring different environments (Menzel 1989). M. campechiensis prefers near shore environments with salinities above 30 ppt (Menzel 1989). M. mercenaria occurs in estuarine areas with salinities ranging from 20 to 30 ppt (Grizzle et al.
19 20 01) It has also been suggested that although the Indian River area contains naturally occurring hybrids, the species remain discrete in other geographic locations where they co occur because of increased seasonality providing different reproductive cues f or each species that prevents hybridization ( Dillion and Manzi 1989, Dillon 1992). Hybrids of northern hard clams and southern hard clams have been successful ly produced and reared in hatchery systems that resulted in a viable and fertile first generation ( F1 ) second generation ( F2 ) third generation ( F3 ) and backcross generations with either parent (Menzel 19 89 ). Hybrids have outperformed pure M. mercenaria for growth and total production in field trials, but had reduced shelf life ( Scarpa et al. 2011 ). Backcrossing has been suggested as a method of genetic improvement in other forms of aquaculture .Backcrossing involves breeding a hybrid with a favorable trait, like heat tolerance, back to a parent al species (Bartley 1997). Backcrossed families of M. mer cenaria x M. campechiensis hybrids have been developed with the goal of preserving the improved heat tolerance of the hybrids and emphasizing the shelf life of the northern hard clams (Sturmer et al. 2012). S ummer temperatures in Florida are thought to be a major factor in summer mortality events, and data on acute upper temperatures will inform growers when their crop is at risk An effort should be made to improve hard clam stocks through breeding techniques t o reduce summer related mortality and increase the maximum aquaculture production in Florida. Backcrossed hard clams could potentially provide farmers with a superior animal for culturing; however more information on their performance under exposure to various stresses is necessary. If ocean temperatu res continue to rise, as expected due to climate change, there could be serious implications for clam growers in Florida, where temperatures are already stressful, emphasizing
20 the need for stock development. The objective s of my research were to 1) det e rmi ne the acute upper temperature limit for cultured hard clams M. mercenaria, and 2) examine the performance of backcrossed hybrid hard clams in laboratory challenges against hard clam controls. It was hypothesized that backcross hybrid hard clams would sur vive longer in the challenge conditions than pure hard clams.
21 CHAPTER 2 MATERIALS AND METHODS Upper Acute Temperature Limit The northern hard clam s M. ercenaria mercenaria (Lennaeus 1 7 5 8 ) used in the acute upper temperature limit experiment were harv ested from culture bags on a Dog Island clam lease in the Gulf of Mexico near Cedar Key, Florida in September 2011 Ambient water temperature was 26C and clams were held overnight at 2 6 C and 25 ppt in the University of Florida Shellfish Aquaculture Resea rch and Education Facility ( SAREF ) in Cedar Key Clams were transported in a cooler to the University of Florida Fisheries and Aquatic Sciences laboratory in Gainesville, Florida, whereupon they were placed in acclimation tanks. Clams were acclimated in 30 gallon tanks of 25 ppt 26 C water. During acclimation temperature was increased 2 C per day until 32 C was reached. Water changes of 50% were conducted daily during acclimation, and clams were observed for mortality. Any dead animals were removed. Salinit y was kept the same throughout the experiment, 25 ppt. Shell lengths were taken from a subsample (n = 25) while clams were acclimating The acute upper temperature limit of Mercenaria mercenaria was determined by expos ing clams to various target temperatur es after being acclimated at 32C and 25 ppt for four days. There were two tanks of each temperature for duplication (Figure 2 1A). The animals were spilt haphazardly into eight groups of 20 each and stocked in to eight 38 L tanks (50.8 x 25.4 x 31.75 cm). Each tank was equipped with a Fisher Scientific memory monitoring temperature probe for reading the temperature, at least one Marineland visi therm 200 or 300 W aquarium heater, and one air stone. The temperature was raised 1C every 30 minutes at the sta rt of the experiment until target temperatures of 32, 34, 36, and 38C were established in each duplicate tank Tank temperatures were increased in a staggered order so that all tanks reached intended
22 test temperature s at the same time. Temperatures were r aised by use of Marineland visi therm 200 or 300 W aquarium heaters (model number ML90443 00, Marineland Aquarium Products, Cincinnati, Ohio); h eaters could not be set above 32C, therefore additional heaters were added to some tank s to reach target temper atures Once intended temperatures were reached, clam viability was monitored every 4 hours until 100% mortality was reached in a single temperature treatment. The n umber of dead animals was recorded every 4 hours and mortalities removed Mortality was det ermined by visually inspecting the tanks for gaping clams. Gaping clams were after manual compression, they were considered dead and removed from the tank. Water changes were conducted every 8 hours; dissolved oxygen was monitored every 12 hours with a 650 MDS YSI meter (Yellow Springs Instruments, Yellow Springs, Ohio) Laboratory Challenges of Backcross H ybrids ( M. mercenaria X M. campech i ensis ) B ackcrossed hybr id hard clams were produced at Harbor Branch Oceanographic Institut e at Florida Atlantic University in Fort Pierce, Florida Backcrossed families were produced by breeding previously developed hybrid hard clam ( M. mercenaria x M. campech iensi s and recipro cal cross) families to M. mercenaria stocks. Each clam family ha d four groups ; Mercenaria mercenaria pure breds as a control group, two backcross groups where the mother of the hybrid animal was M. mercenaria and a single backcross group where the mothe r of the hybrid animal was M. campechiensis G roup numbers (31 42) were used to code for the specific crosses and the heritage of each family and its previous generations For example, i n families D and E a hybrid female were backcrossed to a pure hard clam male Within each family, there were control hard clam groups of pure hard clam females crossed with the same male used to produce the backcrosses (Table 2 1). For f amily F the reciprocal hybrid cross was
23 used for backcrossing. That is, a pure hard c lam female was crossed with a hybrid male, and the control hard clam group used the same female (Table 2 1). All clams were first reared the SAREF in Cedar Key, Florida, before being planted in to commercial lease sites in the Gulf of Mexico near Cedar Ke y. Backcross hybrid hard clams were harvested after 6 months (April 2011 ) and 12 months (October 2011 ) and held no longer than two weeks in the SAREF until they were delivered to the U niversity of F lorida Fisheries and Aquatic Sciences laboratory Gainesvil le, Florida. Once clams were received, subsamples of shell length from 30 individuals in each group were measured. Clams were also labeled on both valves with their group numbers using a Sharpie marker before being placed in to acclimation tanks. Experimen tal design Acclimation of clams was conducted in 30 gallon aquaria for each set of environmental parameters for one week by, increasing salinity at about 2 ppt/day and temperature at about 2C/day (Figure 2 1B) After acclimation to particular challenge co nditions clams were sorted into experimental aquaria with corresponding conditions as described above. Each clam group was haphazard ly divided into sets of 10 to a replicate aquaria resulting in 40 clams per aquaria, a group of 10 individuals fr om each of 4 groups in a family and 2 families per system Backcross h ybrid clam families E and F were t es ted at 6 months of age (April 2011), and families D and F at 12 months (October 2011). Laboratory challenges of backcross hybrids were conducted in systems as d escribed in Hoover (2007) and described briefly as follows Each system consist ed of two 17 L aquaria each connected to a 38 L sump, (Figure 2 2A) Each sump contain ed a 200 W heater and a 120 V 60 Hz Quietone pump (model number 1200, Pentair Aquatics, El Monte, CA) that pump ed water back into the two associated aquaria The aquaria overflow ed back into the sump, thus creating a recirculating system. Each system was replicated
24 16 times resulting in four blocks of treatments with 32 total aquaria T emperatur e was maintained at 32C in all systems In each of the four blocks, e ach s ystem was randomly assigned a salinity treatment of 15, 25 (control) or 35 ppt at normoxia (D.O. > 5 mg/L), or 25 ppt hypoxi a (D.O. < 2 mg/L). Hypoxic conditions were created by bu bbling N 2 into the sumps with air tubing and an air stone from a source liquid N 2 tank. All tanks were insulat ed to minimize fluctuation in water temperature. Seawater was stored in a series of 378.5 L tanks mixed to the appropriate salinities with Oceani c natural sea salt mix (Aquarium Systems, Ohio) and well water. Enough sea water was prepared for daily 30% water changes (Figure 2 2B) Procedure O bservations of mortality and gaping behavior were conducted e very 24 hours (Figure 2 3A) Mortality was ass essed in two ways: failure to remain closed when valves we re manually compressed and unresponsiveness when probed at the mantle edge (Figure 2 3B) Gaping behavior is often indicative of death. If a gaping animal wa s responsive and able to hold itself clos ed it wa s considered to be alive but possibly stressed or close to death. Dead clams were removed A ll tanks were monitored for a minimum of 24 days or until all clams in a t least one treatment expire d Water quality parameters were also monitored daily to ensure temperature, salinity, and dissolved oxygen levels were within acceptable ranges. Tank temperature s w ere monitored continuously with Fisher Scientific digital thermometer s (model number 15 077 8D, Pittsburgh, Pennsylvania), which store d the mini mum and maximum temperatures over the last 24 hour period Sump temperature was also monitored daily before water changes with a 650 MDS YSI sonde (Yellow Springs Instrument, Yellow Springs, Ohio) Salinity was checked daily using a refractometer.
25 Data An alysis The acute upper limit experiment mortality data was entered in a Microsoft Excel spreadsheet. The data where plotted in a graph to show mean (n=2) percent mortality over time in hours at each experimental temperature. Statistical analys e s of the da ta from the backcross hybrid hard clam experiments w ere using JMP and Statsdirect. Statsdirect format ted the daily mortality data binomially into days of mean survival by backcross hybrid group number by assigning each individual with a 0 or 1 respectivel y, for alive or dead on a particular day. This data output was then transferred to JMP as the continuous response variable M ean D ays of S urvival for each group in each treatment, e.g Mean Days of Survival for group 42 in 15 ppt. A two way factorial ANOVA was performed using group number and treatment as independent variables for the dependent variable M ean D ays of S urvival The interaction was not significant in either challenge (p=0.99) so the model was run again with the removal of the interaction as a n additive model. was used to examine the differences between the control (25 ppt normoxia) and the three treatment s (15 ppt normoxia, 35 ppt normoxia and 25 ppt hypoxia) Differences within families or between groups, were examined with Wilcoxon rank sums test s for a difference Results were considered significantly different if p < 0.05.
26 Table 2 1. The group number, parental cross, and families used in the backcross experiments;M= M. mercenaria C= M. campechiensis In parental cross, the first two letters represent the cross of the female parent, and the second two letters represent the cross of the male parent. Families D & F were used at 12 months of age and families E & F were used at six months of age. Group number Parental cross Family 31 MM X MM D 32 MC X MM D 33 MC X MM D 34 CM X MM D 35 MM X MM E 36 MC X MM E 37 MC X MM E 38 CM X MM E 39 MM X MM F 40 MM X MC F 41 MM X MC F 42 MM X CM F
27 A) B) Figure 2 1. Experimental aquaria and the acclimation process. A) T he acute upper thermal limit experiment system. Five 38 liter aquaria equipped with multiple heaters to reach target temperatures. B) View of acclimation tanks used for all experiments. P h oto s courtesy of Melissa Broderick A) B) Figure 2 2. Experimental system s. A) One block of the system used to challenge backcrosses. The 38 liter sumps were equipped with a pump that moved water into the two 17 liter aquari a above where clams were placed. B) View of the wet lab with large sea water reservoirs on the right, acclimation tanks in the back, and experimental challenge systems on the left. Photos courtesy of Melissa Broderick.
28 A) B) Figure 2 3. Backcross experiment daily checks. A) Example of a gaping clam. B) The inside of a aquaria holding all four groups family of backcross hybrid clams in the challenge experiment. Photos courtesy of Melissa Broderick
29 CHAPTER 3 RESULTS Water q uality All water quality parameters stayed near expected values fo r each experiment, with the exception of N 2 (Figure 3 1). During the challenge of t he six month old backcross hybrids and controls there was some difficulty maintaining gradual temperature increase during a cclimation, because of issues with electrical wiring causing frequent power loss. F uses in the laboratory blew continuously during the acclimation process. The fuse s would be reset and the electronics redistributed, but it took several arrangements to find a balance. During this process clams experienced large fluxes in temperature from heaters powering on and off overnight ; mortality was considerable and probably related to the stress of this unstable temperature. In other words, clams were slowly being a djusted to experimental temperature of 32C at a rate of 2C/day, but failing power would shut off heaters, and the water would cool down by as much as 6C overnight. Deceased clams were removed multiple times a day, and 50% water changes were performed da ily. During challenge of 12 month old backcross hybrids and controls, dissolved oxygen in hypoxic tanks peaked above 5 mg/L at times, because of a leak in the nitr ogen delivery line (Figure 3 2). New leaks w ere found throughout the experiment resulting in intermittent hypoxia for those tanks. In an attempt to keep dissolved oxygen low for those tanks no water changes were made until N 2 flow was restored. Upper temperature limit Target temperatures (32, 34, 36, 38 C) were successfully reached in 1 degree in crements within the planned acclimation time (1 C/30 mins) The mean shell length of a subsample of 25 clams was 55.1 mm (S.D. = 3.6 mm). The first mortalities were observed at 16 hours of exposure to 38C. Both r eplicates at 38C experienced similar morta lity ; 4 deaths and 6 deaths out of 20
30 clams/tank for an average of 25% mortality Over the next four hours (i.e., after 20 hours of exposure), in the 38C treatment had another 7 and 10 deaths for a total mortality average of 68% By 28 hours 100% morta lity occurred in both replicat es No mortality was experienced at any other temperature during the 28 hours (Figure 3 3) Backcross hybrid challenges Six month old backcross hybrids Six month old hard clams had a mean shell length of 38.3 mm (S.D. = 1.8 m m n=240). There was no significant difference (p = 0.96) in mean days of survival between backcross hybrid hard clam groups and pure bred hard clams under the conditions tested. Treatment had a significant effect (p<0.001) on mean days of survival for six month old backcross hybrid and pure bred hard clams Mean days of survival was significantly lower in 15 ppt normoxic and in 25 ppt hypoxic conditions (p<0.001) as compared to the control treatment of 25 ppt normoxia (Figure 3 4A) The 35 ppt normoxi a tre atment was not significantly different (p=0.11) from t he control 25 ppt normoxia treatment. The median is often a more appropriate statistic when distributions are non normal, as the Mean Days of Survival for this experiment. The median for the distributio n of mean days of survival in all six month rank sums test was used to evaluate differences between groups in mean days of survival, because the distribution of mean days of survival was non normal. There was no signific ant relationship between mean days of survival and group number (p=0.96 Figure 3 5A) using the indicat es that backcross hybrids and hard clam controls performed similarly; and that there were no group differences, because a family was made up of four specific groups
31 Twelve month old backcross hybrids Twelve month old hard clams had a mean shell length of 47.1 mm (S.D. = 2.6 mm, n=240). Survival during acclimation was noticeably improved in 12 month old backcross hybrids and losses were minimal when compared to 6 month old backcross hybrids The median for the distribution of mean days of survival was 19.7 days for the length of the challenge ent in days of mean survival (p=0.95, Figure 3 4B). Dunnett s method was then used to compare the treatments to the control (25 ppt normoxia Figure 3 4B) The 25 ppt intermittent hypoxia treatment was not significantly different (p=0.34 3 ) from t he control However, mean days of survival in 35 ppt and 15 ppt were found to be significantly different (p<0.001) from the control treatment ; with backcross hybrids in 35 ppt having higher mean days of survival than the control, and backcross hybrids in 15 ppt havi ng lower mean days of survival than the control (Figure 3 4B) There was no significant relationship between mean days of s urvival and group number (p=0.95, Figure 3 5B).
32 Figure 3 1. The daily dissolved oxygen concentration (mg/L) averaged for each tre atment in the six month old backcross hybrid challenge. Dissolved oxygen data was not collected once all clams had died in a treatment. Figure 3 2. The daily dissolved oxygen concentration (mg/L) averaged for each treatment in the 12 month old backcr oss hybrid challenge. Disssolved oxygen data was not collected once all clams had died in a treatment.
33 Figure 3 3. Upper acute temperature limit of M. mercenaria Mean (n = 2) mortality for Florida cultured M. mercenaria (55.1 mm shell length S.D.= 3.6 mm, n = 160) exposed to four temperatures to determine upper acute temperature limit.
34 A) B) Figure 3 4 Mean days of survival across all groups by treatment with standard deviations A) Six month old hard clams; 15 ppt and 25 ppt hypoxia were signifi cantly lower than the control, 25 ppt normoxia. B) Twelve month old clams; 35 ppt was significantly higher days of mean survival than the control (25 ppt normoxia) and 15 ppt was significantly lower days mean survival than the control (25 ppt normoxia).
35 A ) B) Figure 3 5 Box and whisker plot of mean days of survival by group with quantiles to display variability in data A) Six month old backcross hybrids, days of m ean survival was not found to be statistically different among group s Midli ne represents mean days of survival across all groups. B) Twelve month old backcross hybrids, m ean survival was not found to be statistically different among group s Midline represents mean days of survival across all groups.
36 CHAPTER 4 DISCUSSION Far mers in Florida have been suffering heavy losses of hard clams in the summer when environmental conditions are stressful. Water temperatures are high, phytoplankton abundance is low, salinity is variable, and dissolved oxygen can be low. Breeding a hard cl am with improved environmental tolerance is vital for overcoming these summer challenges. In this study, I found that 1) I found that the upper acute temperature for Florida cultured M. mercenaria was 38C, 2) that an increase of only 2C from 36C to 38 C increased mortality from 0 to100%, 3) treatments were a significant factor affecting days of mean survival for backcross hybrid hard clams and controls, 4) backcross hybrid hard clams did not survive challenge conditions differently than control clams. I found that 38 C was the acute upper thermal limit for cultured M. mercenaria All animals at 38C died within 28 hours while at 36 C and lower temperatures, all other clams survived during this interval Clams exposed to 36C were gaping, indicating stres s at the conclusion of the experiment, but upon manual compression were able to hold themselves closed ; making 2 C the difference between 0 and 100% mortality within 28 hours The influence of just 1 C on mortality was noted by Kennedy and Mihursky (1972) who examined critical upper temperatures for bivalves in the Patuxent estuary. Kennedy and Mihursky (1972) also found Gemma gemma acclimated at 25 C had an upper critical temperature of 35.6 C, and Mulinia lateralis acclimated at 25 C had an upper critical temperature of 33.5 C. Hicks and McMahon (2002) acclimated the brown mussel, Perna perna at 15, 20, 25, and 30 C and found the upper thermal limit to be 30 C in long term experiments and 44 C during acute thermal stress. Environmental limits were investi gated in the penshell, Atrina m aura which had an upper
37 temperature of 33.2 C (Leyva Valencia et al. 2001). In a study of the critical thermal maximum of the sea cucumber, Apostichopus japonicas acclimated at 16, 21 and 26 C, critical thermal maximums we re 33.1, 34.1 and 36.6 C, respectively (Wang et al. 2012). Compared to these other aquatic poikilotherms, Florida cultured M. mercenaria have a fairly high thermal limit. It is possible that clam culturists have been unknowingly selecting temperature toler ant animals. Although the Florida coast of the Gulf of Mexico does reach peak water temperatures of 35 C (August 2011 Dog Island Cedar Key, Florida ), and can experience large fluxes in temperature of up to 3 C/30 min ute s (August 2008 Dog Island Cedar Ke y, Florida ) there is a cooling period at night. The natural increase in temperature in the Gulf of Mexico is not as rapid as manipulated in the laboratory. The rapid increase of temperature in the laboratory was used to specifically initiate stress respo nse in the clams. When allowed to acclimate animals can typically survive much higher temperatures ; it has been shown that animals living in warmer waters have higher heat resistance (Henderson 1929, Kennedy and Mihursky 1972). Increasing the temperature as quickly as in th e present experiment prevents further physiological adjustment ; therefore, isolating the effect of temperature stress. There is no consensus in the literature on the appropriate rate of temperature increas e; experiments have increased te mperature in acclimated animals from a range of 1C/ 5 min ute s to 1C/day (Kennedy and Mihursky 1972, Hicks and McMahon 2002, Alexander and McMahon 2003, Peck et al. 2009) Thermal biology is a field of growing interest, because of the implications for globa l climate change. As water temperatures in the Gulf of Mexico annually increase, thermal tolerance on clams and other invertebrates will be an important economic issue for Florida. Work on fruit flies, Drosophilla melanogaste r, confirms that the protocol o f upper temperature limit experiments influences the results, and in some cases can create a bias (Santos et al 2011).
38 Santos et al. (2011) modeled the critical temperature of Drosophilla and compared the results to published protocols revealing inconsis tency and bias in results. Santos et al. (2011) conclude that short term acclimatory responses have a confounding effect in long term critical temperature studies in Drosophilla and that acute studies are preferred. These findings may extend to other ecto therms including bivalves. The procedure followed in t he upper acute temperature limit experiment on Florida cultured hard clams aimed to increase the temperature of acclimated animals by a rate that was perhaps reasonable on the hottest and most stressful day in the Gulf of Mexico to a set of target temperatures that are observed in the summer months ( Florida Department of Agriculture and Consumer Services, Division of Aquaculture, Dog Island, Cedar Key, Florida sonde data). However, in the field temperat ures this extreme would not be sustained, because of diurnal warming and cooling. If this experiment is repeated, it may be improved by extending the experiment to determine how much longer it would take for clams to experience death at the lower temperatu res, and adding a dirurnal cooling period. I would also suggest taking tissue samples and examining histological changes in those animals dying from heat stress. In the laboratory challenge of six month old backcross hybrid hard clams, differences in mean days of survival were found among treatments. Days of mean survival in the control treatment (mean = 6.6 days) was significantly different from the hypoxic treatment and the 15 ppt treatment (mean = 2.2 days). Hard clams are generally found in coastal are as where salinities range from 12 to 30 ppt, with most populations occurring >15 ppt (FAO 2012 ) yet in both experiments full strength sea water treatments (35 ppt) had the highest mean days of survival. It was also surprising that in both challenges, clam s survived poorest in the 15 ppt normoxic treatment. It was expected that hypoxia would be the most stressful treatment, because hard
39 clams resistance to periods of hypoxia is diminished in combination with heat stress (Baker et al. 2002). My results did n ot reflect this effect. The N 2 flux problem in the 12 month old backcross hybrid hard clam challenge should not have had any effect on clams in the other treatment aquaria, yet survival differences remained unapparent between backcrosses and hard clam con trols across treatments. The improved survival (mean = 19.7 days) in the 12 month old backcross hybrids may have been the result of the i ncrease d size and age of animal s. Additionally, 12 month old backcross hybrids were held in Cedar Key for only one nigh t after harvest compared to a few weeks for the six month old animals. During this holding time, it is possible that clams were nutritionally stressed. Although the holding facility was flow through, clams may not have been receiving enough food to reach s atiation. The possibility that older, larger animals handle stress better than younger, smaller individuals has been examined. Yuan et al (2010) found that in the mussel Mytella charruana larger individuals (20 to 24 mm) survived salinity stress better overall than smaller (3 to 19 mm) mussels, but that smaller mussels tolerated a wider range of salinities (2 to 40 ppt) tha n larger mussels. However, in a study of temperature tolerance in A rctic marine phyla, Peck et al (2009) found that across 14 specie s from six phyla small animals survived to warmer temperatures than did larger ones ; the top 10% largest animals in the size distribution for each species failed to survive to the highest temperature of exposure I found; however, that larger clams, survi ved longer than smaller clams under stressful conditions. Indirectly related to size and age is reproductive status which energy stores, with up to 52% of the total organic production released during spawning events in hard clams (Ans ell et al. 1964) An animal has a budget of energy based on how much food and
40 oxygen are available, if energy is being allocated to developing gametes th e n less energy is available for handling environmental stress. It follows that if an animal has stor ed a great deal of energy in reproductive tissues it may be vulnerable to environmental stress after spawning, until energy stores are replenished in the somatic tissues. Upon histological review (courtesy of Dr. Susan Laramore, Harbor Branch Oceanographic Institute, Ft. Pierce, Florida) of a subsample (n = 40) of six month old backcross hybrid hard clams from families E and F, it was d etermined that some individuals were in early gonadal development (25%) or a post spawn state (33%) It may be that the capa city to tolerate stress was reduced in these animals from the start, because of their reproductive status. No differences in mean days of survival were found among groups or between families in either challenge. High mortalities occurred during the acclima tion process for six month old backcross hybrid hard clams due to power issues Clams continued to expire in high numbers until a few days after start ing the experiment. This stress was likely exacerbated by deteriorating water quality as animals died, des pite daily 50% water changes. In the past this recirculating laboratory challenge setup has been successfully used to delineate survival differences between hybrid and pure hard clam families validating the (Baker e t al. 2011) It is possible that backcross hybrids are more similar to the pure hard clams than their hybrid relatives in a way which makes their differences undetectable by this system. If a hybrid is genetically 50% M. campechiensis and 50% M. mercenaria then it follows that backcross hybrids should be 75% M. mercenaria and 25% M. campechiensis Therefore, I may not have detected any advantages of backcross hybridization, because backcross hybrids have genetic composition that is very similar to the pure hard clams to which they are being compared.
41 Field data indicated that the same backcross hybrid families used in this experiment, did have greater survival in the field nursery and grow out than did pure hard clam controls (Sturmer et al. 2012). In the field nursery, survival wa s improved in backcross hybrids (71 to 82%) compared to control hard clam group within a family (65%) and at harvest 65% of backcrossed famil ies yielded higher survival (81 to 91%) compared to hard clams (79%) (Sturmer et al. 20 12 ). From the standpoint of a clam farmer, t he trends being d escribed in the field concerning backcross hybrids are much more relatable; these are the conditions to which farmers will actually be exposing their crop. The laboratory challenge design was in tended to tease apart differences in families exposed to a singl e particular stressor, but perhaps the families are not yet different enough in one generation of backcrossing for this subtle difference to be significant. I hypothesize that, in the field, b ackcrosses are continually exposed to stressful conditions, and any slight genetic advantage makes a difference in survival. Whereas, the laboratory challenge was not stressful enough to illustrate these subtly emerging family differences. Generally one g eneration is enough time to see differences when hybridizing two species ( Bartley 200 1). Hy brids did produce families that outperformed control hard clams in both the field and the laboratory challenges (Baker et al 2011 ). However, Newkirk (1983) notes in his review of shellfish breeding programs that gains are made with each generation, and that real success of a breeding program may take s everal generations Backcross hybrids of M. mercenaria and M. campechien si s should continue to be explored as a produ ct to improve the efficiency and longevity of hard clam aquaculture in Florida. Although, the results of this research effort indicated that there were no differences in backcross hybrid hard clams compared to control hard clams when exposed to stressful e nvironmental conditions, field data is promising. Developing a breeding program is crucial to
42 the continuation of a promising and economically valuable industry in Florida. Further consideration o f the breeding program and future efforts may include resear ch in marker assisted selection efforts which are becoming more widespread in aquaculture. Developing broodstock that surviv e heat challenges may also produce genetically superior stock. In conclusion, I found that the acute upper temperature limit for Flo rida cultured M. mercenaria was 38C and that as little as 2C differential in water temperature can result in mortality. Identifying the acute upper temperature limit can inform the effort to breed heat tolerant hard clams for the aquaculture industry. Th e acute upper temperature limit of Florida cultured northern hard clams can be compared to the acute upper temperature limit of northern hard clams along the U.S. east coast to assess if Florida culture efforts have unknowingly been selecting for heat tole rance. Additionally, I examined survival of backcross hybrid hard clams compared to pure hard clams in potentially stressful summer conditions in laboratory challenges. I found that treatment conditions had a significant effect on mean days of survival for all clams. These indicate that both salinity and dissolved oxygen levels, under summer temperature (32C) have a significant effect on the survival of hard clams. There were no advantages of backcross hybrid hard clams compared to pure hard clams, suggest ing that backcross hybrids and pure hard clams have similar stress tolerance thresholds. Future investigation of backcross hybrids and hard clams should test at more extreme conditions. The hard clam aquaculture industry of Florida may benefit from continu ing to explore other methods of selective breeding.
43 APPENDIX WATER QUALITY DATA Table A 1. Mean water quality values in each replicate of treatment 15 ppt normoxia for 12 month old hard clams. Tank # Water Quality Value Mean (n=26) Standard Deviation Ma ximum Minimum 4 Salinity (ppt) 15.0 0.0 15 15 6 Salinity (ppt) 15.0 0.0 15 15 9 Salinity (ppt) 14.8 0.8 15 12 14 Salinity (ppt) 15.0 0.0 15 15 4 Temperature (C) 32.8 0.3 33.1 32.24 6 Temperature (C) 33.2 2.3 35.8 31.2 9 Temperature (C) 30. 6 1.2 33.1 28.5 14 Temperature (C) 32.5 2.0 34.8 31.1 4 Dissolved Oxygen (mg/L ) 5.0 0.6 5.7 4.3 6 Dissolved Oxygen (mg/L ) 4.0 2.3 5.3 1.3 9 Dissolved Oxygen (mg/L ) 5.3 0.7 6.3 3.7 14 Dissolved Oxygen (mg/L ) 4.2 1.7 5.5 2.3
44 Table A 2. Mean w ater quality values in each replicate of treatment 25 ppt, normoxia for 12 month old hard clams Tank # Water Quality Value Mean (n=26) Standard Deviation Maximum Minimum 3 Salinity (ppt) 24.6 1.6 27 20 5 Salinity (ppt) 23.5 2.3 25 20 11 Salinity (ppt) 20.0 0.0 20 20 15 Salinity (ppt) 24.0 2.0 25 20 3 Temperature (C) 31.6 1.0 34.1 31.0 5 Temperature (C) 31.1 0.6 33.3 30.0 11 Temperature (C) 33.5 1.5 35.3 31.2 15 Temperature (C) 32.2 1.7 34.2 26.6 3 Dissolved Oxygen (mg/L ) 5.1 0.6 5.8 3.4 5 Dissolved Oxygen (mg/L ) 5.4 0.3 6.3 4.8 11 Dissolved Oxygen (mg/L ) 3.9 2.0 5.6 1.4 15 Dissolved Oxygen (mg/L ) 5.2 0.6 6.0 2.6
45 Table A 3. Mean water quality values in each replicate of treatment 25 ppt, hypoxia for 12 month old hard clams. Tank # Water Quality Value Mean (n=26) Standard Deviation Maximum Minimum 2 Salinity (ppt) 23.9 1.9 25 20 8 Salinity (ppt) 24.1 2.3 25 15 12 Salinity (ppt) 21.6 2.1 25 20 16 Salinity (ppt) 24.8 1.0 25 20 2 Temperature (C) 33 .0 0.6 33.3 30.3 8 Temperature (C) 31.1 0.5 32.4 29.5 12 Temperature (C) 33 .0 2 .0 34.7 28.7 16 Temperature (C) 32.8 0.8 33.3 29.3 2 Dissolved Oxygen (mg/L ) 4.1 1.2 5.7 2.6 8 Dissolved Oxygen (mg/L ) 4.4 1.3 6.5 1.8 12 Dissolved Oxygen (mg/L ) 2.9 1.6 5.1 1.1 16 Dissolved Oxygen (mg/L ) 3.3 1.6 5.1 1 .0
46 Table A 4. Mean water quality values in each replicate of treatment 35 ppt 12 month old hard clams. Tank # Water Quality Value Mean (n=26) Standard Deviation Maximum Minimum 1 Salinity (ppt) 32.3 2.2 35 30 7 Salinity (ppt) 31.7 2.1 35 30 10 Salinity (ppt) 32.2 2.2 35 30 13 Salinity (ppt) 32 .0 2.1 35 30 1 Temperature (C) 32.1 0.8 34 .0 29.6 7 Temperature (C) 30.8 1.8 35.4 28.3 10 Temperature (C) 32.6 0.6 33.5 30.1 13 Temperature (C) 31.2 1 .1 34.6 29.2 1 Dissolved Oxygen (mg/L ) 5.6 0.5 6.4 3.4 7 Dissolved Oxygen (mg/L ) 5.3 0.3 5.8 4.5 10 Dissolved Oxygen (mg/L ) 5 .0 0.8 8.3 3.7 13 Dissolved Oxygen (mg/L ) 5.2 0.3 5.5 4.5
47 Table A 5. Mean water quality values in each replicate of treatment 15 ppt for six month old hard clams. Tank # Water Quality Value Mean (n=21) Standard Deviation Maximum Minimum 1 Salinity (ppt) 15 .0 0 .0 15 15 5 Salinity (ppt) 15.3 0. 5 16 15 11 Salinity (ppt) 15. 7 0. 6 16 15 14 Salinity (ppt) 15 .0 0 .0 15 15 1 Temperature (C) 31. 3 0. 3 31.6 31.0 5 Temperature (C) 32. 5 1.3 34.1 29.9 11 Temperature (C) 34. 3 0. 5 34. 7 33. 8 14 Temperature (C) 32.4 0.5 32.9 31. 7 1 Dissolved Oxygen (mg/L ) 6. 1 0.7 6.8 4.9 5 Dissolved Oxygen (mg/L ) 5. 8 0. 9 6.7 4. 6 11 Dissolved Oxygen (mg/L ) 4.5 0. 4 4. 8 4. 1 14 Dissolved Oxygen (mg/L ) 5.3 0. 7 6.4 4.5
48 Table A 6. Mean water quality values in each replicate of treatment 25 ppt normoxia for six month old hard clams. Tank # Water Quality Value Mean (n=21) Standard Deviation Maximum Minimum 3 Salinity (ppt) 25.2 0.7 27 24 7 Salinity (ppt) 24.9 0.3 25 24 9 Salinity (ppt) 23.9 1.8 25 20 15 Salinity (ppt) 23.4 0.9 25 23 3 Temperature (C) 32.5 1.1 33.3 29.9 7 Temperature (C) 33.1 0.8 33.8 29.9 9 Temperatu re (C) 33.6 0.4 34.1 32.8 15 Temperature (C) 33.8 1.3 35.0 32.3 3 Dissolved Oxygen (mg/L ) 6.0 0.3 6.4 5.1 7 Dissolved Oxygen (mg/L ) 5.8 0.5 6.2 4.5 9 Dissolved Oxygen (mg/L ) 5.7 0.6 6.2 4.5 15 Dissolved Oxygen (mg/L ) 4.7 0.6 5.6 4.2
49 Table A 7. Mean water quality values in each replicate of treatment 25 ppt hypoxia for six month old hard clams. Tank # Water Quality Value Mean (n=21) Standard Deviation Maximum Minimum 4 Salinity (ppt) 23.8 0. 8 25 23 8 Salinity (ppt) 24.9 0. 3 25 24 12 Sali nity (ppt) 23. 6 1.7 25 20 13 Salinity (ppt) 24.2 1.1 25 22 4 Temperature (C) 31.7 1. 5 33. 3 29.6 8 Temperature (C) 31.5 1.4 32.7 28.1 12 Temperature (C) 32.5 0. 9 33 .0 30. 5 13 Temperature (C) 33.0 0.6 33.5 32.0 4 Dissolved Oxygen (mg/ L ) 3. 4 1. 9 6. 5 1.7 8 Dissolved Oxygen (mg/L ) 2. 7 2.0 6.4 0. 8 12 Dissolved Oxygen (mg/L ) 3.0 1.9 6.5 1.1 13 Dissolved Oxygen (mg/L ) 4.1 1.5 6.5 2. 1
50 Table A 8. Mean water quality values in each replicate of treatment 35 ppt for six month old hard clam s. Tank # Water Quality Value Mean (n=21) Standard Deviation Maximum Minimum 2 Salinity (ppt) 34.5 1.3 37 32 6 Salinity (ppt) 34. 4 1. 3 37 32 10 Salinity (ppt) 33. 7 1. 7 36 30 16 Salinity (ppt) 30.3 0. 6 31 30 2 Temperature (C) 31.7 1. 6 33.7 27. 7 6 Temperature (C) 31. 9 1.8 34.4 28 .0 10 Temperature (C) 32. 8 1.0 33.6 29. 4 16 Temperature (C) 33. 6 1.0 34.2 32.4 2 Dissolved Oxygen (mg/L ) 5. 9 0.4 6. 5 4. 7 6 Dissolved Oxygen (mg/L ) 5.8 0. 4 6. 4 5. 1 10 Dissolved Oxygen (mg/L ) 5.6 0.3 6.0 4. 8 16 Dissolved Oxygen (mg/L ) 3. 5 2.6 5.7 0.6
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56 BIOGRAPHICAL SKETCH Melissa Ann Broderick was born in the naval base hospital of Bremerton, Washington. After many years of moving, her family settled in Cape May, New Jersey. Melissa has always wanted to be a marine biologist. Growing up on the beach deeply connected her with marine life and a fascination for conservation grew. She received her B.S. in marine sc iences from Rutgers University in 2009. After working in an oyster hatchery as a technician at The Haskin Shellfish Research Laboratory Cape Shore facility for a summer, Melissa moved to Gainesville to pursue a M.S. at the University of Florida. No funding was available, and an aquaculture /agriculture internship opportunity in Walt Disney World strengthened her passion for aquaculture. She began her graduate work at the University of Florida in the summer of 2010, and received her M.S. in the summer of 2012. She left Gainesville to study fr eshwater bivalve conservation in San Antonio, Texas as a field biologist.