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Survivorship, Growth and Pigmentation Responses of the Marine Ornamental Invertebrate Tridacna maxima to Varied Irradian...


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SURVIVORSHIP, GROWTH AND PIGMENTATION RESPONSES OF THE MARINE ORNAMENTAL INVERTEBRATE Tridacna maxima TO VARIED IRRADIANCE LEVELS IN TWO DI FFERENT CULTURE SYSTEMS By MICAH ALO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Micah Alo

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This document is dedicated to the friends and supporters of the marine ornamental hobby.

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iv ACKNOWLEDGMENTS This thesis would not have been possibl e without the help of many individuals. First, I thank all my committee members Dr. Charles Cichra, Dr. Shirley Baker, Dr. Daryl Parkyn and Dr. John Baldwin, for their encour agement and much needed enlightenment. This project was made possible with funding provided by the USDA CS-REES Special Research Grants and funding from the University of Florida’s College of Agricultural and Life Scien ces and Department of Fish eries and Aquatic Sciences. I owe a lot of thanks to the staff and stude nts of the University of Florida’s Tropical Aquaculture Lab. I thank Craig Watson for hi s belief in the advancement of aquaculture and giving this project its di rection. Scott Graves, Robert Leonard, and Tina Crosby all deserve credit for building th e greenhouse and systems. Fellow graduate student and tinkerer Jon Kao was instrumental for aidi ng in tank design and construction, ammonia readings; and most other problems that I en countered during my study. Also, I need to thank Dr. Jeff Hill for his statistical anal ysis and Dr. Roy Yanong for clam disease diagnostics and all the mentori ng they bestowed upon me. I am indebted to the late Jana Col and Dr. Ramon Littell from the Institute of Food and Agricultural Science’s Statistics Department for putting together the st atistical model for my experiment. Many thanks go to Oceans Reefs and Aqua riums who showed much generosity for providing the seed clams at a reduced cost. I am grateful to Florid a Marine Aquaculture for the helpful advice it provided in setting up my culture systems. Thanks go to the

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v Florida Aquarium for allowing me to have water samples analyzed by their ion-coupled plasma spectrophotometer. I thank Dave Watson and Rebecca Varner, of Florida Lakewatch, for the excellent job they did in making my metal halide light maps. Great thanks go to the Florida Lakewatch water chemistry lab for its thorough analysis of water samples. I would like to thank the Ruskin, Florida, NOAA weat her station for providing weather data. I thank Dr. Pam Muller from USF for grac iously lending me her LICOR light meter when mine was out of commission. To John Lucas, I am grateful for his knowledge and generous advice that he supplied on giant clams. I thank Mr. Gary Townsend for providing the US giant clam import data. I would like to thank C.L. Cheshire for his valuable information on giant clam economics. I am thankful to Dr. Charles Adams for sharing with me the economics viewpoint in aquaculture. Lastly, I need to thank my family and girlfriend, Amy Singivipul ya, for their never ending support and love that led me to accomplish all that I have done in life.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES.............................................................................................................x ABSTRACT......................................................................................................................x ii CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 Biology........................................................................................................................ .1 Inland Production..........................................................................................................4 Objectives..................................................................................................................... 5 2 METHODS...................................................................................................................6 Inland Culture Systems.................................................................................................6 Nutrient Additions........................................................................................................7 Water Quality................................................................................................................8 Clams.......................................................................................................................... ..9 Light Treatments.........................................................................................................10 Duration and Sampling...............................................................................................12 Acclimation Period..............................................................................................12 Study Period........................................................................................................12 Light Measurements............................................................................................12 Growth Measurements.........................................................................................13 Color Measurements............................................................................................13 Statistical Analysis......................................................................................................15 3 RESULTS...................................................................................................................22 Survival....................................................................................................................... 22 Shell Growth...............................................................................................................23 Wet Weight.................................................................................................................23 Color.......................................................................................................................... .24

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vii Reference Clams..................................................................................................24 Survivors..............................................................................................................25 Green Switch to Gold (Yellow)...........................................................................25 Light Measurements...................................................................................................26 Solar.....................................................................................................................26 Metal Halide........................................................................................................27 Temperature Profiles..................................................................................................28 Water Quality..............................................................................................................29 4 DISCUSSION.............................................................................................................47 Major Findings............................................................................................................47 Low Survival and Growth..........................................................................................47 Color Analysis............................................................................................................52 Nutrients.....................................................................................................................5 4 5 MANAGEMENT APPLICATIONS..........................................................................63 APPENDIX A CLAM COLOR READINGS.....................................................................................65 B WATER QUALITY...................................................................................................69 LIST OF REFERENCES...................................................................................................76 BIOGRAPHICAL SKETCH.............................................................................................79

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viii LIST OF TABLES Table page 3-1 Two-way analysis of variance ( ANOVA) for survival at day 90 of T. maxima seed clams among two culture systems and three light treatments..........................40 3-2 2 analysis of size class survival of T. maxima seed clams over 90-day study. 2 calculated = 8.07, df = 3, = 0.05, 2 critical = 7.815 (Ott 2001)...........................41 3-3 Split-plot repeated measures analysis of variance (ANOVA) results for shell length data of T. maxima seed clams among two culture systems and three light treatments over 90 days............................................................................................42 3-4 Treatment mean shell growth (percent) of T. maxima seed clams during 90-day study. Standard error (S.E.) is included (n = 6).......................................................43 3-5 Split-plot repeated measures analysis of variance (ANOVA) for weight data of T. maxima seed clams among two culture systems and three light treatments over 90 days.............................................................................................................44 3-6 Ninety-day mean wet weight gain and mean percent weight gain, correcting for biofouling (algae) weight, for T. maxima clams......................................................45 3-7 Mean halide light treatm ent intensity readings (mol/m2s) for all 12 tanks combined..................................................................................................................46 4-1 Mean L*a*b* values for open and shade treatment clams measured for color.......61 4-2 Mean L*a*b* values for three categorie s of color measured clams: blue; green; and gold clams..........................................................................................................62 A-1 Individual color readings for Blue seed size clams..................................................65 A-2 Individual color readings for Green seed size clams................................................67 A-3 Individual color readings for Gold seed size clams.................................................68 B-1 Water Quality for Recirculation System..................................................................69 B-2 Water Quality for Recirculation System..................................................................70 B-3 Mean Water Quality for Rollovers...........................................................................71

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ix B-4 Mean Water Quality for Rollovers...........................................................................72 B-5 Mean, max, and min water temperatures for each recirculatio n treatment tank for 90-day study.............................................................................................................73 B-6 Mean, max, and min water temperatures for each rollover treatment tank for 90day study..................................................................................................................74

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x LIST OF FIGURES Figure page 2-1 Greenhouse layout....................................................................................................16 2-2 Rollover tank............................................................................................................17 2-3 Seed giant clam ( Tridacna maxima ) attached using cyanoacrylate glue to a Floy T-bar tag...................................................................................................................18 2-4 Light treatments seen above in two experimental tanks...........................................19 2-5 Clam length – longest stretch across shell...............................................................20 2-6 Color measurement tank and enlargemen t of spectrophotometer probe and clam position.....................................................................................................................21 3-1 Mean treatment survival after 30, 60 and 90 days (n=6) for all six treatment types.........................................................................................................................3 1 3-2 Survival of clams with initial sizes of 6
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xi 4-1 Daily solar profile taken on day 69 fo r tank 4 open sun treatment shown in dashed line. Mean metal halide intens ity, for all 4 sample periods shown in solid line...................................................................................................................59 4-2 This graph displays spectral shift fr om day one to day 90 for one of the blue clam seed chosen for color measurements (number 715 from the open x recirculation treatment)............................................................................................60 B-1 Mean weekly temperatures for two culture systems. Rollovers are marked by the solid line. Recirculation tanks are marked by the dashed line..........................75

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xii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SURVIVORSHIP, GROWTH AND PIGMENTATION RESPONSES OF THE MARINE ORNAMENTAL INVERTEBRATE Tridacna Maxima TO VARIED IRRADIANCE LEVELS IN TWO DI FFERENT CULTURE SYSTEMS By Micah Alo December, 2005 Chair: Charles Cichra Cochair: Shirley Baker Major Department: Fisher ies and Aquatic Sciences The focus of this study was to determ ine how two environmental variables (irradiance and spectrum) affect in land production of the giant clam Tridacna maxima for the ornamental trade. This study also eval uated two types of land-based closed-system designs, recirculation and rollover systems. Growth of the seed clams was suboptimal, relative to previous studies, in all treatments with a mean of 5% growth in 90 days. Clams grown under metal halide lighting were found to have a mean growth of 3.3% ( 1.0% S.E.) and a significantly ( F 2, 30 = 9.04, P = 0.0008) lower mean survival of 28.5% ( 6.7% S.E.) compared to the two other light treatments (open and shaded). Th is was most likely due to a spike at 365 nm of ultraviolet radiation emitted by the halide lamps. The recirculation culture system had a significantly ( F 1,30 = 17.56, P = 0.0002) higher mean survival of 55.5% ( 5.1% S.E.) compared to the rollover system of 30.9% ( 4.4% S.E.). Mean shell length did not have

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xiii a significant difference between the recirculation system, that had a mean growth of 7.3% ( 0.7% S.E.), and the rollover system, that had a mean grow th of 2.7% ( 0.7% S.E.). Mean weight for system type was significant ( F1,10 = 10.31, P = 0.0093), with the recirculation system showing a mean weight gain of 18.3 3.3% S.E. vs. 2.1 2.9% S.E. for the rollover system. The difference was attributed to the eff ect of the near upper lethal temperatures (34oC) reached in the rollover systems. Only one clam was recorded to change mantle color, from green to ye llow, in the shade recirculation treatment. Qualitative trends of increased darkness and blueness in mantle tissue were recorded in the L* and b* values of clams in th e open and shade treatments over time. The time required to grow seed clams (10-30 mm) to 40-50 mm shell length, a size suitable for sale to the aquarium market, us ing the optimum conditions in this study was calculated to be 5-7 years (mean growth of 9.5% ( 1.0% S.E.) in the open recirculation treatment; mean surviv al of 66.2% ( 6.7% S.E.) in th e shaded recirc ulation culture treatment). It is unlikely that giant clam culture under these conditions would be economically feasible for inland production, unless seed supply became more abundant and less costly.

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1 CHAPTER 1 INTRODUCTION Background Giant clams were put into aquaculture pr oduction for the Asian food market in the early 1980s to prevent overharvest of wild stock. Recent aquaculture production has been refocused towards the ornamental market because of the shor ter growout period to market size and vibrant colora tion (Ellis 2000). Giant clams ( Tridacna spp .) are now a growing segment of the marine ornamental industry, experiencing a 9% annual growth during 1993-2002 (Cheshire and Valeriano 2004). Biology All nine species of giant clams ha rbor the photosynthetic symbionts Symbiodinium microadriaticum within their mantle tissue (Knop 1996) These symbiotic dinoflagellates (i.e., zooxanthellae) supply th e clams with all of their me tabolic carbon requirements in exchange for waste products in the form of ammonia (Fisher et al. 1985). If the zooxanthellae are provided with enough light, the clams can survive in the same low nutrient environments as hermatypic corals. Giant clam populations are limited to cer tain shallow water depths due to the physics of light attenuation and availability of photosynthetically ac tive radiation (PAR), the portion of the electromagnetic spectrum be tween 400-700 nm. In tropical latitudes, PAR at the sea surface peaks around 2500 m ol/m2s (Tomascik 1997). Many giant clams are found intertidally and are able to tolerate full surfac e irradiance (MingoaLicuanan 1993). Tridacna gigas, for example, is capable of photosynthesis while fully

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2 emersed during low tides (Mingoa-Licuanan 199 3). Values of PAR attenuates by 60% after the first meter of depth and by 80% after the first 10 meters of depth in Type I, the clearest, oceanic waters (Gross 1977, Jerlov 1976). This rapid decrease in light intensity is the main factor that dictates the depths at which autotrophic animals, such as giant clams, can live. Studies have shown that li ght tolerance levels are de pendant upon clam size. A respirometry study of T. gigas indicates that, for large juveniles (>10 cm), the light saturation point (light level at which no fu rther increase has an effect on photosynthesis rates) was reached at PAR levels over 2000 mol/m2s (Fisher et al. 1985). Saturation levels decreased with decreasing clam size; some 10-mm clams reached saturation at 500 mol/m2s. However, another study (Lucas et al. 1989) found that 40-mm (1 year old) T. gigas were unable to survive wh en exposed to 298 mol/m2s using 90% shadecloth. The need for stronger light in larger clams is attributed to the self-shading properties of the mantle tissue where the symbiotic algae are harb ored (Fisher et al. 1985 ). More layers of zooxanthellae are contained in the mantle tissue of larger clams. This produces a canopy shading effect as light passe s through the outer zooxanthell ae layers, shading the lower layers. Spectral distribution also varies as light is transmitted through water. In Type I waters, the blue end (300-500 nm) of the electromagnetic spec trum drops off in intensity at a slower rate than the red (>600 nm) portion of the spectrum (Jerl ov 1976). Kinzie et al. (1984) using acrylic light filters found th at corals, grown under blue (400-500 nm) and white light (400-700 nm) of the same intensities (250 mol/m2s), grew at a faster rate than those under green (500-600 nm) or red light (600-700 nm) of similar intensities.

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3 Therefore, blue light may facilitate produc tion of popular marine ornamentals such as corals and giant clams. Clam coloration in shallow depths is hypot hesized to be an adaptation by the clams to shield themselves from harm ful UV rays (Griffiths et al. 1992) The cells responsible for the bright colors found in some clams are called iridocytes and reflect the blue portion of the spectrum including UV-A (320-400 nm ) and UV-B (280-320 nm) (Griffiths et al. 1992). These UV rays can penetrate shallow ma rine waters in tropical zones where the ozone layer is thinner than in temperate zone s (Ishikura et al. 1997). While zooxanthellae outside of the clam cannot photosynthes ize with UV-B wavelengths present, zooxanthellae residing within clam tissues ar e shielded by mycosporine-like amino acids (Ishikura et al. 1997). Regular exposures to these wavelengths may have an effect on the photosynthetic capabilities a nd coloration of tridacnids. The maximum prolonged (6-week) temperat ure threshold for giant clams is 32oC, above which they expel their zooxanthellae a nd eventually starve to death (Buck et al. 2002). If temperatures can be maintained belo w this level, stronger light intensities may improve overall growth and coloration of T. maxima However, smaller clams may not require as much light and may even suffer photoinhibition (i.e., the point when increased light intensity causes damage to the photosynthetic system)(Lawlor 2001) This did not seem to be the case for a clam farmer in Kiribati who grew out T. maxima seed clams (up to 3 cm) in shallow tanks with full sun light exposure (persona l communication, Craig Watson, University of Florida). Most giant clam farms rely on ocean cages and natural sunlight for the majority of growout needs (Ellis 2000). Under these cu lture conditions, clams are highly vulnerable

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4 to predation by gastropods Cymatium spp. and wrasses Thalassoma spp. (Hart et al. 1998). One study in Australia tested the feas ibility of greenhouse recirculation system production of juvenile T. gigas clams (Braley et al. 1992). It showed that young clams, held in greenhouse recirculation systems, grew significantly fa ster and had higher survival than clams held in outdoor flow-through tanks. This was attributed to the ability of greenhouse systems to maintain a 5-7oC higher temperature during winter months within greenhouse systems. Inland Production Greenhouse production of marine ornament als can be an economically viable business (Calfo 2001). Demands for mo re colorful, smaller varieties of Tridacna continue to grow in the aquarium trade (Cheshire and Valeriano 2004). The cost of shipping and the Convention on International Trade in Endangered Species (CITES) certification has increased imported giant clam prices (Bell 1999). Giant clams are listed under Appendix II of CITES. This requires export of giant cl ams to have a permit issued stating “1) The export will not be detrimental to the survival of the the species, 2) The species was not obtained in contravention to the laws of the exporting State 3) The method of export of living species will minimize the risk of injury, damage to health or cruel treatment” (Ellis 2000). Member countries of CITES have to comply with these regulations and ensure that nonmember countries comply with any product, such as giant clams, imported or shipped through a CITES member country. Failure to include the proper permits and documents leads to impound ment or confiscation of the shipment. Currently, average wholesale prices in US dollars are at $10-16, depending on coloration, with larger clams commanding higher prices (Cheshire and Valeriano 2004). This developing market could make inland produc tion of candidate marine ornamental

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5 species, like giant clams, viable in Florida where freshwater tropi cal ornamental fish production already has an established infrastr ucture. However, supplemental artificial light sources may be necessary for light depe ndent invertebrates such as giant clams and coral to maintain adequate grow th and desirable coloration. Objectives The first objective of this study is to co mpare the effects of a blue spectrum artificial metal halide light relative to two le vels of intensity from natural sunlight on the growth, coloration, and survival of giant clam seed. The second objective is to determine effectiveness of two types of closed tank systems for the growout of giant clam seed.

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6 CHAPTER 2 METHODS Inland Culture Systems This study was conducted in a 9 x 22 m gree nhouse, at the University of Florida’s Tropical Aquaculture Laborator y located in Ruskin, Florid a (GPS coordinates 27.71 N latitude, 82.40 W longitude ) (Figure 2-1). The gree nhouse was equipped with an evaporative cooler (American Coolair, Jacksonville, FL) w ith a 1.5 m high x 8 m wide cooling pad on one end and two 1-m wide fa ns on the other end. The greenhouse also had a 225,000-BTU propane heater (Modine, Bu ena Vista, VA). An inflated double layer of plastic, (Sun Selector Type UVA clear, Ginegar Plas tics, Israel), covered the greenhouse. The two layers of plastic were each 150 m thick and transmitted 90% PAR and 20-25% UV-A and UV-B (Ginegar Plastics). Twelve rectangular poly propylene tanks, (267 cm long x 147 cm wide x 56 cm deep) holding approximately 2,250 L each, were used as inland saltwater culture systems. The tanks were operational by July 2004. All 12 tanks had a rollover system design (Figure 2-2) with eight 50 cm deep x 5 cm wide polyvinyl chloride (pvc) airlifts located on one end of each tank to push water across the surface then down and back in a constant loop. Base rock (limestone pieces ranging from 3-15 kg) was positioned along the inside corners of the tank as substrate for biological filtration. Of the 12 tanks, six were randomly selected and connected to the same sump tank of similar dimensions to form a recirc ulation system with a total volume of approximately 16,000 L. This system cont ained a 3/4-hp Jacuzzi Magnum Force pump

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7 (Tipp City, OH), a 65-W UV ster ilizer (Pentair Aquatics, El Monte, CA), a 152 cm tall x 12.5 cm wide venturi-driven protein skimme r (Top Fathom, Hudsonville, MI), an 85-L pneumatic drop bead filter (Polygeyser, Aquaculture Systems Technologies, New Orleans, LA), and a 5-hp chiller (Aqualogi c inc., San Diego, CA). The remaining six tanks were run separately as individual rollover systems. Artificial saltwater was made from Crystal Seas Salt Mix (Marine Enterprises International, Baltimore, MD) and reverse osmosis well water. Two 10% water changes (1,600 L) on the recirculation system were made on days 53 and 57 with Instant Ocean salt mix (Spectrum Brands, Atlanta, GA) due to elevated (1.5 mg/L) nitrite levels. Tank water in the rollover systems was not cha nged during the 90-day st udy. A salinity of 35 ppt was maintained in both system treatments with daily addition of reverse osmosis water. Nutrient Additions Ammonium chloride was administered to each tank twice a week and spiked to concentrations of 50 M. This addition is standard protocol to optimize growth for production of giant clams by supplementi ng nitrogen-limited zooxanthellae with ammonium chloride (Grice and Bell 1999). Rollover tanks were given 1-3 g of ammonium chloride for each dose and recircul ating system tanks were given 3-5 g for each dose. Ammonia readings were taken us ing an Orion ammonia electrode (Beverly, MA) with an Orion EA 940 ion analyzer. On days 53 to 90, recirculation system ammonia was spiked to only 10M due to in creased nitrite concentrations. Weekly supplements of 1 g of yeast (Fleishmann’s Instant Yeast, Fenton, MO) were added to each tank as a source of dissolv ed organic nutrients (Fitt et al. 1984). Algae supplements, of 1 g dried spirulina, were also added w eekly (Spirulina powder, Florida Tropical Fish

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8 Farm Association Store, Apollo Beach, FL ) to each tank. Yeast and spirulina were heated and mixed in 1 L of reverse osomosis wa ter, then spread out evenly into the tanks. Water Quality Weekly monitoring of ammonia, nitrite, nitrate, and alkalinity was conducted using the HACH (Loveland, CO) Saltwater Master test kits. Calcium hardness was measured using either a Seachem (Madison, GA) or La Motte (Chestertown, MD) test kit for the first 30 days. These two tests had high variabil ity, so tests were con tinued with a Salifert (Duiven, Holland) calcium test kit. Calcium hydroxide (Hydrated Lime, Chemical Lime, Brooksville, FL) was added as needed to incr ease calcium concentrations to levels above 400 mg/L. Calcium solutions were prepared by mixing 6 g of calcium hydroxide with 100 mL of acetic acid (Rogers White Dis tilled Vinegar 5% acidity, Speaco Foods, Kansas City, MO) in 4 L of reverse osmosis water. The calcium solution was added to the tanks after 1700 hours. Temperature was l ogged every half hour in all 12 tanks using a YSI model 600 XLM (Yellow Springs, OH) sonde. Once per month, water samples from one randomly selected rollover tank and one recirculation system tank were also analy zed by Severin Trent Laboratories (Tampa, FL) with an inductively-coupled plasma (ICP) mass spectrometer to determine calcium, magnesium, strontium, and molybdenum concen trations (mg/L). In addition, monthly water samples from the recirculation system water and all six rollover tanks were analyzed by the Florida Lakewatch Water Chem istry Laboratory (University of Florida, Department of Fisheries and Aquatic Sciences Gainesville, FL) w ith a spectrophotometer for total nitrogen and total phosphorus. A porta ble 25-W UV sterilizer (Pentair Aquatics) was used on an “as needed” basis to reduce algae blooms inside the rollover tanks. Any excess foam build up was removed manually using fine mesh nets.

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9 Clams Three hundred ninety six ten-month old s eed (7-14 mm) clams were obtained from a giant clam farmer in the South Pacific (ORA Marshall Islands). They were shipped as airline freight cargo with 40 plus hours of travel time. The clams arrived at Tampa International Airport in cooler boxes on Fe bruary 17, 2005. Each shipping box contained 12 plastic bags (15 cm) with eight to 10 clam s each with no form of cooling inside (e.g., ice pack). Each bag was filled halfway with seawater at a salinity of 35 ppt and a pH of 7.8. Upon receipt, clams were acclimated by fl oating the bags until temperatures were equalized. Then, the bags were emptied into two 75-L bins sitting next to the recirculation system sump. A siphon hose sl owly fed water into the bins from the recirculation system’s sump for further acclim ation to water quality parameters. Once acclimated to the system water, the clams were transferred into six trays partially submerged inside the sump of the recirculat ion system with water flowing into each individual tray. The Marshall Islands clam farm kept the clams under 73% shadecloth and were slowly acclimated to ambient greenhouse light levels. This was replicated by using two layers of 30% shadecloth in addition to th e greenhouse plastic (25% shade) to cover the trays in the sump for nine days after arrival. Light intensity in PAR was measured using a LICOR (Lincoln, NE) LI-1000 datalogger a nd underwater quantum sensor LI-192SA. PAR irradiance was measured in mol/m2s. Without the shadeclot h, light levels in the sump trays holding the clams reached levels up to 1500 mol/m2s between 1200-1300 hours. This was 500 mol/m2s below the readings found out side of the greenhouse in full sunlight during the same time. Each la yer of shadecloth lowered midday readings by ~400 mol/m2s. One layer of shadecloth was used for an additional nine days. The

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10 second layer of shadecloth was removed (no sh adecloth) for the final nine days, exposing the clams to full sun within the greenhouse. After clams were acclimated to greenhous e light levels (March 16, 2005), they were mounted on top of 2.5-cm square ceramic tiles. A 3-mm cone-shaped dimple was drilled into the center of each tile to cradle the umbo portion of the clam shell. A T-bar fish tag (Floy tags, Seattle, WA) was used to fasten each clam to its tile (Figure 2-3). This was done by applying Loctite and Supe rglue brand cyanoacr ylate (Henkel, Avon, OH) to each end of the tag and attaching the tag to both the valve of the clam and the edge of the tile. Care was taken not to gl ue too close to the shell opening to prevent mantle tissue irritation. Light Treatments Three light treatments were used within each tank to produce varying irradiance and spectral levels (Figure 2-4). Light tr eatments included the fo llowing: 1.) sunlight within greenhouse (double layer of 150-m pl astic), 2.) sunlight within greenhouse and 30% shadecloth (this was changed to 55% on day 48 due to increasi ng light levels), and 3.) sunlight within greenhouse in conjunction wi th artificial, metal halide lighting set on a 12-hour photoperiod – (0700 to 1900 hours both be fore and after daylight savings time change occurred on day 3). The metal ha lide lights had 400-W 10,000-K mogul bulbs (XM, Orange, CA) with a 400-W electronic ba llast (Coralvue, Kenner, LA) as a power source. These high Kelvin rated bulbs were chosen for producing peak wavelengths in the blue portion of the spectrum. Each bulb was positioned 20 cm above the surface of the water. Light from the bulb was projecte d through a light trap st raight down into the treatment group. Light traps consisted of four black plastic walls hung from a 40 x 30 cm pvc pipe frame, extending down to the water le vel (20 cm) to shield all four sides below

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11 the metal halide reflector canopy. The light traps blocked any metal halide light from reaching the non-metal halide treatment groups ; light reaching adjacent treatment racks was measured at less than 2 mol/m2s. Bulbs were cleaned weekly with fresh water and a wash cloth to remove any salt buildup. These treatments were randomly assigned to one of three positions in each tank (Figures 2-1 and 2-4). Each tank was divide d into three equal areas (89 x 147 cm) for the three treatments. Clams were set upon trea tment racks (50 cm long x 50 cm wide at a depth of 12 cm) made of eggcrate light diffu ser material and supported by a pvc frame. Each rack held nine or more seed clams. Treatment racks had equal numbers of clams from sizes ranging seven to 14 mm. There were a total of six treatments (three sources of light x two culture systems) with six repli cations of each treatmen t, for a total of 36 experimental units. During the experime nt, six tanks had a metal halide light malfunction. On days 22 to 29, the ballast in tank 4 was out of order; on days 32 to 34, the ballast in tank 9 was out of order; on days 52 to 57, ta nk 1 had a burned-out bulb and the ballasts in tank 1 and 4 were out of orde r; on days 55 to 57, the ballast in tank 12 was out of order; on days 61 to 68 the ballast in tank 2 was out of or der; and on days 74-80, the ballast in tank 7 was out of order. The halide treatment racks were placed in full sun until the ballasts/bulbs were replaced and back online. On day 4 of the experiment, four or more coral fragments, each approximately 2-10 cm in length, salvaged from a pier in Key West FL were placed into the shaded treatment racks of all 12 experimental tanks for other research purposes. These corals included the following species : Montastrea cavernosa Stephanocoenia intersepta Porites porites and Diploria clivosa.

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12 Duration and Sampling Acclimation Period Clams were acclimated to the light treatments for 15 days before the study began (March 17 – March 31, 2005). The metal halid e lights were initially set for 5 hours on and 19 hours off, and adjusted daily to a dd 30 minutes more light until it reached 12 hours on 12 hours off. Study Period The study ran for 90 days (April 1 – June 29, 2005). On day 1, six tiles with leashes but no clams were placed in the treatme nt closest to the airlifts of all twelve tanks. Weights were taken for the blank tiles on day 1 and day 90. Light Measurements PAR irradiance was measured daily dur ing midday (1200 to 1300 hours) in an unobstructed area outside the gr eenhouse using the LICOR irradiance sensor. Daily peak readings were not recorded for two weeks (day 27 to 39) due to nece ssary repair work for the LICOR. Amount of daily sunshine minutes was recorded by the National Weather Service Station (National Oceanic and Atmo spheric Association, Ruskin, FL) located 30 meters from the experimental greenhouse. Every 30 days, spectrum and intensity in PAR for each metal halide treatment was measured. An Ocean Optics (Dunedin, FL) USB 2000 spectrophotometer, with a #3 grating (350-800 nm range) and a 200-nm op tic fiber with a CC-3 Cosine Corrector, was used to measure spectral distribution in the cent er of the clam treatment rack. To test for changes in bulb performance, the LICOR was used to measure light intensity in a 5-cm2 grid at clam depth (12 cm). Both of th ese measurements were taken at night so no sunlight was included in the recording. During the second month, a LICOR model LI-

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13 250 light meter was used with the underwater quantum sensor LI-192SA, while the other LICOR was in repair. Light irradiance in the full sun and shaded light treatments was measured in PAR with the LICOR sensor at the depth where cl ams were held (12 cm). This was conducted from 1200 to 1300 hours, taking one reading per tank per hour. These measurements required cloudless days and were therefore conducted three times: three days prior to the study; on day 25; on day 69. Passing clouds alte red light readings from one minute to the next making light readings unrel iable. The peak angle of the sun was recorded on days 5, 48 and 85. This measured the shortest le ngth of the shadow of a 45-cm ruler perpendicular to a level base from 1200 to 1300 hours. Growth Measurements Growth was measured every 30 days for each clam using wet weight (g) and shell length (mm) (Figure 2-5). Length measuremen ts were taken using calipers. Width and height were not accessible due to the clam tag and the tile. Clams were allowed to drip dry for 10 s, while taking the length measurem ent, and the tile bottoms were blotted dry before being placed on the scale for wet weight measurements. Color Measurements Clam mantle color was measured every 30 days. Measurements were taken inside of a light-sealed 30-L tank (out side was painted black) filled with 20 L of saltwater. A 6.35-mm OD barrel was attached to a 200-nm op tic fiber (Figure 2-6). An Ocean Optics WS-1 diffuse reflectance standard was us ed in conjunction with a 50-W xenon halogen bulb (Duralamp Fullerton, CA) and a Dimensi ons F27 halogen track light (Hampton Bay, Atlanta, GA). The reference standard was used before taking each color sample in order to calculate the amount of light reflected into the spectrophotometer. Digital images were

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14 taken with a Nikon D70 (East Rutherford, NJ,) digital camera with its lens placed on a sealed slot entering the top of the color measurement tank. Twenty six clams were subsampled for color measurement on days 3, 33, 63 and 88 of the experiment. Low numbers of colorful clams did not allow for hi gher sampling sizes. Nine more clams, one green and eight brown, were added for color measurement on day 60 due to mortality of some of the previously sampled clams. At day 1, at least two clams from each light treatment were selected for each color mor ph; blue, green, and gold (brown/yellow). Each clam was placed into the color measurement tank and positioned underneath the spectrophotometer probe underwater. Measur ements were not taken until the mantle tissue was exposed. The angle of incide nce from the light to the clam was 45o and the end of the spectrophotometer probe was held at 45o to the clam with a 5 to 6-mm distance from the probe to the mantle tissue (Fi gure 2-6). This measured a 3 to 4-mm2 area of mantle tissue. Three measurements of the same portion of mantle tissue were taken for each clam. Color measurements were also take n with the clam mantle retracted to correct for shell reflectance. Prior to taking the sh ell reflectance measurement, the clam was startled into closing its valv es by sticking a pvc pipe inside the box. Three additional measurements of 5-cm blue color clams we re taken on day 60. These were used to compare to smaller sized clam mantle readi ngs. Results were analyzed using the Ocean Optics OOIIRAD program which measures spectral reflectance and the CIE L*a*b* color space which uses the three variables—L* is the light-dark axis (light positive, dark negative), a* is the red-green axis (red posit ive, green negative), and b* is the blueyellow axis (yellow positive, blue negative).

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15 Statistical Analysis All statistical analyses were conducted at a significance level (T ype I error rate) of = 0.05. Two-way analysis of variance (ANOVA) was used to detect differences in survival proportions at day 90, using light and system treat ments as main effects (Proc GLM; SAS Institute, Cary, North Carolina) Proportions of surviving clams were arcsine-square root transformed prior to an alysis. Split-plot re peated measures ANOVA was used to detect differences in length a nd weight data for all four clam measurement periods, using light and system treatments as main effects (Proc Mixed; SAS Institute, Cary, North Carolina). Signi ficant ANOVAs were followed by the least squares means multiple comparison procedure. A chi-square analysis of survival at day 90 was conducted on four different size groups of clams to determine if smaller size classes had lower survival over time. The initial size groups used at the start of the experiment for these clams were: 6-9 mm; 10 mm; 11 mm; and 12-14 mm. Proportions of size class survival were compared using the original clam size. Means for L*a*b* values were collected fr om survivors of the 26 original clams measured from the first sample period. Thes e did not include the nine clams added on the third sample period.

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16 Sump Bead Filter 1 Syst S = Shaded, O = Open, M = Metal Halide Fan Fan Evaporative Coole r Door Greenhouse Dimensions : 22 X 9.1 meters N UV Sterilizer 2 Syst 3 Syst 4 Roll 5 Roll 6 Syst 7 Syst 8 Roll 9 Syst 10 Roll 11 Roll 12 Roll Shade cover OSM OMS MOS OSM MOS MOS MOS OSM OMS SOM MOS MOS OSM Chiller Tank Treatments Protein Skimmer Airlift rack Water flow Treatment racks M S O Enlargement of Tank 9 aerial view Figure 2-1. Greenhouse layout. Twelve experimental tanks are numbered on upper left corner along with culture system treatment (Roll = Rollover, Syst = Recirculation System) on upper right co rner. Enlarged tank shows position of light treatment racks inside of one of the tanks.

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17 Figure 2-2. Rollover tank. Ei ght airlifts on the right side of tank draw up water and pushes it across the surface of the tank to circulate the water in a rollover fashion.

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18 Figure 2-3. Seed giant clam ( Tridacna maxima ) attached using cyanoacrylate glue to a Floy T-bar tag (marked by arrow). The Floy T-bar tag was attached to a 2.5cm2 ceramic tile using cyanoacrylate glue.

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19 Figure 2-4. Light treatments s een above in two experimental tanks. Note metal halide lights with heavy plastic covering to k eep artificial light from reaching other treatments. Shaded treatments included some coral fragments around edges of rack with clams grouped in the center.

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20 umbo length byssal opening Figure 2-5. Clam length – l ongest stretch across shell.

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21 xenon halogen bulb probe tip clam Ex p loded view of p robe and cla m probe stand camera slot 30 degree angle tile with cradle space light source color area measured barrel probe tip clam 4 mm distance 1-2 mm distance Figure 2-6. Color measurement tank and en largement of spectrophotometer probe and clam position. Top of tank was sealed with an acrylic cover and heavy drape cloth. Tank sides and bottom are c overed with black acrylic paint.

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22 CHAPTER 3 RESULTS Survival Clams had a mean 74.6% monthly survival (43.4% for all 90 days) during the experiment. There were 172 clams at the end of the experiment. Among the six treatment types, mean surviv al over 90 days was highest in both the shaded x recirculation treatment with a 66.2% ( 6.7% S.E.) and the open x recirculation treatment, with a survival ra te of 56.9% ( 10.7% S.E.) (Figur e 3-1). The treatment with the lowest survival after 90 days was halide x rollover, with a surviv al rate of 13.2% ( 5.8% S.E.). Two of the six replicate rack s of the halide x rollover treatment had no surviving clams after 90 days. Mean survival for each treatment type is as follows: open treatment was 47.7% ( 7.3% S.E.); shaded treatment was 55.1% ( 6.3% S.E.); halide was 28.5% ( 7.2% S.E.); recirculation system was 55.5% ( 5.4% S. E.); and rollover was 30.9% ( 4.7% S.E.). The two-way ANOVA found that system type ( F 1,30 = 17.56, P = 0.0002) and light source ( F 2,30 = 9.04, P = 0.0008) each had a significant effect on clam survival (Table 3-1). The rollover systems had signifi cantly lower survival than the recirculation systems. No significant difference was f ound between clam survival in the open and shaded treatments (LS Means, P = 1.000). The halide treatmen t had significantly lower survival than both open (LS Means, P = 0.0076) and shaded ( P = 0.0012) treatments. A Chi-square analysis showed significant differences ( 2 = 8.07, df = 3, P = 0.05) in survival of size groups (Table 3-2). Sma ller sized clams (6-9 mm) had a survival rate

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23 of 29.3% compared to larger size groups su rvival of 50.6% for 10 mm, 42.7% for 11 mm, and 50.9% for 12-14 mm (Figure 3-2). Shell Growth Split-plot repeated measures ANOVA found no significant effect of light or system type on shell growth (Table 3-3). Clams that survived from day 1 to day 90, had a mean growth of 0.58 mm with a mean initial length of 11.17 mm. This equals a mean growth of 5% in 90 days. Mean growth over 90 days for surviving clams was as follows: recirculation system treatment was 0.8 mm (7.3%); rollover treat ment was 0.3 mm (2.7%); open treatment was 0.7 mm (6.4%); shaded treatment wa s 0.6 mm (5.6%); halide treatment was 0.4 mm (3.3%); open x recirculation treatment was 1.1 mm (9.5%); shade x recirculation treatment was 0.8 mm (7.1%); halide x recirc ulation treatment was 0.6 mm (5.4%); open x rollover treatment was 0.3 mm (3.1%); shad e x rollover treatment was 0.5 mm (4.1%); and halide x rollover treatment was 0.0 mm (0%).(Table 3-4). Nine clams (two blue and seven gold) gr ew 2 mm or more during the 90-day study. Four of these were in open x recircula tion treatments, three were in the shaded recirculation system treatment, and one was in the metal halide recirculation system treatment. One gold clam (#313) in the ope n x recirculation treatment had a maximum total growth of 4.7 mm. It had a maximum monthly growth of 2.4 mm during the last 30 days of the study. Wet Weight Culture system had a significant effect on mean weight ( F 1,10 = 10.31, P = 0.0093) (Table 3-5). Mean weight gain for all blank tiles was 0.04 g, while mean weight gain was 0.062 g and 0.019 g for tiles in the reci rculation and rollover tanks, respectively.

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24 Noticeably more biofouling (mainly unidentif ied diatoms and filamentous algae) had settled upon the tiles from th e recirculation system than upon those from the rollover tanks. Therefore, clam wet weight values were calculated by subt racting mean weight gain for blank tiles each culture system. Table 3-6 summarizes mean wet weight gain for clams in each treatment surviving to day 90. Mean clam wet weight for the six treatment types was 0.300 0.008 S.E. g at the beginning of the study. Mean wet weight gain for clams surviving to day 90 was 0.026 g (8.7%). Mean wet weight gain or loss for each treatment was as follows; recirculation sy stem treatment was 0.057 g (18.3%); rollover treatment was -0.006 g (-2.1%); open treatmen t was 0.029 g (9.4%); shade treatment was 0.032 g (10.7%); and halide treatment was 0.023 g (7.6%). Color Giant clam mantle coloration is an important factor for determining market price. Clams with bright blue or gr een colored mantles are termed “ultra” and fetch the highest prices. However, the majority of giant cl ams have a brown or ye llow mantle tissue and are termed “gold”. The clams in this study we re divided into three categories for ease of color quantification: blues, greens and golds. Individual clam read ings are listed in Appendix A. During the acclimation period, 14 clams w ith green mantle tissue and two clams with blue mantle tissue changed to gold ma ntle tissue by the start of the experiment. Since these changes took place prior to th e start of the experiment, there is no spectrophotometer record of the color change. Reference clams Three 5-cm blue “ultra” T. maxima clams were obtained from an online retail store after the second color sample pe riod and measured as a blue and green color reference for

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25 ideal marketable color. One 5-cm clam had dark blue mantle tissue and showed a lower percent spectral reflectance signature compared to the other two 5-cm clams (peak 400 nm, 2% reflectance) (Figure 3-3). Another 5cm clam had light bl ue mantle tissue and showed a high percent spectral reflectance signature (peak 441nm, 20% reflectance) (Figure 3-4). The third 5-cm clam had a mi xed blue-green pattern on its mantle tissue and had a spectral reflectance signature showing a shift to the blue-green portion of the spectrum (peak 481 nm, 7.5% reflectance). The light and dark blue clams showed a high blue b* value (-15.5, -10.8) while the mixed gr een and blue clam showed a high green a* value of -3.4 and a blue b* value of 4.9 (l ower numbers are higher values for both green and blue in CIE L*a*b*). Survivors Only eleven clams out of 396 were visually found to have blue color within the mantle tissue. Out of these, only thr ee from the open treatment (clam # 112, 1111, and 715), two from the shade treatment (8211 a nd 9210), and one from the halide treatment (1237), survived until the end of the experiment. Three blue clams died after the first color sample. One blue clam from the shade treatment died after the second color sample (823). One blue clam from the shade treatmen t died after the third color sample (522). Only two green clams out of seven survived until the end of the study (311 and 7110). A third green clam (7212) died after the third sample period. Only three gold clams out of eight survived until the e nd of the study (225, 1212, and 3110). Green switch to Gold (Yellow) One clam switched from green to gold dur ing the experiment. Clam 721 from the shade x recirculation treatment was one of nine clams added midway during the experiment for color sampling due to losses of other color sampled clams. Overall

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26 spectral reflectance decreased except for th e lower portion of the blue region (400-415 nm) (Figure 3-5). Both green a* and yellow b* values decreased from the third to the fourth sample period. The photograph shows a green mantle tissue in the third sample period and a smaller sized yellow mantle tissu e in the fourth sample period. Although the photographs agree with the a* values, it was contradicted by the decreased b* values. This may have been due to a decrease in mantle size. Light Measurements Solar Photosynthetic Active Radiation (PAR) m easured outside of the greenhouse in open air between 1200 and 1300 hours had a maxima of 2400 mol/m2s. This was recorded on day 15 with partly cloudy skies. Only when clouds are positioned right beside the sun, but not fully bloc king it, is sunlight reflecte d at higher intensities than on clear days. Peak PAR on clear days was 2100 mol/m2s. Mean daily peak PAR was 1620 mol/m2s. A slight decrease in daily peak PAR readings was found to occur during the study (Figure 3-6). This was mainly due to an increase in cloudy days during the second half of the study as seen in the percent po ssible sunshine minutes (Figure 3-7). However, underwater measurements at clam depth did display an increase in midday PAR readings over the course of this study. This was attributed to increased midday sunlight angle. Mid-day sun light angle increased from 65 o three days prior to the experiment to 85 o at day 90. Summer solstice occu rred on day 82. Winter solstice in Ruskin, FL had an angle of 30 o. Day length increased from 750 min (12.5 h) on day 1 to 833 min (13.9 h) on day 90. Three days prior to the study, mean peak intensity of the open treatments was 1304 mol/m2s. This increased to a mean of 1414 mol/m2s on day 25. The final reading

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27 found a mean peak intensity of 1412 mol/m2s on day 69. Maximum intensity was recorded in tanks 3, 4, 5 and 10 with 1500 mol/m2s on days 25 and 69. The peak wavelength was 588 nm. Intensity rose sharpl y before 460 nm, leveled off, then dropped after 700 nm (Figure 3-8). Three days prior to the study, peak inte nsity in the shaded treatment was 911 mol/m2s. This increased to a mean peak of 987 mol/m2s on day 25. Shadecloth percentage was raised from 30% to 55% on day 48 to decrease rising intensity levels in the shade treatment. The final r eading found a mean peak of 719 mol/m2s on day 69. Shaded treatments shared the same peak wavelengths as the open treatments with a peak wavelength at 588 nm, and a plateau runni ng from 460 nm to 700 nm (Figure 3-8). Metal Halide Mean intensities rang ed from 660-758 mol/m2s in the halide light treatments during the 90 day study (Table 3-7). Only one halide treatment (Tank 1) had highly variable readings (Figure 3-9). Tank 1 had the maximum intensity recorded (1494 mol/m2s during week five) and the minimu m recorded intensity (140 mol/m2s during week 13; its maximum intensit y during week 13 was 280 mol/m2s). Tank 1 mean intensity decreased respectively by 484 and 417 mol/m2s from weeks five to nine and weeks nine to thirteen. These changes in inte nsity were most likely due to fluctuations in voltage to the ballasts. Peak wavelengths of the metal halide treatm ents for all tanks were similar and did not vary greatly over the 90-day study (Figur e 3-8). The peak wavelength was measured at 420 nm, the second highest peak was at 403 nm, and the third peak was at 435 nm. A smaller peak at 365 nm appeared as high as the 435 nm peak on a few occasions. This happened in tank 4, 6, and 9 during week 5. It also appeared in tanks 1, 7, and 9 during

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28 week 9 and finally in tank 1 on week 13. Tank 1 had a major spectrum shift on the final week of the study (week 13) with even dist ribution between the th ree peaks at 365, 435, and 403 nm. Six halide lights malfuncti oned at some time during the study due to the high humidity greenhouse environment and fluctuatin g voltage. Ballasts were placed in the greenhouse five months before the start of the experiment and were exposed to high humidity at that time. Th ese lights ran for less than 100 hours before the acclimation period. A period of low voltage occurred durin g the experiment due to a problem with the Florida Power and Light company (FPL) se rvice reaching the greenhouse. Electronic metal halide ballasts are known to be sensitiv e to drops in voltage and may have been damaged due to these conditions. The voltage issue was resolved by FPL on day 47 but may have had lasting effects on a few ballasts. Temperature profiles Higher than ideal temperatures (>30 oC) (Ellis 2000) occurred in all rollover tanks. This was due to the inability of the evaporat ive cooler to keep midday air temperatures inside the greenhouse below 38 oC from day 30 on. This raised temperatures in rollovers to a maxima of 34.66 oC in tank 4. The remaining five rollover tanks reached temperatures above 33 oC. Recirculation system tanks stayed below 30 oC throughout the study with the aid of the chiller. Weather data gathered by the local w eather station indica ted a minimum outdoor air temperature of 10 oC during the first and third weeks of the experiment. After the fourth week, day 28, the heater was left o ff and the windows for the evaporative cooler pads were left open. A maximu m outdoor air temperature of 33 oC was reached on day 60 of the study (NOAA weather station, Ruskin, FL).

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29 Water Quality Total nitrogen in tanks reached a maximum of 1.8 mg/L in the rollover tanks and a maximum of 2.8 mg/L in the recirculation sy stem tanks according to spectrophotometer readings by Florida Lakewatch. The HACH te st kit was not sensitive enough to detect these low levels. The only high nitrogen r eading from the HACH test kit came from a nitrite spike during week 8. Rollover tanks accumulated large amounts of foam each day. This was caused by the presence of high organics in the tank wa ter. Insects attracted to the metal halide lights during evening hours became trapped on the water surface. This added to the input of nutrients which turned into dissolved orga nic matter that was late r separated out of the recirculation system tanks by the protein skimme r. Constant collection of foam from the rollover tanks was not enough to keep the trea tments within these tanks from receiving less light due to the shadowi ng effect of the foam. The foam layer reduced light transmittance by up to 150 mol/m2s in rollover tank treatments. The recirculation system was unable to keep nitrites from spik ing. Nitrite reached 1.5 mg/L on week 8. More algae was observe d inside of recirculation system tanks compared to rollover tanks. Three times as much algae weight was recorded on the blank tiles from the recirculation tanks than from the rollover tanks, a m ean weight gain of 0.062 g compared to 0.019 g. Calcium levels were low in both rollovers and recirculation systems during the first sample period (370 mg/L). The LaMotte and Seachem Calcium test kits gave faulty readings thereby contributing to the suboptimal levels. Once the Salifert test kits were used, calcium readings stabi lized at 400 mg/L or greater according to the ICP mass spectrophotometer readings for the remainder of the study. Alkalinity readings ranged

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30 from 171 to 205.2 mg/L. Total phosphate levels for all four sample periods were less than 20 g/L in all tanks.

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31 0 10 20 30 40 50 60 70 80 90 100 306090 Time in daysMean Percent Survival open recirc shade recirc halide recirc open roll shade roll halide roll Figure 3-1. Mean treatment su rvival after 30, 60 and 90 days (n=6) for all six treatment types: open x recirculation; shade x recirculation; ha lide x recirculation; open x rollover; shade x rollover; and halide x rollover. See methods for specific details of the treatments. Standard error bars included.

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32 0 10 20 30 40 50 60 Day 90Percent 6
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33 0 0.5 1 1.5 2 2.5 3398 416 434 452 470 488 506 523 541 558 576 593 610 627 644 661 678 694Wavelength (nm)Reflectance (%) Figure 3-3. Spectral refl ectance (%) for a 5-cm T. maxima clam measured on the third sample period. Its dark blue mantle tissue showed low spectral reflectance (2%) in comparison to the light blue mantle 5-cm T. maxima reference clam in Figure 10 (20%).

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34 0 5 10 15 20 25400 417 433 449 465 481 497 513 529 545 561 576 592 607 622 638 653 668 683 698Wavelength (nm)Reflectance (%) Figure 3-4. Spectral refl ectance (%) for a 5-cm T. maxima clam measured on the third sample period. Its light blue colored ma ntle tissue was used to compare to the smaller sized clams. The peak wavele ngth is found in the blue region at 440 nm, consistent with what was found in smaller blue seed-size clams. This bright blue is the color in de mand for the ornamental trade.

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35 0 1 2 3 4 5 6 7 8 9390 408 427 445 464 482 500 518 536 554 572 590 607 625 642 659 676 693Wavelength (nm)Reflectance (%) Figure 3-5. Spectral reflectance of one of the clams added for color sampling for the third sample period (clam 721 from the sh ade x recirculation system). This was the only clam recorded to have ch anged from a dark green color to a yellow colored mantle. Spectral reflect ance for the color change is shown above from day 60 (solid line), to da y 90 (dashed line). Photographs taken show mantle switching colors from a dark green on day 60 to a yellow color on day 90. Mantle tissue diminished in size on day 90 and resulted in a low spectral reflectance

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36 0 500 1000 1500 2000 2500 12345678910111213 Weekmol m-2 s-1 Figure 3-6. Mean weekly peak PAR. Calcul ated from daily peak PAR readings taken outside of the greenhouse. S.E. bars included (n = 7, week 13 n = 6). Weeks 4 to 6 are missing due to meter malfunctions

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37 0 20 40 60 80 100 120 1611162126313641465156616671768186 DayPercent per Day Figure 3-7. Percent possible s unshine. Percentage of daytime during which the direct solar radiation exceeds the level set by NOAA. This activates a sunshine recorder to log each sunshine minute between sunrise and sunset. Data provided by NOAA weather sta tion in Ruskin, FL. Last 30 days showed an increase in cloudy weather.

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38 0.00 0.05 0.10 0.15 0.20339 376 411 447 482 517 551 585 619 652 685 717 749 781 812 842Wavelength (nm)Watts/cm2/ms Halide treatment Shaded treatment Open Treatment Full sun outside greenhouse 365 nm Figure 3-8. Spectral power di stribution for all three light treatments at midday. A reading of full sun outside of the greenhouse was taken at midday. Shade treatment was measured with 30% shadecl oth. Halide treatment had a spectral distribution concentrated in the blue wavelengths and included a UV-A spike at 365 nm.

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39 TANK 1 WEEK 1 TANK 1 WEEK 5 TANK 1 WEEK 9 TANK 1 WEEK 13 Figure 3-9. Light maps in mol/m2s for halide treatment in Tank 1 during week one, five, nine, and 13. High light readings were recorded in week five. Light readings decreased in weeks nine and 13. Twin peaks for maximum intensity were normal for all other halide lights. Intensity decrea sed around the edges of the treatment racks.

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40 Table 3-1. Two-way analysis of varian ce (ANOVA) for survival at day 90 of T. maxima seed clams among two culture systems and three light treatments. Source DF SS Mean Square F P SYSTEM 1 0.75207574 0.75207574 17.56 0.0002 *** LIGHT 2 0.77437041 0.38718520 9.04 0.0008 *** SYSTEM*LIGHT 2 0.07369967 0.03684984 0.86 0.4331 ERROR 30 1.28453669 0.04281789 TOTAL 35 R-Square = 0.55 *** significant difference among means (P < 0.001)

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41 Table 3-2. 2 analysis of size class survival of T. maxima seed clams over 90-day study. 2 calculated = 8.07, df = 3, = 0.05, 2 critical = 7.815 (Ott 2001). Survival after 90 days size class (mm) expected observed 6-9 42.6 28 10 36.9 44 11 41.7 40 12-14 50.8 60

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42 Table 3-3. Split-plot repeated measures analysis of variance (ANOVA) results for shell length data of T. maxima seed clams among two culture systems and three light treatments over 90 days. Num Den Effect DF DF F Value Pr > F SYSTEM 1 10 2.15 0.1736 LIGHT 2 20 0.07 0.9364 SYSTEM*LIGHT 2 20 0.73 0.4964 TIME 3 30 12.66 <0.0001 *** SYSTEM*TIME 3 30 2.10 0.1207 LIGHT*TIME 6 1023 0.27 0.9514 SYSTEM*LIGHT*TIME 6 1023 0.26 0.9562 *** significant difference among means (P < 0.001)

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43 Table 3-4. Treatment mean shell growth (percent) of T. maxima seed clams during 90day study. Standard error (S .E.) is included (n = 6). Treatment Percent growth S.E. open x roll 3.1 1.0 shade x roll 4.1 1.2 halide x roll 0.0 0.8 open x recirc 9.5 1.0 shade x recirc 7.1 1.1 halide x recirc 5.4 0.9

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44 Table 3-5. Split-plot repeated measures analysis of variance (ANOVA) for weight data of T. maxima seed clams among two culture systems and three light treatments over 90 days. Num Den Effect DF DF F Value Pr > F SYSTEM 1 10 10.31 0.0093 ** LIGHT 2 20 0.73 0.4944 SYSTEM*LIGHT 2 20 2.58 0.1005 TIME 3 30 24.53 <0.0001 *** SYSTEM*TIME 3 30 7.47 0.0007 *** LIGHT*TIME 6 1023 0.32 0.9250 SYSTEM*LIGHT*TIME 6 1023 1.01 0.4143 *** significant difference among means (P < 0.001) ** significant difference among means (P < 0.01)

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45 Table 3-6. Ninety-day mean wet weight gain and mean pe rcent weight gain, correcting for biofouling (algae) weight, for T. maxima clams. Rollovers show weight loss. Wet weight gain (g) S.E. Percent weight gain (%) S.E. clam weight gain with algae weight included open x roll -0.002 0.016 -0.7 5.9 shade x roll 0.029 0.012 9.8 4.2 halide x roll 0.014 0.010 4.5 3.3 clam weight gain after correction for algae weight (1) open x roll -0.021 0.016 -7.6 5.9 shade x roll 0.010 0.012 3.3 4.2 halide x roll -0.005 0.010 -1.8 3.3 clam weight gain with algae weight included open x recirc 0.142 0.023 42.0 6.9 shade x recirc 0.117 0.007 38.8 2.5 halide x recirc 0.097 0.015 33.0 5.1 clam weight gain after correction for algae weight (2) open x recirc 0.080 0.023 23.6 6.9 shade x recirc 0.055 0.007 18.3 2.5 halide x recirc 0.035 0.015 11.9 5.1 1 Mean algal weight gain of 0.019 g (S.E. = 0.005) was subtracted from mean rollover clam weight gain 2 Mean algal weight gain of 0.062 g (S.E. = 0.008) was subtracted from mean recirculation clam weight gain

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46 Table 3-7. Mean halide light tr eatment intensity readings (mol/m2s) for all 12 tanks combined. Maximum and minimum readi ngs from all 12 tanks recorded as well as mean maximums and mean mini mums of all 12 tanks. Numbers in parenthesis are S.E. (n = 6). Time after start of experiment week 1 week 5 week 9 week 13 mean 747 (19) 758 (37) 708 (25) 660 (45) maximum 1295 1494 1250 1060 minimum 370 355 345 140 mean maximum 1045 (29) 1019 (54) 971 (40) 883 (58) mean minimum 468 (22) 485 (29) 437 (22) 460 (34)

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47 CHAPTER 4 DISCUSSION Major Findings In this study, I found that 1) The recircul ation system treatment had significantly higher survival compared to the rollover treat ment, 2) the open and shade treatments had significantly higher survival compared to the halide treatment, and 3) the recirculation treatment had significantly higher weight ga in compared to the rollover treatment. This is the first study to examine inland production of T. maxima and also is the first study to test an artificial source of light on giant clams. Low Survival and Growth Excessive handling during attachment to th e clam leashes before the experiment added to shipping stress, may ha ve contributed to low survival and stunted clam growth. The 66% survival rate in the shaded x reci rculation treatment ove r the 90-day study is comparable to the 75% survival rate found in most clam farm operations for clams at that stage for the same duration (Ellis 2000). The recirculation system had better surviv al than the rollover system, with 55.5 vs. 30.9% survival. This can be attributed to the chiller’s capability to maintain temperatures below 30 oC. Evaporative coolers were unable to keep temperatures from approaching lethal tolerance levels in the rollover culture system because they do not function at relative humidity levels appr oaching saturation. However, a greenhouse with heavy shadecloth (>50% shade) may be able to main tain temperatures within the ideal range with the help of an evaporative cooler. However, caution should be taken to avoid

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48 blocking too much light with the shadecloth for clams to survive. Even with 50% shadecloth, slight differences in growth were found in the study by Lucas et al. (1989). Most of the clams under the metal halides showed signs of stress by not fully extending their mantle tissue. This was possibly a sign of photoinhibition due to UV wavelengths given off by the halide lights. Spectral peaks at 365 nm confirmed that UVA was reaching the clams (Figure 3-8). Open (47.7%) and shaded (55.1%) treatments both had much higher survival rates compared to the metal halide treatment ( 28.5%). This was most likely due to the combination of constant, high intensity light and the portion of energy from the metal halide lights found in the UV-A (320-400 nm) range. Only three percent of sunli ght is composed of UV wavele ngths (200-400 nm). This portion of the spectrum is responsible for damage to zooxanthellae (Jokiel and York 1984). Jokiel and York (1984) used UV-blocking films that transmitted up to 92% of full surface radiation and did not find severe photoinhibition in zooxanthellae, while zooxanthellae in another treatm ent without the UV-blocking film could not tolerate levels of light higher than 20% of full surface radiation. The performance of blue spectrum metal halides could be improved by using a UVblocking film, such as OP-3 acrylic (Cyro Plastics, USA), to shield UV from reaching culture tank organisms such as clams and corals. This shield would need to be kept at a large enough distance from the ha lide light so that it does no t melt from the intense heat produced by the bulb. Clams in this study grew slowly in all 6 treatments. Although no statistically significant differences in growth were found among treatments, mean growth rates were

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49 higher for clams in the open treatments w ith 6.3 % growth, compared to 5.6% in the shaded and 3.3% in the halide treatments. Only one clam from the open recirculation treatment exhibited a growth that would reac h marketable size (4-5 cm) in less than 2 years. This is comparable to the growth found in clam cage culture which has abundant amounts of algae available for the clams to feed on (Hart et al. 1998). Besides reduced heterotrophic energy sour ces, the slow growth rate found in the majority of the clams may be attributed to the unusually small mean size (for given age) of clams used for the experiment. One-year-old T. maxima seed should be 10-30 mm in length (Ellis 2000). All of the clams used in this study were below the median size of 15 mm for 1-year-old seed clams. These may have been the culls of a spawn and had a genetically inferior growth rate compared to the larger individuals of the batch. Multiple disturbances were made when attaching clams to tiles and monitoring growth. These could have further contributed to the slow growth. Ellis (2000) found that clams removed from their substrate more than once every three months would be stunted due to energy expended to lay down new byssal threads. Future giant clam growth studies should use longer tim e intervals between measurem ents or find some way to measure in-situ. Another possible reason for sl ow growth may have been due to substrate choice. Clams were attached to the T-bar tag/leash in such a way to increase their probability of attaching to the ceramic tile vi a byssal threads. The ceramic tiles used were non-porous, unlike the coral heads that T. maxima are usually settled on in the wild. During the study, some clams managed to detach themselves from their plastic leashes. Thirty-five clams were reglued to their leashes in the middle of the experiment. Some of the clams did not

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50 have byssal threads attached to the tiles duri ng measurement periods and their shells were lifted from the tile so that a small gap c ould be seen. These clams may have lifted themselves from the tile by movements made by their foot before the glue was fully cured. In the majority of weakened clams (clams without fully extended mantle tissue), copepods were found underneath the clam. They appeared to be feeding on clam tissue through the byssal opening. Heavy mucous s ecretions from the byssal opening were found in many clams, probably to protect th em from opportunistic gammarid amphipods. The large byssal opening of giant clams l eaves them vulnerable if kept open and unprotected by a suitable substrate. Unexpectedly, blank tiles showed greater weight gain from biofouling than tile weight loss from erosion. The larger weight gain in the recirculat ion system blank tiles was likely due to the nitrite spike during week nine. Tile algal layers were thicker in recirculation system tanks versus rollove r tanks. Algae weight did not mask the differences in weight gain between the recirc ulation and rollover systems. The corrected clam weight gain after subtracting mean algae weight from each system treatment showed more weight gain in the recirculati on system (Table 3-6). No major differences of algae were observed among li ght treatments. Blank tiles, placed into different light treatments within the recirculation treatment did not show large differences in mean algal weight gain: open was 0.065 g; shaded wa s 0.055 g; and halide was 0.062 g. Rollover tanks had mean blank tile algal weight gain of 0.025 g in the open treatments, 0.016 g in the halide treatments, but no tiles placed in the shade treatments. This was because no shade treatments were placed next to the airlifts of the rollover tanks.

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51 Stress from transport, combined with the possibility that clams may have not have been able to acclimate to the various treatment intensities, may have further weakened the clams. T. gigas juveniles at 10 mm were repor ted to saturate at 500 mol/m2s (Fisher et al. 1985). T. maxima is known to tolerate higher levels of light than T. gigas and shows no saturation up to 1900 mol/m2s; however, no clam size was reported for this study (Ralph et al. 1999). More studies need to be conducted on the irradiation levels necessary to saturate the growth capacity of seed-size T. maxima juveniles. Metal halides were chosen to augment th e blue (actinic) end of the spectrum (400500 nm). The mean intensity recorded in the metal halide treatments was 718 mol/m2s which is less than peak intensities (1500 mol/m2s) in the open treatment. However, halide treatments received a constant light intensity for 12 h per day (except for the 15 minutes that are needed for halides to reach full intensity from startup). Therefore, intensity in the halide treatments exceeded that in the open sun treatment before 1000 hours and after 1600 hours, for a daily total of 6 hours of higher intensity light (measured on day 69) (Figure 4-1). There is some debate as to whether blue light is needed to promote more vibrant colors in both clams and corals. No increase in coloration was observed for the clams underneath a ny of the metal halide treatments. Instead of promoting growth as expected, halide treatments had lower mean growth than open treatments (0.4 mm compared to 0.7 mm). One exception was a clam in the halide x recirculation treatment that grew 2.1 mm (16% growth) over the 90 days. This clam may have acquired a stra in of zooxanthellae with in creased resistance to high irradiance in the blue portion of the spect ra as suggested by some studies on different

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52 strains of zooxanthellae (Fitt and Warner 1993). No bright mantle color was noted for this clam, only a dull brown. Color Analysis Due to low numbers of colorful clams and low survival, a full analysis for effects due to treatments was not possible. A qualitative trend of darker and increased blue mantle tissue from the first to the third sa mple periods was found for the clams in both the open and shaded treatments. This change was seen in both L* and b* values (Table 4-1 and 4-2) as well as in the spectral refl ectance (Figure 4-2). The b* values in all clams, except for one gold clam (3110), decreas ed from the first to the second sample period, indicating an increase in blue. This was clearly seen in the photographs of the clams categorized as blue but not those of green and gold clams. The green and one gold clams had minimal amounts of small blue portions of mantle tissue (clam 3110 days 3090, clam 311 day one). Between the third and the fourth sampling pe riods, the majority of b* values increased, indicating a decrease in blue. This again was evident in the photos of the blue clams but not in the green or the gold. Yellow color inside the mantle tissue was usually shaded out by dark brown and was difficult for the spectrophotometer to measure in b* values. One clam had a dramatic color change from dark green to yellow (721). The high spectral reflectance peak in the blue portion of spectrum may have been responsible for the green found in its mantle tissue (Fi gure 3-5). Possibly, a mixture of brown zooxanthellae and light blue tissue, most lik ely caused by iridocytes (Griffiths et al. 1992), created the greenish shade that was repla ced by yellow tissue. Another possibility is that different colored strains of zooxant hellae are selected for certain portions of mantle tissue. Carlos et al. (2000), showed that multiple strains of zooxanthellae have

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53 been found inside giant clams. If one of th ese strains contain less peridinin, an orange based pigment, then it would appear gr een, due to the remaining chlorophyll a and c2 pigments. Other zooxanthellae strains contai ning more peridinin would appear brown as a result of the combination of orange and green pigments. This “brown strain” of zooxanthellae may have been what rema ined in the mantle of clam 721. Only clams from the rollover treatments experienced tissue bleaching due to mass zooxanthellae expulsion as i ndicated by photographs. This was likely due to loss of brown zooxanthellae from the mantle tissue fr om increased temperatures (Buck et al. 2002). The remaining color left behind was a light cream/peach mantle with some light blue tissue colored by iridocytes as seen in clam 8211. This was also found in clam 438; the photograph showed minute amounts of gree n besides a very light brown. However, bleaching did not occur in all clams from roll over tanks. For example, clam 412 showed no signs of bleaching at the end of the study. This was unexpected since it was in the treatment with the highest amount of light a nd heat (open x rollove r). The zooxanthellae in this clam may have acquired a toleran ce to high light, prev enting photoinhibition. Only two clams with large blue mantles were recorded at the end of the study. These were clams 715 from the open x recirc ulation treatment and 9210 from the shade x recirculation treatment. Clam 715 had a higher blue spectral peak, while 9210 had a lower blue spectral peak. This difference in blue was also evident in the photographs taken of the clams 9210 and 715. Although clam 8211 from the shade x rollover treatment had a large area of light blue dur ing the fourth sample period, it was not as vivid a blue as that f ound in both clams 715 and 9210.

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54 Overall, L* and b* values showed more differences over time while a* values stayed fairly constant. Two qualitative tre nds appeared in this study for both the open and shade treatments according to L* and b* values (Table 4-1). The first qualitative trend showed an increase in darkness from the first to the second sample period. The second qualitative trend showed an increased bl ueness from the first to the third sample period. This increase in dark pigment and blue color for the first three sample may suggest that the clams from these two tr eatments were increasing the amount of iridocytes and zooxanthellae density in response to e nvironmental conditions (e.g., increase in sunlight). Only two of eight bl ue clams did not have photographs that agreed with b* values. This was less problematic for the larger 5-cm clams. This may have been due to larger, more uniform mantle ti ssue area of the 5-cm clams compared to the smaller seed clams. Slight changes due to mantle tissue deformity would have greater effects on readings with the smaller cl ams compared to the larger clams. Although the measurements taken with th e spectrophotometer were difficult to perform and interpret for the seed size clam s, color differences were obtainable when mantle tissue did not vary in size or shap e over time. Further i nvestigation using the spectrophotometer as an objective means to m easure clam color is needed to optimize environmental parameters for improved color in giant clams. This could be applied to increase production of clams that exhibit brilliant blues and gr eens, allowing greater economic success of ornamental giant clam producers. Nutrients Normal background levels of dissolved inorganic nitrogen as the sum of ammonium nitrate and nitrite in non macroalgae dominated r eefs is <15 g/L (Lapointe et al. 1993). Bivalves appear to have the capacity to tolerate high concentrations of

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55 nitrogen. The bivalves Mercenaria mercenaria and Crassostrea virginica have 96-h mean lethal tolerance limits of 110-880 mg/L, and 1081 to 2415 mg/L for ammonia and nitrite, respectively (Epifanio and Srna 1975). One study of giant clams grown in aquaculture effluent showed no adverse affects to T. maxima with exposures of nitrite up to 1.3 mg/L (Sparsis et al. 2001). In the cu rrent study, clams in the recirculation system showed no obvious detrimental effects of exposur e to the increased levels of nitrite (1.5 mg/L) after week 9. Regular heavy dos ing of ammonia did not occur until the experiment began. This may explain why th e establishment of a sufficient population of Nitrobacter for biofiltration was delayed. Both amm onium and nitrite levels were spiked using ammonium chloride and sodium nitrite in the months previous to the experiment, but may have been insufficient to maintain a sufficiently large Nitrobacter colony capable of keeping nitr ite levels in check. Total phosphate was kept below 20 g/L in all tanks. Normal background levels of soluble reactive phosphate found in non-macroalg ae dominated coral reefs is less than 3 g/L (Lapointe et al. 1993). A study on T. maxima juveniles (Estacion et al 1986), found that clams fed Isochrysis and Tetraselmis once every other day had signi ficantly higher growth than unfed clams. Feeding live algae may impr ove growth rates enough for inland culture of juvenile clams to be economically viable. Ho wever, this means added cost for the grower to provide adequate amounts of algae. Nu trient buildup may be another concern with algae feeding. Despite appare ntly high tolerance levels of giant clams to certain nutrients, culture system reactions to increased amounts of nutrient loading need to be examined more thoroughly.

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56 Small clam species may be poor candidates for ocean growout and better suited for inland production. Hart et al (1998) studied the two smalle st, but most colorful clam species, T. maxima and T. crocea as well as the larger clam T. derasa He determined that 35-mm standard length (SL) T. crocea and 50-mm (SL) T. maxima are in greatest demand in the aquarium trade. However, due to their smaller and weaker shells, these clams are at higher risk for preda tion than larger species such as T. derasa Hart et al. (1998) found a mean growth rate in offshor e cage culture of 2.9 (S.D.=0.6) mm/month in juvenile T. maxima over a 16-month period starting fr om 8-month-old seed ranging from 10-30 mm SL. T. maxima reached 50 mm in 9 months, while T. derasa seed (10-30 mm) grew to a size of 80 mm in the same time period and under the same conditions. Poor survival rates (~40%) were recorded for T. maxima during the growout period in the ocean cages compared to ~90% survival in T. derasa for the same study. The smaller T. maxima species is a poor candidate for ocean growout and better suited for inland production where it is out of reach from such pr edators as rice snails and wrasses that can find their way through clam cage openings. Greater protection from environmental conditions such as storms and anthropologica l factors such as po achers and pollution can more easily be maintained in inland culture systems. In conclusion, the two most favorable trea tments in this study were the shaded x recirculation system and the open x recirculati on system. Survival rate was significantly (F 2,30 = 9.04, P = 0.0008) higher for these two light s (47.7% and 55.5% vs. 28.5%) than for the halide. Also mean weight was significant ( F1,10 = 10.31, P = 0.0093) for the recirculation system (mean weight gain of 18.3%) compared to the rollover (mean weight loss of -2.1%). No significant differences were found among treatments for shell growth,

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57 but the open x recirculation system treatment had a slight advantage over the shade x recirculation system treatment for the highe st growth (9.5% vs. 7.1%). Lucas et al. (1989) also found lower growth in the shade tr eatments, compared to sunlight treatments, but survival was higher in the shade treatment s. These two treatments produced the only two healthy clams that exhibited blue mantle at the end of the experiment, while the rest of the clams displayed a dull green or gold (yellow/brown) mantle tissue. These two clams were in tanks that had a larger than average number of coral fragments inside the shaded treatment racks. More coral fragme nts were introduced into these tanks because their shade treatments were closer to the airl ifts than other tanks and provided a higher flow that corals need relative to clams. Clam s have the ability to dr aw in water with their gills, while corals have to rely on a current fo r food or expelling waste. Whether an extra supply of zooxanthellae provided by the corals may have helped pr oduce better color in these two clams is not clear. An unpublished study on coloration of juvenile T. maxima clams showed that light intensity was not as much a factor as was keeping the clams in their natural oceanic environment (personal communicat ion, Lynette Kumar, Universi ty of the South Pacific, unpublished, masters thesis). This study found th at clams planted in different depths in a longline nursery 50 m off the r eef, had better coloration at mo st depths than clams grown in onshore tanks that used varying levels of shadecloth to simulate the depths of the experimental clams out on the sea. More studies need to be conducted on inla nd culturing of giant clams. Despite the problems found in this study, it could ease the ov erharvesting of wild stocks of marine ornamentals throughout the world’s coral reefs. Overharvesting has already eradicated

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58 certain species of giant clams in the Philippines and is a growing problem with wild stocks in Vietnam (Cheshire and Valeria no 2004). Cultivation technology for marine ornamentals has been advancing and should be adopted by the industry before further collapse in wild stocks can occur.

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59 0 200 400 600 800 1000 1200 1400 1600500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100Hourmol m-2 s-1 Tank 4 open treatment intensity Mean halide intensity Figure 4-1. Daily solar profile taken on day 69 for tank 4 open sun treatment shown in dashed line. Mean metal halide intens ity, for all 4 sample periods shown in solid line.

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60 0 1 2 3 4 5 6 7 8 9390 405 420 434 449 464 478 493 508 522 536 551 565 579 593 607 621 635 649 662 676 690 703Wavelength (nm)Reflection (%) Day 1 Day 30 Day 60 Day 90 Figure 4-2. This graph displays spectral shift from day one to day 90 for one of the blue clam seed chosen for color measurements (number 715 from the open x recirculation treatment). Blue p eaks around 440 nm increases in percent reflection while green (500-560 nm) and brown (yellow 560-600, orange 600650) decrease in percent reflection. This was seen in photographic documentation at those time periods w ith increased portions of the mantle tissue turning blue over time.

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61 Table 4-1. Mean L*a*b* values for open and shade treatment clams measured for color. S.E. included in parenthesis (n=6, n=4 for sample period 3 in shade clams). Only clam 1237 survived from the ha lide clams measured for color (n=1). Open Shade Halide sample period L* a* b* L* a* b* L* a* b* 0 -3.5(1.4) 0.7(0.5) 6.0(1.2) -2.4(1.3) 0.7(0.4) 5.1(1.7) -5.4 1.0 3.8 1 -9.1(2.5) 1.3(0.9) 5.3(1.7) -6.7(1.3) 1.3(0.7) 5.8(2.0) 4.3 -0.1 2.2 2 -2.5(0.9) 0.8(0.2) 0.5(1.4) -4.0(2.2) 0.6(0.7) 0.2(1.5) -8.1 -3.1 -0.3 3 -3.3(1.1) 0.3(0.3) 2.2(0.8) -3.7(1.8) 1.1(0.6) 1.1(0.9) -3.5 0.8 2.0

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62 Table 4-2. Mean L*a*b* values for three categories of color measured clams: blue; green; and gold clams. S.E. included in parenthesis (n = 6 for blue clams, n = 3 for green and gold clams). blue clam mean green clam mean gold clam mean sample period L* a* b* L* a* b* L* a* b* 0 -3.5(1.3) 1.6(0.1) 4.2(1.3) -1.4(0.1) -0.5(0.1) 3.6(0.5) -5.2(2.8) 0.0(0.4) 9.1(0.1) 1 -8.5(2.5) 2.5(0.8) 3.2(1.4) -5.9(2.7) -0.5(0.5) 3.4(0.4) -7.9(2.2) 0.4(0.6) 11.5(0.6) 2 -4.3(1.4) 0.2(0.9) -2.3(1.2) -1.4(1.5) 0.3(0.4) 2.1(1.8) -6.1(3.2) 0.4(0.1) 2.5(0.4) 3 -4.2(0.8) 0.9(0.2) 1.4(0.7) -0.4(0.1) 0.2(0.1) 0.6(0.5) -3.9(2.7) 0.4(1.1) 3.3(1.4)

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63 CHAPTER 5 MANAGEMENT APPLICATIONS Based on this research, the two optimal tr eatments for growing out T. maxima seed are the open x recirculation system and shade x recirculation system. The combination of evaporative coolers with the rollover syst em was insufficient for controlling high temperatures during the peak hours of the day leading to increased mortality. The halide treatment had high mortality from harmful UV radiation and also proved difficult to maintain in the high humidity conditions of a greenhouse. Cost of clam seed was the main limiting f actor of this projec t due to lack of production and transport stress. For this st udy, clam seed was obtained at $1 per clam including shipping. Currently, th ere are no sources of clam s eed in Florida. Technology to produce clam seed in Florida would need to be developed for a more consistent supply of seed. This technology could be carried over from well-established bivalve hatcheries such as those used to produce the hard clam Mercenaria mercenaria A local source of seed may allow increased survival sin ce shipping is probably very stressful. Care must be taken so that no potential diseases are transferred over to the hard clam production facilities such as the Perkinsus strain found in some giant clams (Williams and Bunkley-Williams 1990). The nega tive impacts of culturing exotic species can be avoided by practicing the standard Be st Management Practices (BMPs) of the aquaculture industry. Another aspect that needs to be addressed is selection of a suitable substrate for growing out seed clams. The substrate should be more porous than ceramic tiles. Some

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64 of the currently used substrates for giant cl am production are concre te trays and basalt chips. These may not be as practical as othe r types of substrate th at can be acquired and handled with more ease such as plastic or fiberboard. Established marine ornamental producer s, who already have inland production tanks or systems in place, can add clams in for polyculture growout. This would lower economic risk since there is less start up costs. Corals may be grown in conjunction with these clams and may be beneficial for a s ource of differing strains of zooxanthellae. Lowering the price of clam seed to the prices that are paid for Mercenaria seed would allow for longer grow-out periods. More suitable substrate may also allow higher survival. Only if these two challenges are addressed, can the culture techniques utilized in this study become profitable for a sm all-scale giant clam production facility.

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65 APPENDIX A CLAM COLOR READINGS Table A-1. Individual color readings for Bl ue seed size clams. A* values are more negative with more green color and more positive with more red. B* values are more negative with more blue color and more positive with more yellow. Spectral peaks are also listed for the blue (400-500 nm), green (500-560 nm), and brown (yellow/orange) (560-650 nm ) regions of the color spectrum. Clam Color Readings Blues clam ID # t(0) t(1) t(2) t(3) Wavelength peak in blue region (nm) 112 440 415 442 437 Reflection (%) 112 2.9 1.4 8.3 3 Wavelength peak in green/brown region (nm) 112 573 605 573 573 Reflection (%) 112 3.2 0.8 7.4 1.8 B* 112 4.2 0.9 2 2.7 Wavelength peak in blue region (nm) 1111 433 441 435 425 Reflection (%) 1111 2.9 7.6 2.1 5.9 Wavelength peak in green/brown region (nm) 1111 605 605 605 573 Reflection (%) 1111 4.7 4.4 1.1 1.2 B* 1111 4.0 2 -1.2 0.5 Wavelength peak in blue region (nm) 715 442 443 437 439 Reflection (%) 715 4.6 5.2 5.4 5.8 Wavelength peak in green/brown region (nm) 715 573 573 573 573 Reflection (%) 715 5.9 4.8 1.1 2.1 B* 715 10.5 9.7 -4.9 3.7 Wavelength peak in blue region (nm) 522 443 442-469 439-500 dead Reflection (%) 522 4 3.5 2-2.3 dead Wavelength peak in green/brown region (nm) 522 573 605 573-605 dead Reflection (%) 522 5 4.8 2.4-2.5 dead B* 522 10.5 9.7 -4.9 dead Wavelength peak in blue region (nm) 8211 432 438 415 427 Reflection (%) 8211 2.7 6.8 10.3 3.9 Wavelength peak in green/brown region (nm) 8211 573 605 605 605 Reflection (%) 8211 1 5.8 2.9 1.8 B* 8211 1.6 0.5 -5.9 -0.4 Wavelength peak in blue region (nm) 9210 417 442 412 406 Reflection (%) 9210 2 4.5 5.3 3.82 Wavelength peak in green/brown region (nm) 9210 573 608 573 573 Reflection (%) 9210 2 2.9 1.6 1 B* 9210 1.3 3.8 -3.4 -0.1 Wavelength peak in blue region (nm) 1237 443 463 433 410 Reflection (%) 1237 1.6 2.3 4.2 1.6 Wavelength peak in green/brown region (nm) 1237 573 605 573 653

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66 Table A-1. Continued. Clam Color Readings Blues clam ID # t(0) t(1) t(2) t(3) Reflection (%) 1237 3.6 2 1 0.9 B* 1237 3.8 2.2 -0.3 2 Wavelength peak in blue region (nm) 823 400500 447 dead dead Reflection (%) 823 1.1 9.3 dead dead Wavelength peak in green/brown region (nm) 823 573605 605 dead dead Reflection (%) 823 1.8 8.2 dead dead B* 823 1.3 0.6 dead dead

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67 Table A-2. Individual color readings for Green seed size clams. A* values are more negative with more green color and more positive with more red. B* values are more negative with more blue color and more positive with more yellow. Spectral peaks are also listed for the blue (400-500 nm), green (500-560 nm), and brown (yellow/orange) (560-650 nm ) regions of the color spectrum. Clam Color Readings Greens clam ID # t(0) t(1) t(2) t(3) Wavelength peak in blue region (nm) 721 N/A N/A 455 400 Reflection (%) 721 N/A N/A 3.7 4 Wavelength peak in green/brown region (nm) 721 N/A N/A 573 573 Reflection (%) 721 N/A N/A 1.3 0.6 A* 721 N/A N/A -0.6 0 Wavelength peak in blue region (nm) 311 495 461 400 490 Reflection (%) 311 3.6 3.2 2.7 1.3 Wavelength peak in green/brown region (nm) 311 573 573 603 573 Reflection (%) 311 6.8 1.1 3 1 A* 311 -0.3 -0.9 1.1 0.3 Wavelength peak in blue region (nm) 7110 495 485 485 443 Reflection (%) 7110 4.1 3.2 2.6 1.3 Wavelength peak in green/brown region (nm) 7110 573 573 573 573 Reflection (%) 7110 5.5 3.9 4.3 1.6 A* 7110 -0.8 0.6 0.3 0 Wavelength peak in blue region (nm) 7212 490 495 475 dead Reflection (%) 7212 2.7 3.1 2.3 dead Wavelength peak in green/brown region (nm) 7212 573 573 500-600 dead Reflection (%) 7212 4.1 4 2.1 dead A* 7212 -0.5 -1.1 -0.4 dead

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68 Table A-3. Individual color readings for Gold seed size clams. A* values are more negative with more green color and more positive with more red. B* values are more negative with more blue color and more positive with more yellow. Spectral peaks are also listed for the blue (400-500 nm), green (500-560 nm), and brown (yellow/orange) (560-650 nm ) regions of the color spectrum. Clam Color Readings Golds clam ID # t(0) t(1) t(2) t(3) Wavelength peak in blue region (nm) 225 485 495 440 440 Reflection (%) 225 2.5 4.5 1.2 1.5 Wavelength peak in green/brown region (nm) 225 573 573 573 573 Reflection (%) 225 7.4 6.3 1.8 1.1 B* 225 9.3 10.4 2.3 0.7 Wavelength peak in blue region (nm) 1212 443 443 468 426 Reflection (%) 1212 2.5 8.3 4.5 2.5 Wavelength peak in green/brown region (nm) 1212 573 604 573 607 Reflection (%) 1212 3.5 9.7 6.3 0.9 B* 1212 8.8 12.5 3.3 4.1 Wavelength peak in blue region (nm) 3110 495 495 435 495 Reflection (%) 3110 1.3 2.5 2.1 1.5 Wavelength peak in green/brown region (nm) 3110 573 573 573 573 Reflection (%) 3110 5.2 3.7 0.9 2.7 B* 3110 9.1 11.6 1.8 5.2

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69 APPENDIX B WATER QUALITY Table B-1. Water Quality for Recirculati on System (mg/L except for TN = g/L). Date Week AmmoniumNH3 NO2 NO3 TN 04/01/2005 0 5 <0.6 ppm <0.15 <10 545 04/08/2005 1 5 <0.6 ppm <0.15 <10 04/15/2005 2 5 <0.6 ppm <0.15 <10 04/22/2005 3 40 <0.6 ppm <0.15 <10 04/29/2005 4 50 <0.6 ppm <0.15 <10 05/06/2005 5 60 <0.6 ppm <0.15 <10 2645 05/13/2005 6 55 <0.6 ppm <0.15 <10 05/20/2005 7 60 <0.6 ppm <0.15 <10 05/27/2005 8 60 <0.6 ppm 1.5 <10 06/03/2005 9 20 <0.6 ppm 0.75 <10 2750 06/10/2005 10 10 <0.6 ppm 0.6 <10 06/17/2005 11 5 <0.6 ppm 0.45 <10 06/24/2005 12 10 <0.6 ppm 0.3 <10 06/27/2005 13 10 <0.6 ppm <.15 <10 1690

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70 Table B-2. Water Quality for Recirculati on System (mg/L except for TP = g/L). Date Week CA test kits CA ICP Alkalinity TP salinity ppt 04/01/2005 0 438 370 171 13 35 04/08/2005 1 438 171 35 04/15/2005 2 412 171 35 04/22/2005 3 400 171 35 04/29/2005 4 420 171 35 05/06/2005 5 420 400 188 13 35 05/13/2005 6 420 205 35 05/20/2005 7 450 205 35 05/27/2005 8 475 222 35 06/03/2005 9 420 400 205 9 35 06/10/2005 10 400 171 35 06/17/2005 11 400 205 35 06/24/2005 12 420 188 35 06/27/2005 13 420 400 205 5 35

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71 Table B-3. Mean Water Quality for Roll overs (mg/L except for TN = g/L). Date Week AmmoniumNH3 NO2 NO3 TN 04/01/2005 0 5 <0.6 ppm <0.15<10 559 04/08/2005 1 5 <0.6 ppm <0.15<10 04/15/2005 2 5 <0.6 ppm <0.15<10 04/22/2005 3 55 <0.6 ppm <0.15<10 04/29/2005 4 45 <0.6 ppm <0.15<10 05/06/2005 5 45 <0.6 ppm <0.15<10 1883 05/13/2005 6 55 <0.6 ppm <0.15<10 05/20/2005 7 45 <0.6 ppm <0.15<10 05/27/2005 8 40 <0.6 ppm <0.15<10 06/03/2005 9 40 <0.6 ppm <0.15<10 1387 06/10/2005 10 40 <0.6 ppm <0.15<10 06/17/2005 11 50 <0.6 ppm <0.15<10 06/24/2005 12 45 <0.6 ppm <0.15<10 06/27/2005 13 50 <0.6 ppm <0.15<10 1688

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72 Table B-4. Mean Water Quality for Roll overs (mg/L except for TP = g/L). Date Week CA test kits CA ICP AlkalinitySalinity ppt TP 04/01/2005 0 423 370 171 35 11 04/08/2005 1 417 171 35 04/15/2005 2 422 171 35 04/22/2005 3 417 188 35 04/29/2005 4 407 197 35 05/06/2005 5 407 400 200 35 13 05/13/2005 6 413 202 35 05/20/2005 7 412 202 35 05/27/2005 8 407 211 35 06/03/2005 9 412 410 214 35 12 06/10/2005 10 413 211 35 06/17/2005 11 412 208 35 06/24/2005 12 423 217 35 06/27/2005 13 422 420 217 35 9

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73 Table B-5. Mean, max, and min water temperat ures for each recircul ation treatment tank for 90-day study. Recirculation Temperatures (C) Tank mean max min 1 26.3 29.4 23.5 2 26.2 29.5 23.5 3 26.3 29.6 23.6 6 26.3 30.4 23.6 7 26.2 30.2 23.6 9 26.0 29.3 23.4

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74 Table B-6. Mean, max, and min water temperatures for each rollover treatment tank for 90-day study. Rollover Temperatures (C) Tank mean max min 4 28.3 34.7 23.5 5 27.1 33.2 22.8 8 28.1 34.3 23.5 10 27.3 33.2 22.8 11 27.3 33.1 22.6 12 27.6 33.7 22.6

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75 25 26 27 28 29 30 31 32 12345678910111213 WeekTemperature (C) Figure B-1. Mean weekly temperatures for tw o culture systems. Rollovers are marked by the solid line. Recirculation ta nks are marked by the dashed line.

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76 LIST OF REFERENCES Bell, J. D. 1999. New species for coasta l aquaculture in the tropical Pacific – constraints, prospects and considerati ons. Aquaculture International 7:207-223. Braley, R. D., D. Sutton, S. S. M. Mi ngoa, and P. C. Southgate. 1992. Passive greenhouse heating, recirculation, and nutrient addition for nursery phase Tridacna gigas : growth boost during winter months. Aquaculture 108:29-50. Buck, B. H., H. Rosenthal, and U. Saint-Paul 2002. Effect of increased irradiance and thermal stress on the symbiosis of Symbiodinium microadriaticum and Tridacna gigas Aquatic Living Resources 15:107-117. Calfo, A. R. 2001. Book of coral propa gation. Volume One. Reading Trees, Monroeville, PA. Carlos, A. A., B. K. Baillie, and T. Maruyama. 2000. Diversity of dinoflagellate symbionts (zooxanthellae) in a host indivi dual. Marine Ecology Progress Series 195:93-100. Cheshire, C., and S. Valeriano. 2004. Live clam market in the United States. Pacific Business Center Program, Sea Grant, University of Hawaii, Honolulu, HI. Ellis, S. 2000. Nursery and grow-out techni ques for giant clams (Bivalvia: Tridacnidae). Center for Tropical and Subtropical A quaculture Publication No. 143. Waimanalo, HI. Epifanio, C. E., and R. F. Srna. 1975. Toxic ity of ammonia, nitrite ion, nitrate ion, and orthophosphate to Mercenaria mercenaria and Crassostrea virginica Marine Biology 33:241-246. Estacion, J., E. Solis, and L. Fabro. 1986. A preliminary study of the effect of supplementary feeding on the growth of Tridacna maxima (Roding) (Bivalvia: Tridacnidae). Silliman Journal 33:111-116. Fisher, C. R., W. K. Fitt, and R. K. Tren ch. 1985. Photosynthesis and respiration in Tridacna gigas as a function of irradiance and size. Biological Bulletin 169:230245. Fitt, W. K., C. R. Fisher, and R. K. Trenc h. 1984. Larval biology of Tridacnid clams. Aquaculture 39:181-195.

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77 Fitt, W. K., and M. E. Warner. 1993. Bleach ing patterns of four species of Caribbean reef corals. Biologi cal Bulletin 189:298-307. Grice, A. M., and J. D. Bell. 1999. Applicat ion of ammonium to e nhance the growth of giant clams ( Tridacna maxima ) in the land-based nursery: effects of size class, stocking density and nutrient co ncentration. Aquaculture 170:17-28. Griffiths, D. J., H. Winsor, and T. Luong-Va n. 1992. Iridiphores in the mantle of giant clams. Australian Journal of Zoology 40:319-326. Gross, M. G. 1977. Oceanography: A view of the earth. Prentice Hall, Inc., Englewood Cliffs, NJ. Hart, A. M., J. D. Bell, and T. P. Foyle. 1998. Growth and survival of the giant clams, Tridacna derasa, T. maxima and T. crocea at village farms in the Solomon Islands. Aquaculture 165:203-220. Ishikura, M., C. Kato, and T. Maruya ma. 1997. UV-absorbing substances in zooxanthellate and azooxanthellate clams. Marine Biology 128:649-655. Jerlov, N. G. 1976. Marine optics. Elsevi er Scientific Publishing., Co., Amsterdam ; New York, NY. Jokiel, P. L., and R. H. York. 1984. Importance of ultrav iolet radiation in photoinhibition of microalgal growth Limnology and Oceanography 29:192-199. Kinzie, R. A., P. L. Jokiel, and R. York. 1984. Effects of light of altered spectral composition on coral zooxanthell ae. Marine Biology 78:239-248. Knop, D. 1996. Giant clams: A comprehensiv e guide to the identification and care of Tridacnid clams. Dahne Verlag GmbH, Ettlingen, Germany. Lapointe, B. E., M. M. Littler, and D. S. Littler. 1993. Modification of benthic community structure by natural eutrophicati on: the Belize barrier reef. Proceedings of the Seventh International Cora l Reefs Symposium, Guam, 1992. 1:323-334. Lawlor, D. W. 2001. Photosynthesis. Sp ringer-Verlag New York, Inc., New York, NY. Lucas, J. S. 1994. The biology, exploita tion, and mariculture of giant clams (Tridacnidae). Reviews in Fisheries Science 2:181-223. Lucas, J. S., W. J. Nash, C. M. Crawford, and R. D. Braley. 1989. Environmental influences on growth and survival duri ng ocean-nursery rearing of giant clams, Tridacna gigas Aquaculture 80:45-61. Mingoa-Licuanan, S. S. 1993. Oxygen consump tion and ammonia excretion in juvenile Tridacna gigas (Linne, 1758): effects of emersion. Journal of Experimental Marine Biology and Ecology 171:119-137.

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78 Ott R. L., and M. Longnecker. 2001. An in troduction to statistical methods and data analysis. Wadsworth Group. Pacific Grove, CA. Ralph, P. J., R. Gademann, A. W. D. La rkum, and U. Schreiber. 1999. In situ underwater measurements of photosynthetic activity of coral zooxanthellae and other reef dwelling dinoflag ellate endosymbionts. Marine Ecology Progress Series 180:139-147. Sparsis, M., J. Lin, and R. W. Hagood. 2001. Growth, survivorship, and nutrient uptake of giant clams (Tridacna) in aquaculture effluent. Journal of Shellfish Research 20:171-176. Tomascik, T. 1997. The ecology of the Indonesi an Seas Part One. Periplus Editions, Hong Kong. Williams, E. H., and L. Bunkley-Williams. 1990. Helpline for giant clams. Nature 345:119.

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79 BIOGRAPHICAL SKETCH Micah Alo was born in Manila, the capitol of the Philippines, in 1977. His family soon immigrated to the United States in 1984 and settled in Plains boro, New Jersey, for his formative school years. Numerous family vacations to the Florida Keys had ignited a passion for aquatic biology which drove Micah to receive his AA degree at the Florida Keys Community College. From there he we nt on to finish his bachelor’s degree in marine biology at Florida Atlantic Univers ity in Boca Raton, Florida, earning his degree cum laude and being initiated into Phi Thet a Kappa in 2000. He then worked at the University of Florida’s Tropical Aquaculture Laboratory and the Whitney Laboratory for Marine Bioscience for 4 years. Afterwards, he elected to pursue a Master of Science degree in the Universi ty of Florida’s Department of Fisheries and Aquatic Sciences, which he hopes will help him to pursue a career in the field of aquaculture.


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Permanent Link: http://ufdc.ufl.edu/UFE0012200/00001

Material Information

Title: Survivorship, Growth and Pigmentation Responses of the Marine Ornamental Invertebrate Tridacna maxima to Varied Irradiance Levels in Two Different Culture Systems
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0012200:00001

Permanent Link: http://ufdc.ufl.edu/UFE0012200/00001

Material Information

Title: Survivorship, Growth and Pigmentation Responses of the Marine Ornamental Invertebrate Tridacna maxima to Varied Irradiance Levels in Two Different Culture Systems
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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SURVIVORSHIP, GROWTH AND PIGMENTATION RESPONSES OF THE
MARINE ORNAMENTAL INVERTEBRATE Tridacna maxima TO VARIED
IRRADIANCE LEVELS IN TWO DIFFERENT CULTURE SYSTEMS















By

MICAH ALO


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


2005

































Copyright 2005

by

Micah Alo

































This document is dedicated to the friends and supporters of the marine ornamental hobby.















ACKNOWLEDGMENTS

This thesis would not have been possible without the help of many individuals.

First, I thank all my committee members Dr. Charles Cichra, Dr. Shirley Baker, Dr. Daryl

Parkyn and Dr. John Baldwin, for their encouragement and much needed enlightenment.

This project was made possible with funding provided by the USDA CS-REES

Special Research Grants and funding from the University of Florida's College of

Agricultural and Life Sciences and Department of Fisheries and Aquatic Sciences.

I owe a lot of thanks to the staff and students of the University of Florida's Tropical

Aquaculture Lab. I thank Craig Watson for his belief in the advancement of aquaculture

and giving this project its direction. Scott Graves, Robert Leonard, and Tina Crosby all

deserve credit for building the greenhouse and systems. Fellow graduate student and

tinkerer Jon Kao was instrumental for aiding in tank design and construction, ammonia

readings; and most other problems that I encountered during my study. Also, I need to

thank Dr. Jeff Hill for his statistical analysis and Dr. Roy Yanong for clam disease

diagnostics and all the mentoring they bestowed upon me. I am indebted to the late Jana

Col and Dr. Ramon Littell from the Institute of Food and Agricultural Science's Statistics

Department for putting together the statistical model for my experiment.

Many thanks go to Oceans Reefs and Aquariums who showed much generosity for

providing the seed clams at a reduced cost. I am grateful to Florida Marine Aquaculture

for the helpful advice it provided in setting up my culture systems. Thanks go to the









Florida Aquarium for allowing me to have water samples analyzed by their ion-coupled

plasma spectrophotometer.

I thank Dave Watson and Rebecca Varner, of Florida Lakewatch, for the excellent

job they did in making my metal halide light maps. Great thanks go to the Florida

Lakewatch water chemistry lab for its thorough analysis of water samples. I would like

to thank the Ruskin, Florida, NOAA weather station for providing weather data.

I thank Dr. Pam Muller from USF for graciously lending me her LICOR light meter

when mine was out of commission. To John Lucas, I am grateful for his knowledge and

generous advice that he supplied on giant clams. I thank Mr. Gary Townsend for

providing the US giant clam import data. I would like to thank C.L. Cheshire for his

valuable information on giant clam economics. I am thankful to Dr. Charles Adams for

sharing with me the economics viewpoint in aquaculture.

Lastly, I need to thank my family and girlfriend, Amy Singivipulya, for their never

ending support and love that led me to accomplish all that I have done in life.
















TABLE OF CONTENTS

page

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

LIST OF TABLES .............. ..... ................ ...... ................ .. viii

LIST OF FIGURES ................................. .. ... .. ..................x

ABSTRACT ........ .............. ............. ........ .......... .......... xii

CHAPTER

1 IN TR O D U C T IO N ............................................................. .. ......... ...... .....

B ack g rou n d ...................................... .............................. ... .......... ....... .
B io lo g y ................................................................. 1
Inland Production.............................................. 4
O objectives .............. .......................................... .... ... ....... .. ...... ..

2 M E TH O D S .................................................................6

Inland C culture System s .............................................................. ...................
N utrient A additions ....................... ........................ .......... .......7.
W after Q u ality ................................................ 8
C la m s ....................................................... 9
L ig h t T re atm en ts .................................................................................................... 10
D u ration an d S am pling ................................................................................. 12
A cclim ation P eriod ........................................................................ 12
S tu d y P erio d ................................................................12
L eight M easurem ents .................................................................................... .......12
G row th M easurem ents ........................................................................................ 13
C olor M easu rem ents ....................................................................................... 13
S statistic al A n aly sis ................................................................................................. 1 5

3 R E S U L T S .............................................................................2 2

S u rv iv a l ......................................................................................................2 2
S h e ll G ro w th .......................................................................................................... 2 3
W et W e ig h t ............................................................................................................ 2 3
C o lo r ...................................................................................................................... 2 4









Reference Clams....................... ..................... ......... 24
Survivors ................................... ............................... ........ 25
G reen Sw itch to G old (Y ellow )......................................... ........................ 25
Light M easurem ents ...................................................... ........... 26
S o la r ................................................................................................................ 2 6
M etal H alid e ................................................................2 7
T em perature Profiles ........................ .................. ................. .. ....... ..28
W after Q uality................................................. 29

4 D ISC U S SIO N ............................................................................... 47

Maj or Findings............... .....................................................47
Low Survival and G row th ............................................... ............... 47
Color A analysis ...................... ........................ .... .. ........ .......... 52
N u trie n ts ...........................................................................................................5 4

5 MANAGEMENT APPLICATIONS ........................................ ........................ 63

APPENDIX

A CLAM COLOR READINGS ............................................................................. 65

B W A T E R Q U A L IT Y .......................................................................... ....................69

L IST O F R E FE R E N C E S ............................................................................. .............. 76

B IO G R A PH IC A L SK E TCH ...................................................................... ..................79















LIST OF TABLES


Table pge

3-1 Two-way analysis of variance (ANOVA) for survival at day 90 of T. maxima
seed clams among two culture systems and three light treatments........................40

3-2 x2 analysis of size class survival of T maxima seed clams over 90-day study. X2
calculated = 8.07, df = 3, a = 0.05, X2 critical = 7.815 (Ott 2001)...........................41

3-3 Split-plot repeated measures analysis of variance (ANOVA) results for shell
length data of T maxima seed clams among two culture systems and three light
treatm ents ov er 90 day s ............................................................................. ..... 42

3-4 Treatment mean shell growth (percent) of T maxima seed clams during 90-day
study. Standard error (S.E.) is included (n = 6).................................................... 43

3-5 Split-plot repeated measures analysis of variance (ANOVA) for weight data of
T. maxima seed clams among two culture systems and three light treatments
over 90 days. .........................................................................44

3-6 Ninety-day mean wet weight gain and mean percent weight gain, correcting for
biofouling (algae) weight, for T. maxima clams. ......................... ..................45

3-7 Mean halide light treatment intensity readings (tmol/m2s) for all 12 tanks
com bined. .............................................................................46

4-1 Mean L*a*b* values for open and shade treatment clams measured for color .......61

4-2 Mean L*a*b* values for three categories of color measured clams: blue; green;
and gold clam s .................................................. ................. 62

A-i Individual color readings for Blue seed size clams .............................................65

A-2 Individual color readings for Green seed size clams.................................... 67

A-3 Individual color readings for Gold seed size clams.. ............................................ 68

B-l W ater Quality for Recirculation System. ...................................... ............... 69

B-2 W ater Quality for Recirculation System. ...................................... ............... 70

B-3 M ean W ater Quality for Rollovers......... ............................. .................. 71









B-4 M ean W ater Quality for Rollovers............ ....... .......................... ............... 72

B-5 Mean, max, and min water temperatures for each recirculation treatment tank for
90-day study .........................................................................73

B-6 Mean, max, and min water temperatures for each rollover treatment tank for 90-
d ay stu dy ......................................................... ................ 74
















LIST OF FIGURES


Figure page

2-1 G greenhouse layout .................. ........................................ .. ............ 16

2-2 R ollover tank. .................................................................17

2-3 Seed giant clam (Tridacna maxima) attached using cyanoacrylate glue to a Floy
T -bar tag. ................................................................... 18

2-4 Light treatments seen above in two experimental tanks........................................19

2-5 Clam length longest stretch across shell. ................................... ............... 20

2-6 Color measurement tank and enlargement of spectrophotometer probe and clam
position .................. .......... .... ............ .. ........... ........... 21

3-1 Mean treatment survival after 30, 60 and 90 days (n=6) for all six treatment
types. .................................................................. 3 1

3-2 Survival of clams with initial sizes of 6 12
3-3 Spectral reflectance (%) for a 5-cm T. maxima clam (dark blue) measured on the
third sam ple period .............................................. .. ... ... .. ........ .... 33

3-4 Spectral reflectance (%) for a 5-cm T. maxima clam (light blue) measured on the
third sam ple period ............ .............................................................. ...... ..... .. 34

3-5 Spectral reflectance of one of the clams added for color sampling for the third
sample period (clam 721 from the shade x recirculation system)............................35

3-6 Mean weekly peak PAR. Calculated from daily peak PAR readings taken
outside of the greenhouse. ..................................... ........ .. .... ............... 36

3-7 Percent possible sunshine. Percentage of daytime during which the direct solar
radiation exceeds the level set by NOAA. .................................... .................37

3-8 Spectral power distribution for all three light treatments at midday........................38

3-9 Light maps in imol/m2s for halide treatment in Tank 1 during week one, five,
nine, and 13. ...........................................................................39









4-1 Daily solar profile taken on day 69 for tank 4 open sun treatment shown in
dashed line. Mean metal halide intensity, for all 4 sample periods shown in
solid line. ............................................................................59

4-2 This graph displays spectral shift from day one to day 90 for one of the blue
clam seed chosen for color measurements (number 715 from the open x
recirculation treatm ent). ................................................ ............................... 60

B-l Mean weekly temperatures for two culture systems. Rollovers are marked by
the solid line. Recirculation tanks are marked by the dashed line ........................75















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

SURVIVORSHIP, GROWTH AND PIGMENTATION RESPONSES OF THE
MARINE ORNAMENTAL INVERTEBRATE Tridacna Maxima TO VARIED
IRRADIANCE LEVELS IN TWO DIFFERENT CULTURE SYSTEMS

By

Micah Alo

December, 2005

Chair: Charles Cichra
Cochair: Shirley Baker
Major Department: Fisheries and Aquatic Sciences

The focus of this study was to determine how two environmental variables

irradiancee and spectrum) affect inland production of the giant clam Tridacna maxima for

the ornamental trade. This study also evaluated two types of land-based closed-system

designs, recirculation and rollover systems.

Growth of the seed clams was suboptimal, relative to previous studies, in all

treatments with a mean of 5% growth in 90 days. Clams grown under metal halide

lighting were found to have a mean growth of 3.3% ( 1.0% S.E.) and a significantly (F 2,

30 = 9.04, P = 0.0008) lower mean survival of 28.5% ( 6.7% S.E.) compared to the two

other light treatments (open and shaded). This was most likely due to a spike at 365 nm

of ultraviolet radiation emitted by the halide lamps. The recirculation culture system had

a significantly (F 1,30 = 17.56, P = 0.0002) higher mean survival of 55.5% ( 5.1% S.E.)

compared to the rollover system of 30.9% ( 4.4% S.E.). Mean shell length did not have









a significant difference between the recirculation system, that had a mean growth of 7.3%

( 0.7% S.E.), and the rollover system, that had a mean growth of 2.7% ( 0.7% S.E.).

Mean weight for system type was significant (F1,io = 10.31, P = 0.0093), with the

recirculation system showing a mean weight gain of 18.3 3.3% S.E. vs. 2.1 2.9% S.E.

for the rollover system. The difference was attributed to the effect of the near upper

lethal temperatures (34C) reached in the rollover systems. Only one clam was recorded

to change mantle color, from green to yellow, in the shade recirculation treatment.

Qualitative trends of increased darkness and blueness in mantle tissue were recorded in

the L* and b* values of clams in the open and shade treatments over time.

The time required to grow seed clams (10-30 mm) to 40-50 mm shell length, a size

suitable for sale to the aquarium market, using the optimum conditions in this study was

calculated to be 5-7 years (mean growth of 9.5% ( 1.0% S.E.) in the open recirculation

treatment; mean survival of 66.2% ( 6.7% S.E.) in the shaded recirculation culture

treatment). It is unlikely that giant clam culture under these conditions would be

economically feasible for inland production, unless seed supply became more abundant

and less costly.














CHAPTER 1
INTRODUCTION

Background

Giant clams were put into aquaculture production for the Asian food market in the

early 1980s to prevent overharvest of wild stock. Recent aquaculture production has

been refocused towards the ornamental market because of the shorter growout period to

market size and vibrant coloration (Ellis 2000). Giant clams (Tridacna spp.) are now a

growing segment of the marine ornamental industry, experiencing a 9% annual growth

during 1993-2002 (Cheshire and Valeriano 2004).

Biology

All nine species of giant clams harbor the photosynthetic symbionts Symbiodinium

microadriaticum within their mantle tissue (Knop 1996). These symbiotic dinoflagellates

(i.e., zooxanthellae) supply the clams with all of their metabolic carbon requirements in

exchange for waste products in the form of ammonia (Fisher et al. 1985). If the

zooxanthellae are provided with enough light, the clams can survive in the same low

nutrient environments as hermatypic corals.

Giant clam populations are limited to certain shallow water depths due to the

physics of light attenuation and availability of photosynthetically active radiation (PAR),

the portion of the electromagnetic spectrum between 400-700 nm. In tropical latitudes,

PAR at the sea surface peaks around 2500 imol/m2s (Tomascik 1997). Many giant

clams are found intertidally and are able to tolerate full surface irradiance (Mingoa-

Licuanan 1993). Tridacna gigas, for example, is capable of photosynthesis while fully









emersed during low tides (Mingoa-Licuanan 1993). Values of PAR attenuates by 60%

after the first meter of depth and by 80% after the first 10 meters of depth in Type I, the

clearest, oceanic waters (Gross 1977, Jerlov 1976). This rapid decrease in light intensity

is the main factor that dictates the depths at which autotrophic animals, such as giant

clams, can live.

Studies have shown that light tolerance levels are dependant upon clam size. A

respirometry study of T. gigas indicates that, for large juveniles (>10 cm), the light

saturation point (light level at which no further increase has an effect on photosynthesis

rates) was reached at PAR levels over 2000 imol/m2s (Fisher et al. 1985). Saturation

levels decreased with decreasing clam size; some 10-mm clams reached saturation at 500

[mol/m2s. However, another study (Lucas et al. 1989) found that 40-mm (1 year old) T.

gigas, were unable to survive when exposed to 298 rmol/m2s using 90% shadecloth. The

need for stronger light in larger clams is attributed to the self-shading properties of the

mantle tissue where the symbiotic algae are harbored (Fisher et al. 1985). More layers of

zooxanthellae are contained in the mantle tissue of larger clams. This produces a canopy

shading effect as light passes through the outer zooxanthellae layers, shading the lower

layers.

Spectral distribution also varies as light is transmitted through water. In Type I

waters, the blue end (300-500 nm) of the electromagnetic spectrum drops off in intensity

at a slower rate than the red (>600 nm) portion of the spectrum (Jerlov 1976). Kinzie et

al. (1984) using acrylic light filters found that corals, grown under blue (400-500 nm) and

white light (400-700 nm) of the same intensities (250 imol/m2s), grew at a faster rate

than those under green (500-600 nm) or red light (600-700 nm) of similar intensities.









Therefore, blue light may facilitate production of popular marine ornamentals such as

corals and giant clams.

Clam coloration in shallow depths is hypothesized to be an adaptation by the clams

to shield themselves from harmful UV rays (Griffiths et al. 1992). The cells responsible

for the bright colors found in some clams are called iridocytes and reflect the blue portion

of the spectrum including UV-A (320-400 nm) and UV-B (280-320 nm) (Griffiths et al.

1992). These UV rays can penetrate shallow marine waters in tropical zones where the

ozone layer is thinner than in temperate zones (Ishikura et al. 1997). While zooxanthellae

outside of the clam cannot photosynthesize with UV-B wavelengths present,

zooxanthellae residing within clam tissues are shielded by mycosporine-like amino acids

(Ishikura et al. 1997). Regular exposures to these wavelengths may have an effect on the

photosynthetic capabilities and coloration of tridacnids.

The maximum prolonged (6-week) temperature threshold for giant clams is 32C,

above which they expel their zooxanthellae and eventually starve to death (Buck et al.

2002). If temperatures can be maintained below this level, stronger light intensities may

improve overall growth and coloration of T maxima. However, smaller clams may not

require as much light and may even suffer photoinhibition (i.e., the point when increased

light intensity causes damage to the photosynthetic system)(Lawlor 2001). This did not

seem to be the case for a clam farmer in Kiribati who grew out T maxima seed clams (up

to 3 cm) in shallow tanks with full sunlight exposure (personal communication, Craig

Watson, University of Florida).

Most giant clam farms rely on ocean cages and natural sunlight for the majority of

growout needs (Ellis 2000). Under these culture conditions, clams are highly vulnerable









to predation by gastropods Cymatium spp. and wrasses Thalassoma spp. (Hart et al.

1998). One study in Australia tested the feasibility of greenhouse recirculation system

production of juvenile T. gigas clams (Braley et al. 1992). It showed that young clams,

held in greenhouse recirculation systems, grew significantly faster and had higher

survival than clams held in outdoor flow-through tanks. This was attributed to the ability

of greenhouse systems to maintain a 5-7C higher temperature during winter months

within greenhouse systems.

Inland Production

Greenhouse production of marine ornamentals can be an economically viable

business (Calfo 2001). Demands for more colorful, smaller varieties of Tridacna

continue to grow in the aquarium trade (Cheshire and Valeriano 2004). The cost of

shipping and the Convention on International Trade in Endangered Species (CITES)

certification has increased imported giant clam prices (Bell 1999). Giant clams are listed

under Appendix II of CITES. This requires export of giant clams to have a permit issued

stating "1) The export will not be detrimental to the survival of the the species, 2) The

species was not obtained in contravention to the laws of the exporting State 3) The

method of export of living species will minimize the risk of injury, damage to health or

cruel treatment" (Ellis 2000). Member countries of CITES have to comply with these

regulations and ensure that nonmember countries comply with any product, such as giant

clams, imported or shipped through a CITES member country. Failure to include the

proper permits and documents leads to impoundment or confiscation of the shipment.

Currently, average wholesale prices in US dollars are at $10-16, depending on coloration,

with larger clams commanding higher prices (Cheshire and Valeriano 2004). This

developing market could make inland production of candidate marine ornamental









species, like giant clams, viable in Florida where freshwater tropical ornamental fish

production already has an established infrastructure. However, supplemental artificial

light sources may be necessary for light dependent invertebrates such as giant clams and

coral to maintain adequate growth and desirable coloration.

Objectives

The first objective of this study is to compare the effects of a blue spectrum

artificial metal halide light relative to two levels of intensity from natural sunlight on the

growth, coloration, and survival of giant clam seed. The second objective is to determine

effectiveness of two types of closed tank systems for the growout of giant clam seed.














CHAPTER 2
METHODS

Inland Culture Systems

This study was conducted in a 9 x 22 m greenhouse, at the University of Florida's

Tropical Aquaculture Laboratory located in Ruskin, Florida (GPS coordinates 27.71 N

latitude, 82.40 0 W longitude) (Figure 2-1). The greenhouse was equipped with an

evaporative cooler (American Coolair, Jacksonville, FL) with a 1.5 m high x 8 m wide

cooling pad on one end and two 1-m wide fans on the other end. The greenhouse also

had a 225,000-BTU propane heater (Modine, Buena Vista, VA). An inflated double

layer of plastic, (Sun Selector Type UVA clear, Ginegar Plastics, Israel), covered the

greenhouse. The two layers of plastic were each 150 [im thick and transmitted 90% PAR

and 20-25% UV-A and UV-B (Ginegar Plastics).

Twelve rectangular polypropylene tanks, (267 cm long x 147 cm wide x 56 cm

deep) holding approximately 2,250 L each, were used as inland saltwater culture systems.

The tanks were operational by July 2004. All 12 tanks had a rollover system design

(Figure 2-2) with eight 50 cm deep x 5 cm wide polyvinyl chloride (pvc) airlifts located

on one end of each tank to push water across the surface then down and back in a

constant loop. Base rock (limestone pieces ranging from 3-15 kg) was positioned along

the inside corners of the tank as substrate for biological filtration.

Of the 12 tanks, six were randomly selected and connected to the same sump tank

of similar dimensions to form a recirculation system with a total volume of

approximately 16,000 L. This system contained a 3/4-hp Jacuzzi Magnum Force pump









(Tipp City, OH), a 65-W UV sterilizer (Pentair Aquatics, El Monte, CA), a 152 cm tall x

12.5 cm wide venturi-driven protein skimmer (Top Fathom, Hudsonville, MI), an 85-L

pneumatic drop bead filter (Polygeyser, Aquaculture Systems Technologies, New

Orleans, LA), and a 5-hp chiller (Aqualogic inc., San Diego, CA). The remaining six

tanks were run separately as individual rollover systems.

Artificial saltwater was made from Crystal Seas Salt Mix (Marine Enterprises

International, Baltimore, MD) and reverse osmosis well water. Two 10% water changes

(1,600 L) on the recirculation system were made on days 53 and 57 with Instant Ocean

salt mix (Spectrum Brands, Atlanta, GA) due to elevated (1.5 mg/L) nitrite levels. Tank

water in the rollover systems was not changed during the 90-day study. A salinity of 35

ppt was maintained in both system treatments with daily addition of reverse osmosis

water.

Nutrient Additions

Ammonium chloride was administered to each tank twice a week and spiked to

concentrations of 50 itM. This addition is standard protocol to optimize growth for

production of giant clams by supplementing nitrogen-limited zooxanthellae with

ammonium chloride (Grice and Bell 1999). Rollover tanks were given 1-3 g of

ammonium chloride for each dose and recirculating system tanks were given 3-5 g for

each dose. Ammonia readings were taken using an Orion ammonia electrode (Beverly,

MA) with an Orion EA 940 ion analyzer. On days 53 to 90, recirculation system

ammonia was spiked to only 10tM due to increased nitrite concentrations. Weekly

supplements of 1 g of yeast (Fleishmann's Instant Yeast, Fenton, MO) were added to

each tank as a source of dissolved organic nutrients (Fitt et al. 1984). Algae supplements,

of 1 g dried spirulina, were also added weekly (Spirulina powder, Florida Tropical Fish









Farm Association Store, Apollo Beach, FL) to each tank. Yeast and spirulina were

heated and mixed in 1 L of reverse osomosis water, then spread out evenly into the tanks.

Water Quality

Weekly monitoring of ammonia, nitrite, nitrate, and alkalinity was conducted using

the HACH (Loveland, CO) Saltwater Master test kits. Calcium hardness was measured

using either a Seachem (Madison, GA) or LaMotte (Chestertown, MD) test kit for the

first 30 days. These two tests had high variability, so tests were continued with a Salifert

(Duiven, Holland) calcium test kit. Calcium hydroxide (Hydrated Lime, Chemical Lime,

Brooksville, FL) was added as needed to increase calcium concentrations to levels above

400 mg/L. Calcium solutions were prepared by mixing 6 g of calcium hydroxide with

100 mL of acetic acid (Rogers White Distilled Vinegar 5% acidity, Speaco Foods,

Kansas City, MO) in 4 L of reverse osmosis water. The calcium solution was added to

the tanks after 1700 hours. Temperature was logged every half hour in all 12 tanks using

a YSI model 600 XLM (Yellow Springs, OH) sonde.

Once per month, water samples from one randomly selected rollover tank and one

recirculation system tank were also analyzed by Severin Trent Laboratories (Tampa, FL)

with an inductively-coupled plasma (ICP) mass spectrometer to determine calcium,

magnesium, strontium, and molybdenum concentrations (mg/L). In addition, monthly

water samples from the recirculation system water and all six rollover tanks were

analyzed by the Florida Lakewatch Water Chemistry Laboratory (University of Florida,

Department of Fisheries and Aquatic Sciences, Gainesville, FL) with a spectrophotometer

for total nitrogen and total phosphorus. A portable 25-W UV sterilizer (Pentair Aquatics)

was used on an "as needed" basis to reduce algae blooms inside the rollover tanks. Any

excess foam build up was removed manually using fine mesh nets.









Clams

Three hundred ninety six ten-month old seed (7-14 mm) clams were obtained from

a giant clam farmer in the South Pacific (ORA, Marshall Islands). They were shipped as

airline freight cargo with 40 plus hours of travel time. The clams arrived at Tampa

International Airport in cooler boxes on February 17, 2005. Each shipping box contained

12 plastic bags (15 cm) with eight to 10 clams each with no form of cooling inside (e.g.,

ice pack). Each bag was filled halfway with seawater at a salinity of 35 ppt and a pH of

7.8. Upon receipt, clams were acclimated by floating the bags until temperatures were

equalized. Then, the bags were emptied into two 75-L bins sitting next to the

recirculation system sump. A siphon hose slowly fed water into the bins from the

recirculation system's sump for further acclimation to water quality parameters. Once

acclimated to the system water, the clams were transferred into six trays partially

submerged inside the sump of the recirculation system with water flowing into each

individual tray.

The Marshall Islands clam farm kept the clams under 73% shadecloth and were

slowly acclimated to ambient greenhouse light levels. This was replicated by using two

layers of 30% shadecloth in addition to the greenhouse plastic (25% shade) to cover the

trays in the sump for nine days after arrival. Light intensity in PAR was measured using

a LICOR (Lincoln, NE) LI-1000 datalogger and underwater quantum sensor LI-192SA.

PAR irradiance was measured in imol/m2s. Without the shadecloth, light levels in the

sump trays holding the clams reached levels up to 1500 imol/m2s between 1200-1300

hours. This was 500 imol/m2s below the readings found outside of the greenhouse in

full sunlight during the same time. Each layer of shadecloth lowered midday readings by

-400 imol/m2s. One layer of shadecloth was used for an additional nine days. The









second layer of shadecloth was removed (no shadecloth) for the final nine days, exposing

the clams to full sun within the greenhouse.

After clams were acclimated to greenhouse light levels (March 16, 2005), they

were mounted on top of 2.5-cm square ceramic tiles. A 3-mm cone-shaped dimple was

drilled into the center of each tile to cradle the umbo portion of the clam shell. A T-bar

fish tag (Floy tags, Seattle, WA) was used to fasten each clam to its tile (Figure 2-3).

This was done by applying Loctite and Superglue brand cyanoacrylate (Henkel, Avon,

OH) to each end of the tag and attaching the tag to both the valve of the clam and the

edge of the tile. Care was taken not to glue too close to the shell opening to prevent

mantle tissue irritation.

Light Treatments

Three light treatments were used within each tank to produce varying irradiance

and spectral levels (Figure 2-4). Light treatments included the following: 1.) sunlight

within greenhouse (double layer of 150-pm plastic), 2.) sunlight within greenhouse and

30% shadecloth (this was changed to 55% on day 48 due to increasing light levels), and

3.) sunlight within greenhouse in conjunction with artificial, metal halide lighting set on a

12-hour photoperiod (0700 to 1900 hours both before and after daylight savings time

change occurred on day 3). The metal halide lights had 400-W 10,000-K mogul bulbs

(XM, Orange, CA) with a 400-W electronic ballast (Coralvue, Kenner, LA) as a power

source. These high Kelvin rated bulbs were chosen for producing peak wavelengths in

the blue portion of the spectrum. Each bulb was positioned 20 cm above the surface of

the water. Light from the bulb was projected through a light trap straight down into the

treatment group. Light traps consisted of four black plastic walls hung from a 40 x 30 cm

pvc pipe frame, extending down to the water level (20 cm) to shield all four sides below









the metal halide reflector canopy. The light traps blocked any metal halide light from

reaching the non-metal halide treatment groups; light reaching adjacent treatment racks

was measured at less than 2 imol/m2s. Bulbs were cleaned weekly with fresh water and

a wash cloth to remove any salt buildup.

These treatments were randomly assigned to one of three positions in each tank

(Figures 2-1 and 2-4). Each tank was divided into three equal areas (89 x 147 cm) for the

three treatments. Clams were set upon treatment racks (50 cm long x 50 cm wide at a

depth of 12 cm) made of eggcrate light diffuser material and supported by a pvc frame.

Each rack held nine or more seed clams. Treatment racks had equal numbers of clams

from sizes ranging seven to 14 mm. There were a total of six treatments (three sources of

light x two culture systems) with six replications of each treatment, for a total of 36

experimental units. During the experiment, six tanks had a metal halide light

malfunction. On days 22 to 29, the ballast in tank 4 was out of order; on days 32 to 34,

the ballast in tank 9 was out of order; on days 52 to 57, tank 1 had a burned-out bulb and

the ballasts in tank 1 and 4 were out of order; on days 55 to 57, the ballast in tank 12 was

out of order; on days 61 to 68 the ballast in tank 2 was out of order; and on days 74-80,

the ballast in tank 7 was out of order. The halide treatment racks were placed in full sun

until the ballasts/bulbs were replaced and back online.

On day 4 of the experiment, four or more coral fragments, each approximately 2-10

cm in length, salvaged from a pier in Key West, FL were placed into the shaded treatment

racks of all 12 experimental tanks for other research purposes. These corals included the

following species : Montastrea cavernosa, Stephanocoenia intersepta, Poritesporites,

and Diploria clivosa.









Duration and Sampling

Acclimation Period

Clams were acclimated to the light treatments for 15 days before the study began

(March 17 March 31, 2005). The metal halide lights were initially set for 5 hours on

and 19 hours off, and adjusted daily to add 30 minutes more light until it reached 12

hours on 12 hours off

Study Period

The study ran for 90 days (April 1 June 29, 2005). On day 1, six tiles with

leashes but no clams were placed in the treatment closest to the airlifts of all twelve

tanks. Weights were taken for the blank tiles on day 1 and day 90.

Light Measurements

PAR irradiance was measured daily during midday (1200 to 1300 hours) in an

unobstructed area outside the greenhouse using the LICOR irradiance sensor. Daily peak

readings were not recorded for two weeks (day 27 to 39) due to necessary repair work for

the LICOR. Amount of daily sunshine minutes was recorded by the National Weather

Service Station (National Oceanic and Atmospheric Association, Ruskin, FL) located 30

meters from the experimental greenhouse.

Every 30 days, spectrum and intensity in PAR for each metal halide treatment was

measured. An Ocean Optics (Dunedin, FL) USB 2000 spectrophotometer, with a #3

grating (350-800 nm range) and a 200-nm optic fiber with a CC-3 Cosine Corrector, was

used to measure spectral distribution in the center of the clam treatment rack. To test for

changes in bulb performance, the LICOR was used to measure light intensity in a 5-cm2

grid at clam depth (12 cm). Both of these measurements were taken at night so no

sunlight was included in the recording. During the second month, a LICOR model LI-









250 light meter was used with the underwater quantum sensor LI-192SA, while the other

LICOR was in repair.

Light irradiance in the full sun and shaded light treatments was measured in PAR

with the LICOR sensor at the depth where clams were held (12 cm). This was conducted

from 1200 to 1300 hours, taking one reading per tank per hour. These measurements

required cloudless days and were therefore conducted three times: three days prior to the

study; on day 25; on day 69. Passing clouds altered light readings from one minute to the

next making light readings unreliable. The peak angle of the sun was recorded on days 5,

48 and 85. This measured the shortest length of the shadow of a 45-cm ruler

perpendicular to a level base from 1200 to 1300 hours.

Growth Measurements

Growth was measured every 30 days for each clam using wet weight (g) and shell

length (mm) (Figure 2-5). Length measurements were taken using calipers. Width and

height were not accessible due to the clam tag and the tile. Clams were allowed to drip

dry for 10 s, while taking the length measurement, and the tile bottoms were blotted dry

before being placed on the scale for wet weight measurements.

Color Measurements

Clam mantle color was measured every 30 days. Measurements were taken inside

of a light-sealed 30-L tank (outside was painted black) filled with 20 L of saltwater. A

6.35-mm OD barrel was attached to a 200-nm optic fiber (Figure 2-6). An Ocean Optics

WS-1 diffuse reflectance standard was used in conjunction with a 50-W xenon halogen

bulb (Duralamp Fullerton, CA) and a Dimensions F27 halogen track light (Hampton Bay,

Atlanta, GA). The reference standard was used before taking each color sample in order

to calculate the amount of light reflected into the spectrophotometer. Digital images were









taken with a Nikon D70 (East Rutherford, NJ,) digital camera with its lens placed on a

sealed slot entering the top of the color measurement tank. Twenty six clams were

subsampled for color measurement on days 3, 33, 63 and 88 of the experiment. Low

numbers of colorful clams did not allow for higher sampling sizes. Nine more clams, one

green and eight brown, were added for color measurement on day 60 due to mortality of

some of the previously sampled clams. At day 1, at least two clams from each light

treatment were selected for each color morph; blue, green, and gold (brown/yellow).

Each clam was placed into the color measurement tank and positioned underneath the

spectrophotometer probe underwater. Measurements were not taken until the mantle

tissue was exposed. The angle of incidence from the light to the clam was 45 and the

end of the spectrophotometer probe was held at 450 to the clam with a 5 to 6-mm distance

from the probe to the mantle tissue (Figure 2-6). This measured a 3 to 4-mm2 area of

mantle tissue. Three measurements of the same portion of mantle tissue were taken for

each clam. Color measurements were also taken with the clam mantle retracted to correct

for shell reflectance. Prior to taking the shell reflectance measurement, the clam was

startled into closing its valves by sticking a pvc pipe inside the box. Three additional

measurements of 5-cm blue color clams were taken on day 60. These were used to

compare to smaller sized clam mantle readings. Results were analyzed using the Ocean

Optics OOIIRAD program which measures spectral reflectance and the CIE L*a*b*

color space which uses the three variables-L* is the light-dark axis (light positive, dark

negative), a* is the red-green axis (red positive, green negative), and b* is the blue-

yellow axis (yellow positive, blue negative).









Statistical Analysis

All statistical analyses were conducted at a significance level (Type I error rate) of

a = 0.05. Two-way analysis of variance (ANOVA) was used to detect differences in

survival proportions at day 90, using light and system treatments as main effects (Proc

GLM; SAS Institute, Cary, North Carolina). Proportions of surviving clams were

arcsine-square root transformed prior to analysis. Split-plot repeated measures ANOVA

was used to detect differences in length and weight data for all four clam measurement

periods, using light and system treatments as main effects (Proc Mixed; SAS Institute,

Cary, North Carolina). Significant ANOVAs were followed by the least squares means

multiple comparison procedure.

A chi-square analysis of survival at day 90 was conducted on four different size

groups of clams to determine if smaller size classes had lower survival over time. The

initial size groups used at the start of the experiment for these clams were: 6-9 mm; 10

mm; 11 mm; and 12-14 mm. Proportions of size class survival were compared using the

original clam size.

Means for L*a*b* values were collected from survivors of the 26 original clams

measured from the first sample period. These did not include the nine clams added on

the third sample period.












Evaporative Cooler


Chiller
Protein Skimmer


Bead Filter


\ OM\UV Sterilizei
12 Roll 8 Roll 4 Roll
OSM OMS MOS *
11 Roll 7yst 3 Syst
MOS OSM MOS

10 Roll 6Syst Syst um
MOS MOS OMS
9 Syst 5 Roll 1 Syst
SOM MOS OSM

Water flow


ST 0 M

Treatment racks


Enlargement of Tank 9 aerial view
Tank Treatments
S = Shaded, O = Open, M = Metal Halide


Airlift rack


4-


Greenhouse Dimensions : 22 X 9.1 meters
Figure 2-1. Greenhouse layout. Twelve experimental tanks are numbered on upper left
corner along with culture system treatment (Roll = Rollover, Syst =
Recirculation System) on upper right corer. Enlarged tank shows position of
light treatment racks inside of one of the tanks.


Fan




Door




Fan








Air supply line


Airlifts

Figure 2-2. Rollover tank. Eight airlifts on the right side of tank draw up water and
pushes it across the surface of the tank to circulate the water in a rollover
fashion.


Water Flow \



O Q ^K^C(TO- 7D :: (-




























Figure 2-3. Seed giant clam (Tridacna maxima) attached using cyanoacrylate glue to a
Floy T-bar tag (marked by arrow). The Floy T-bar tag was attached to a 2.5-
cm2 ceramic tile using cyanoacrylate glue.










Treatment Types


Shade


Metal Halide


Open


Z/ Ulill IIII CelliI UL ItiKU
Corals placed around edge of shade treatment rack


Shade Open Halide
Figure 2-4. Light treatments seen above in two experimental tanks. Note metal halide
lights with heavy plastic covering to keep artificial light from reaching other
treatments. Shaded treatments included some coral fragments around edges of
rack with clams grouped in the center.

















length



byssal opening
umbo


Figure 2-5. Clam length


longest stretch across shell.














xenon halogen bulb
camera slot



probe stand
clam

probe tip



Exploded view of probe and clam

barrel


probe tip

30 degree angle


-light source

4 mm distance


1-2 mm distance

color area measured


clam


tile with cradle space
Figure 2-6. Color measurement tank and enlargement of spectrophotometer probe and
clam position. Top of tank was sealed with an acrylic cover and heavy drape
cloth. Tank sides and bottom are covered with black acrylic paint.














CHAPTER 3
RESULTS

Survival

Clams had a mean 74.6% monthly survival (43.4% for all 90 days) during the

experiment. There were 172 clams at the end of the experiment.

Among the six treatment types, mean survival over 90 days was highest in both the

shaded x recirculation treatment with a 66.2% ( 6.7% S.E.) and the open x recirculation

treatment, with a survival rate of 56.9% ( 10.7% S.E.) (Figure 3-1). The treatment with

the lowest survival after 90 days was halide x rollover, with a survival rate of 13.2% (+

5.8% S.E.). Two of the six replicate racks of the halide x rollover treatment had no

surviving clams after 90 days.

Mean survival for each treatment type is as follows: open treatment was 47.7% (+

7.3% S.E.); shaded treatment was 55.1% (+ 6.3% S.E.); halide was 28.5% ( 7.2% S.E.);

recirculation system was 55.5% ( 5.4% S.E.); and rollover was 30.9% ( 4.7% S.E.).

The two-way ANOVA found that system type (F 1,30 = 17.56, P = 0.0002) and

light source (F 2,30 = 9.04, P = 0.0008) each had a significant effect on clam survival

(Table 3-1). The rollover systems had significantly lower survival than the recirculation

systems. No significant difference was found between clam survival in the open and

shaded treatments (LS Means, P = 1.000). The halide treatment had significantly lower

survival than both open (LS Means, P = 0.0076) and shaded (P = 0.0012) treatments.

A Chi-square analysis showed significant differences (x2 = 8.07, df = 3, P = 0.05)

in survival of size groups (Table 3-2). Smaller sized clams (6-9 mm) had a survival rate









of 29.3% compared to larger size groups survival of 50.6% for 10 mm, 42.7% for 11 mm,

and 50.9% for 12-14 mm (Figure 3-2).

Shell Growth

Split-plot repeated measures ANOVA found no significant effect of light or system

type on shell growth (Table 3-3). Clams that survived from day 1 to day 90, had a mean

growth of 0.58 mm with a mean initial length of 11.17 mm. This equals a mean growth

of 5% in 90 days.

Mean growth over 90 days for surviving clams was as follows: recirculation system

treatment was 0.8 mm (7.3%); rollover treatment was 0.3 mm (2.7%); open treatment

was 0.7 mm (6.4%); shaded treatment was 0.6 mm (5.6%); halide treatment was 0.4 mm

(3.3%); open x recirculation treatment was 1.1 mm (9.5%); shade x recirculation

treatment was 0.8 mm (7.1%); halide x recirculation treatment was 0.6 mm (5.4%); open

x rollover treatment was 0.3 mm (3.1%); shade x rollover treatment was 0.5 mm (4.1%);

and halide x rollover treatment was 0.0 mm (0%).(Table 3-4).

Nine clams (two blue and seven gold) grew 2 mm or more during the 90-day study.

Four of these were in open x recirculation treatments, three were in the shaded

recirculation system treatment, and one was in the metal halide recirculation system

treatment. One gold clam (#313) in the open x recirculation treatment had a maximum

total growth of 4.7 mm. It had a maximum monthly growth of 2.4 mm during the last 30

days of the study.

Wet Weight

Culture system had a significant effect on mean weight (F 1,10 = 10.31, P =

0.0093) (Table 3-5). Mean weight gain for all blank tiles was 0.04 g, while mean weight

gain was 0.062 g and 0.019 g for tiles in the recirculation and rollover tanks, respectively.









Noticeably more biofouling (mainly unidentified diatoms and filamentous algae) had

settled upon the tiles from the recirculation system than upon those from the rollover

tanks. Therefore, clam wet weight values were calculated by subtracting mean weight

gain for blank tiles each culture system. Table 3-6 summarizes mean wet weight gain for

clams in each treatment surviving to day 90. Mean clam wet weight for the six treatment

types was 0.300 + 0.008 S.E. g at the beginning of the study. Mean wet weight gain for

clams surviving to day 90 was 0.026 g (8.7%). Mean wet weight gain or loss for each

treatment was as follows; recirculation system treatment was 0.057 g (18.3%); rollover

treatment was -0.006 g (-2.1%); open treatment was 0.029 g (9.4%); shade treatment was

0.032 g (10.7%); and halide treatment was 0.023 g (7.6%).

Color

Giant clam mantle coloration is an important factor for determining market price.

Clams with bright blue or green colored mantles are termed "ultra" and fetch the highest

prices. However, the majority of giant clams have a brown or yellow mantle tissue and

are termed "gold". The clams in this study were divided into three categories for ease of

color quantification: blues, greens and golds. Individual clam readings are listed in

Appendix A.

During the acclimation period, 14 clams with green mantle tissue and two clams

with blue mantle tissue changed to gold mantle tissue by the start of the experiment.

Since these changes took place prior to the start of the experiment, there is no

spectrophotometer record of the color change.

Reference clams

Three 5-cm blue "ultra" T. maxima clams were obtained from an online retail store

after the second color sample period and measured as a blue and green color reference for









ideal marketable color. One 5-cm clam had dark blue mantle tissue and showed a lower

percent spectral reflectance signature compared to the other two 5-cm clams (peak 400

nm, 2% reflectance) (Figure 3-3). Another 5-cm clam had light blue mantle tissue and

showed a high percent spectral reflectance signature (peak 441nm, 20% reflectance)

(Figure 3-4). The third 5-cm clam had a mixed blue-green pattern on its mantle tissue

and had a spectral reflectance signature showing a shift to the blue-green portion of the

spectrum (peak 481 nm, 7.5% reflectance). The light and dark blue clams showed a high

blue b* value (-15.5, -10.8) while the mixed green and blue clam showed a high green a*

value of -3.4 and a blue b* value of 4.9 (lower numbers are higher values for both green

and blue in CIE L*a*b*).

Survivors

Only eleven clams out of 396 were visually found to have blue color within the

mantle tissue. Out of these, only three from the open treatment (clam # 112, 1111, and

715), two from the shade treatment (8211 and 9210), and one from the halide treatment

(1237), survived until the end of the experiment. Three blue clams died after the first

color sample. One blue clam from the shade treatment died after the second color sample

(823). One blue clam from the shade treatment died after the third color sample (522).

Only two green clams out of seven survived until the end of the study (311 and 7110). A

third green clam (7212) died after the third sample period. Only three gold clams out of

eight survived until the end of the study (225, 1212, and 3110).

Green switch to Gold (Yellow)

One clam switched from green to gold during the experiment. Clam 721 from the

shade x recirculation treatment was one of nine clams added midway during the

experiment for color sampling due to losses of other color sampled clams. Overall









spectral reflectance decreased except for the lower portion of the blue region (400-415

nm) (Figure 3-5). Both green a* and yellow b* values decreased from the third to the

fourth sample period. The photograph shows a green mantle tissue in the third sample

period and a smaller sized yellow mantle tissue in the fourth sample period. Although the

photographs agree with the a* values, it was contradicted by the decreased b* values.

This may have been due to a decrease in mantle size.

Light Measurements

Solar

Photosynthetic Active Radiation (PAR) measured outside of the greenhouse in

open air between 1200 and 1300 hours had a maxima of 2400 imol/m2s. This was

recorded on day 15 with partly cloudy skies. Only when clouds are positioned right

beside the sun, but not fully blocking it, is sunlight reflected at higher intensities than on

clear days. Peak PAR on clear days was 2100 imol/m2s. Mean daily peak PAR was

1620 imol/m2s. A slight decrease in daily peak PAR readings was found to occur during

the study (Figure 3-6). This was mainly due to an increase in cloudy days during the

second half of the study as seen in the percent possible sunshine minutes (Figure 3-7).

However, underwater measurements at clam depth did display an increase in midday

PAR readings over the course of this study. This was attributed to increased midday

sunlight angle. Mid-day sunlight angle increased from 65 three days prior to the

experiment to 85 0 at day 90. Summer solstice occurred on day 82. Winter solstice in

Ruskin, FL had an angle of 30 Day length increased from 750 min (12.5 h) on day 1 to

833 min (13.9 h) on day 90.

Three days prior to the study, mean peak intensity of the open treatments was 1304

imol/m2s. This increased to a mean of 1414 imol/m2s on day 25. The final reading









found a mean peak intensity of 1412 imol/m2s on day 69. Maximum intensity was

recorded in tanks 3, 4, 5 and 10 with 1500 imool/m2s on days 25 and 69. The peak

wavelength was 588 nm. Intensity rose sharply before 460 nm, leveled off, then dropped

after 700 nm (Figure 3-8).

Three days prior to the study, peak intensity in the shaded treatment was 911

imol/m2s. This increased to a mean peak of 987 imol/m2s on day 25. Shadecloth

percentage was raised from 30% to 55% on day 48 to decrease rising intensity levels in

the shade treatment. The final reading found a mean peak of 719 imol/m2s on day 69.

Shaded treatments shared the same peak wavelengths as the open treatments with a peak

wavelength at 588 nm, and a plateau running from 460 nm to 700 nm (Figure 3-8).

Metal Halide

Mean intensities ranged from 660-758 imol/m2s in the halide light treatments

during the 90 day study (Table 3-7). Only one halide treatment (Tank 1) had highly

variable readings (Figure 3-9). Tank 1 had the maximum intensity recorded (1494

imol/m2s during week five) and the minimum recorded intensity (140 imol/m2s during

week 13; its maximum intensity during week 13 was 280 imol/m2s). Tank 1 mean

intensity decreased respectively by 484 and 417 imol/m2s from weeks five to nine and

weeks nine to thirteen. These changes in intensity were most likely due to fluctuations in

voltage to the ballasts.

Peak wavelengths of the metal halide treatments for all tanks were similar and did

not vary greatly over the 90-day study (Figure 3-8). The peak wavelength was measured

at 420 nm, the second highest peak was at 403 nm, and the third peak was at 435 nm. A

smaller peak at 365 nm appeared as high as the 435 nm peak on a few occasions. This

happened in tank 4, 6, and 9 during week 5. It also appeared in tanks 1, 7, and 9 during









week 9 and finally in tank 1 on week 13. Tank 1 had a major spectrum shift on the final

week of the study (week 13) with even distribution between the three peaks at 365, 435,

and 403 nm.

Six halide lights malfunctioned at some time during the study due to the high

humidity greenhouse environment and fluctuating voltage. Ballasts were placed in the

greenhouse five months before the start of the experiment and were exposed to high

humidity at that time. These lights ran for less than 100 hours before the acclimation

period. A period of low voltage occurred during the experiment due to a problem with

the Florida Power and Light company (FPL) service reaching the greenhouse. Electronic

metal halide ballasts are known to be sensitive to drops in voltage and may have been

damaged due to these conditions. The voltage issue was resolved by FPL on day 47 but

may have had lasting effects on a few ballasts.

Temperature profiles

Higher than ideal temperatures (>30 C) (Ellis 2000) occurred in all rollover tanks.

This was due to the inability of the evaporative cooler to keep midday air temperatures

inside the greenhouse below 38 C from day 30 on. This raised temperatures in rollovers

to a maxima of 34.66 C in tank 4. The remaining five rollover tanks reached

temperatures above 33 C. Recirculation system tanks stayed below 30 C throughout the

study with the aid of the chiller.

Weather data gathered by the local weather station indicated a minimum outdoor

air temperature of 10 C during the first and third weeks of the experiment. After the

fourth week, day 28, the heater was left off and the windows for the evaporative cooler

pads were left open. A maximum outdoor air temperature of 33 C was reached on day

60 of the study (NOAA weather station, Ruskin, FL).









Water Quality

Total nitrogen in tanks reached a maximum of 1.8 mg/L in the rollover tanks and a

maximum of 2.8 mg/L in the recirculation system tanks according to spectrophotometer

readings by Florida Lakewatch. The HACH test kit was not sensitive enough to detect

these low levels. The only high nitrogen reading from the HACH test kit came from a

nitrite spike during week 8.

Rollover tanks accumulated large amounts of foam each day. This was caused by

the presence of high organic in the tank water. Insects attracted to the metal halide

lights during evening hours became trapped on the water surface. This added to the input

of nutrients which turned into dissolved organic matter that was later separated out of the

recirculation system tanks by the protein skimmer. Constant collection of foam from the

rollover tanks was not enough to keep the treatments within these tanks from receiving

less light due to the shadowing effect of the foam. The foam layer reduced light

transmittance by up to 150 gmol/m2s in rollover tank treatments.

The recirculation system was unable to keep nitrites from spiking. Nitrite reached

1.5 mg/L on week 8. More algae was observed inside of recirculation system tanks

compared to rollover tanks. Three times as much algae weight was recorded on the blank

tiles from the recirculation tanks than from the rollover tanks, a mean weight gain of

0.062 g compared to 0.019 g.

Calcium levels were low in both rollovers and recirculation systems during the first

sample period (370 mg/L). The LaMotte and Seachem Calcium test kits gave faulty

readings thereby contributing to the suboptimal levels. Once the Salifert test kits were

used, calcium readings stabilized at 400 mg/L or greater according to the ICP mass

spectrophotometer readings for the remainder of the study. Alkalinity readings ranged






30


from 171 to 205.2 mg/L. Total phosphate levels for all four sample periods were less

than 20 tg/L in all tanks.











100


90


80


70


60


a 50


40


30


20


10 .


0
30 60 90
Time in days

open recirc U shade recirc D halide recirc B open roll E5 shade roll R halide roll

Figure 3-1. Mean treatment survival after 30, 60 and 90 days (n=6) for all six treatment
types: open x recirculation; shade x recirculation; halide x recirculation; open
x rollover; shade x rollover; and halide x rollover. See methods for specific
details of the treatments. Standard error bars included.

































0


Day 90


SM6

Figure 3-2. Survival of clams with initial sizes of 6 12












2.5

2

S1.5 -




0.5 -

0
O0 0 t C(40 O CO (0 CO '- CO (0 CO C) I0 9T -- 9O
M '- CO LO I CO 0 C '9T LO -I O) '- C04 ( 0 I- OM
Cm i i j j z LO LO LO LO LO LO 0 (0 0 (0 0 (0
Wavelength (nm)



Figure 3-3. Spectral reflectance (%) for a 5-cm T. maxima clam measured on the third
sample period. Its dark blue mantle tissue showed low spectral reflectance
(2%) in comparison to the light blue mantle 5-cm T. maxima reference clam in
Figure 10 (20%).











25



20



15

(,
S10 -



5 -



0
o-C o L I C ) COo) LO (0 N Ir- N CO CO c0 CO O0
O C -D (DCO 0 M (N cor- 0) 0 (M Co N lC (DO 0 M
.*I- 't ' Wavelength (nm)



Figure 3-4. Spectral reflectance (%) for a 5-cm T. maxima clam measured on the third
sample period. Its light blue colored mantle tissue was used to compare to the
smaller sized clams. The peak wavelength is found in the blue region at 440
nm, consistent with what was found in smaller blue seed-size clams. This
bright blue is the color in demand for the ornamental trade.











9-
8

7-

6
05






0 ......

0 0O r1- LO 'q Cj 0 O0 (D 'q C4 0 r- LO C14 0) (D CO
0) 0 C14 I (D OO O 0 CO LO r- 0) 0 C14 I LO r1- 0)
CO IO Iq I I Iq LO LO LO LO LO LO ID QD (D O' (,O 0
Wavelength (nm)



Figure 3-5. Spectral reflectance of one of the clams added for color sampling for the
third sample period (clam 721 from the shade x recirculation system). This
was the only clam recorded to have changed from a dark green color to a
yellow colored mantle. Spectral reflectance for the color change is shown
above from day 60 (solid line), to day 90 (dashed line). Photographs taken
show mantle switching colors from a dark green on day 60 to a yellow color
on day 90. Mantle tissue diminished in size on day 90 and resulted in a low
spectral reflectance










2500

2000


' 1500


E
o 1000


500

0


1 2 3 4 5 6


7 8 9 10 11 12 13


Week


Figure 3-6. Mean weekly peak PAR. Calculated from daily peak PAR readings taken
outside of the greenhouse. S.E. bars included (n = 7, week 13 n = 6). Weeks
4 to 6 are missing due to meter malfunctions










120

100

S80

60 -

S40

20



1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86
Day


Figure 3-7. Percent possible sunshine. Percentage of daytime during which the direct
solar radiation exceeds the level set by NOAA. This activates a sunshine
recorder to log each sunshine minute between sunrise and sunset. Data
provided by NOAA weather station in Ruskin, FL. Last 30 days showed an
increase in cloudy weather.










0.20 Full sun outside greenhouse

I

0.15 /
I I
E 1 Open .
c,4 Treatment I'J I
0.10 / .
",1 Shaded 'oI
S365 nm
=L 3-/ treatment i

treatmentHalide treatment



0.00


Wavelength (nm)


Figure 3-8. Spectral power distribution for all three light treatments at midday. A
reading of full sun outside of the greenhouse was taken at midday. Shade
treatment was measured with 30% shadecloth. Halide treatment had a spectral
distribution concentrated in the blue wavelengths and included a UV-A spike
at 365 nm.










TANK 1 WEEK 1 TANK 1 WEEK 5






















TANK WEEK 9 TANK WEEK





&250

















Figure 3-9. Light maps in gmol/m2s for halide treatment in Tank 1 during week one,
five, nine, and 13. High light readings were recorded in week five. Light
readings decreased in weeks nine and 13. Twin peaks for maximum intensity
were normal for all other halide lights. Intensity decreased around the edges
of the treatment racks.









Table 3-1. Two-way analysis of variance (ANOVA) for survival at day 90 of T maxima
seed clams among two culture systems and three light treatments.


Mean Sauare


SYSTEM
0.0002 ***
LIGHT
0.0008 ***
SYSTEM*LIGHT
0.4331
ERROR

TOTAL


0.75207574

0.77437041

0.07369967

1.28453669


0.75207574

0.38718520

0.03684984

0.04281789


R-Square = 0.55
*** significant difference among means (P < 0.001)


Source


17.56

9.04

0.86






41


Table 3-2. x2 analysis of size class survival of T maxima seed clams over 90-day study.
x2 calculated = 8.07, df = 3, a = 0.05, X2 critical = 7.815 (Ott 2001).
Survival after 90 days
size class
(mm) expected observed
6-9 42.6 28
10 36.9 44
11 41.7 40
12-14 50.8 60









Table 3-3. Split-plot repeated measures analysis of variance (ANOVA) results for shell
length data of T. maxima seed clams among two culture systems and three
light treatments over 90 days.
Num Den
Effect DF DF F Value Pr > F

SYSTEM 1 10 2.15 0.1736
LIGHT 2 20 0.07 0.9364
SYSTEM*LIGHT 2 20 0.73 0.4964
TIME 3 30 12.66 <0.0001 ***
SYSTEM*TIME 3 30 2.10 0.1207
LIGHT*TIME 6 1023 0.27 0.9514
SYSTEM*LIGHT*TIME 6 1023 0.26 0.9562

*** significant difference among means (P < 0.001)






43


Table 3-4. Treatment mean shell growth (percent) of T maxima seed clams during 90-
day study. Standard error (S.E.) is included (n = 6).
Treatment Percent growth S.E.
open x roll 3.1 1.0
shade x roll 4.1 1.2
halide x roll 0.0 0.8
open x recirc 9.5 1.0
shade x recirc 7.1 1.1
halide x recirc 5.4 0.9









Table 3-5. Split-plot repeated measures analysis of variance (ANOVA) for weight data
of T. maxima seed clams among two culture systems and three light
treatments over 90 days.
Num Den
Effect DF DF F Value Pr > F


SYSTEM
LIGHT
SYSTEM*LIGHT
TIME
SYSTEM*TIME
LIGHT*TIME
SYSTEM*LIGHT*TIME


10
20
20
30
30
1023
1023


10.31
0.73
2.58
24.53
7.47
0.32
1.01


0.0093 **
0.4944
0.1005
<0.0001 ***
0.0007 ***
0.9250
0.4143


*** significant difference among means (P < 0.001)
** significant difference among means (P < 0.01)










Table 3-6. Ninety-day mean wet weight gain and mean percent weight gain, correcting
for biofouling (algae) weight, for T. maxima clams. Rollovers show weight
loss.
Wet weight gain (g) S.E. Percent weight gain (%) S.E.
clam weight gain with algae weight included
open x roll -0.002 0.016 -0.7 5.9
shade x roll 0.029 0.012 9.8 4.2
halide x roll 0.014 0.010 4.5 3.3

clam weight gain after correction for algae weight (1)
open x roll -0.021 0.016 -7.6 5.9
shade x roll 0.010 0.012 3.3 4.2
halide x roll -0.005 0.010 -1.8 3.3

clam weight gain with algae weight included
open x recirc 0.142 0.023 42.0 6.9
shade x recirc 0.117 0.007 38.8 2.5
halide x recirc 0.097 0.015 33.0 5.1

clam weight gain after correction for algae weight (2)


open x recirc 0.080 0.023
shade x recirc 0.055 0.007
halide x recirc 0.035 0.015


23.6
18.3
11.9


1 Mean algal weight gain of 0.019 g (S.E. = 0.005) was subtracted from mean rollover
clam weight gain
2 Mean algal weight gain of 0.062 g (S.E. = 0.008) was subtracted from mean recirculation
clam weight gain







46


Table 3-7. Mean halide light treatment intensity readings (rmol/m2s) for all 12 tanks
combined. Maximum and minimum readings from all 12 tanks recorded as
well as mean maximums and mean minimums of all 12 tanks. Numbers in
parenthesis are S.E. (n = 6).
Time after start of experiment


mean
maximum
minimum
mean maximum
mean minimum


week 1
747 (19)
1295
370


week 5


week 9


758 (37) 708 (25)


1494
355


1250
345


1045 (29) 1019 (54) 971 (40)


468 (22)


485 (29) 437 (22)


week 13
660 (45)
1060
140
883 (58)
460 (34)














CHAPTER 4
DISCUSSION

Major Findings

In this study, I found that 1) The recirculation system treatment had significantly

higher survival compared to the rollover treatment, 2) the open and shade treatments had

significantly higher survival compared to the halide treatment, and 3) the recirculation

treatment had significantly higher weight gain compared to the rollover treatment.

This is the first study to examine inland production of T maxima and also is the

first study to test an artificial source of light on giant clams.

Low Survival and Growth

Excessive handling during attachment to the clam leashes before the experiment

added to shipping stress, may have contributed to low survival and stunted clam growth.

The 66% survival rate in the shaded x recirculation treatment over the 90-day study is

comparable to the 75% survival rate found in most clam farm operations for clams at that

stage for the same duration (Ellis 2000).

The recirculation system had better survival than the rollover system, with 55.5 vs.

30.9% survival. This can be attributed to the chiller's capability to maintain temperatures

below 30 C. Evaporative coolers were unable to keep temperatures from approaching

lethal tolerance levels in the rollover culture system because they do not function at

relative humidity levels approaching saturation. However, a greenhouse with heavy

shadecloth (>50% shade) may be able to maintain temperatures within the ideal range

with the help of an evaporative cooler. However, caution should be taken to avoid









blocking too much light with the shadecloth for clams to survive. Even with 50%

shadecloth, slight differences in growth were found in the study by Lucas et al. (1989).

Most of the clams under the metal halides showed signs of stress by not fully

extending their mantle tissue. This was possibly a sign of photoinhibition due to UV

wavelengths given off by the halide lights. Spectral peaks at 365 nm confirmed that UV-

A was reaching the clams (Figure 3-8).

Open (47.7%) and shaded (55.1%) treatments both had much higher survival rates

compared to the metal halide treatment (28.5%). This was most likely due to the

combination of constant, high intensity light and the portion of energy from the metal

halide lights found in the UV-A (320-400 nm) range.

Only three percent of sunlight is composed of UV wavelengths (200-400 nm). This

portion of the spectrum is responsible for damage to zooxanthellae (Jokiel and York

1984). Jokiel and York (1984) used UV-blocking films that transmitted up to 92% of full

surface radiation and did not find severe photoinhibition in zooxanthellae, while

zooxanthellae in another treatment without the UV-blocking film could not tolerate levels

of light higher than 20% of full surface radiation.

The performance of blue spectrum metal halides could be improved by using a UV-

blocking film, such as OP-3 acrylic (Cyro Plastics, USA), to shield UV from reaching

culture tank organisms such as clams and corals. This shield would need to be kept at a

large enough distance from the halide light so that it does not melt from the intense heat

produced by the bulb.

Clams in this study grew slowly in all 6 treatments. Although no statistically

significant differences in growth were found among treatments, mean growth rates were









higher for clams in the open treatments with 6.3 % growth, compared to 5.6% in the

shaded and 3.3% in the halide treatments. Only one clam from the open recirculation

treatment exhibited a growth that would reach marketable size (4-5 cm) in less than 2

years. This is comparable to the growth found in clam cage culture which has abundant

amounts of algae available for the clams to feed on (Hart et al. 1998).

Besides reduced heterotrophic energy sources, the slow growth rate found in the

majority of the clams may be attributed to the unusually small mean size (for given age)

of clams used for the experiment. One-year-old T maxima seed should be 10-30 mm in

length (Ellis 2000). All of the clams used in this study were below the median size of 15

mm for 1-year-old seed clams. These may have been the culls of a spawn and had a

genetically inferior growth rate compared to the larger individuals of the batch.

Multiple disturbances were made when attaching clams to tiles and monitoring

growth. These could have further contributed to the slow growth. Ellis (2000) found that

clams removed from their substrate more than once every three months would be stunted

due to energy expended to lay down new byssal threads. Future giant clam growth

studies should use longer time intervals between measurements or find some way to

measure in-situ.

Another possible reason for slow growth may have been due to substrate choice.

Clams were attached to the T-bar tag/leash in such a way to increase their probability of

attaching to the ceramic tile via byssal threads. The ceramic tiles used were non-porous,

unlike the coral heads that T maxima are usually settled on in the wild. During the study,

some clams managed to detach themselves from their plastic leashes. Thirty-five clams

were reglued to their leashes in the middle of the experiment. Some of the clams did not









have byssal threads attached to the tiles during measurement periods and their shells were

lifted from the tile so that a small gap could be seen. These clams may have lifted

themselves from the tile by movements made by their foot before the glue was fully

cured.

In the majority of weakened clams (clams without fully extended mantle tissue),

copepods were found underneath the clam. They appeared to be feeding on clam tissue

through the byssal opening. Heavy mucous secretions from the byssal opening were

found in many clams, probably to protect them from opportunistic gammarid amphipods.

The large byssal opening of giant clams leaves them vulnerable if kept open and

unprotected by a suitable substrate.

Unexpectedly, blank tiles showed greater weight gain from biofouling than tile

weight loss from erosion. The larger weight gain in the recirculation system blank tiles

was likely due to the nitrite spike during week nine. Tile algal layers were thicker in

recirculation system tanks versus rollover tanks. Algae weight did not mask the

differences in weight gain between the recirculation and rollover systems. The corrected

clam weight gain after subtracting mean algae weight from each system treatment

showed more weight gain in the recirculation system (Table 3-6). No major differences

of algae were observed among light treatments. Blank tiles, placed into different light

treatments within the recirculation treatment did not show large differences in mean algal

weight gain: open was 0.065 g; shaded was 0.055 g; and halide was 0.062 g. Rollover

tanks had mean blank tile algal weight gain of 0.025 g in the open treatments, 0.016 g in

the halide treatments, but no tiles placed in the shade treatments. This was because no

shade treatments were placed next to the airlifts of the rollover tanks.









Stress from transport, combined with the possibility that clams may have not have

been able to acclimate to the various treatment intensities, may have further weakened the

clams. T gigas juveniles at 10 mm were reported to saturate at 500 gmol/m2s (Fisher et

al. 1985). T. maxima is known to tolerate higher levels of light than T gigas and shows

no saturation up to 1900 gmol/m2s; however, no clam size was reported for this study

(Ralph et al. 1999). More studies need to be conducted on the irradiation levels

necessary to saturate the growth capacity of seed-size T maxima juveniles.

Metal halides were chosen to augment the blue (actinic) end of the spectrum (400-

500 nm). The mean intensity recorded in the metal halide treatments was 718 gmol/m2s

which is less than peak intensities (1500 gmol/m2s) in the open treatment. However,

halide treatments received a constant light intensity for 12 h per day (except for the 15

minutes that are needed for halides to reach full intensity from startup). Therefore,

intensity in the halide treatments exceeded that in the open sun treatment before 1000

hours and after 1600 hours, for a daily total of 6 hours of higher intensity light (measured

on day 69) (Figure 4-1). There is some debate as to whether blue light is needed to

promote more vibrant colors in both clams and corals. No increase in coloration was

observed for the clams underneath any of the metal halide treatments.

Instead of promoting growth as expected, halide treatments had lower mean growth

than open treatments (0.4 mm compared to 0.7 mm). One exception was a clam in the

halide x recirculation treatment that grew 2.1 mm (16% growth) over the 90 days. This

clam may have acquired a strain of zooxanthellae with increased resistance to high

irradiance in the blue portion of the spectra as suggested by some studies on different









strains of zooxanthellae (Fitt and Warner 1993). No bright mantle color was noted for

this clam, only a dull brown.

Color Analysis

Due to low numbers of colorful clams and low survival, a full analysis for effects

due to treatments was not possible. A qualitative trend of darker and increased blue

mantle tissue from the first to the third sample periods was found for the clams in both

the open and shaded treatments. This change was seen in both L* and b* values (Table

4-1 and 4-2) as well as in the spectral reflectance (Figure 4-2). The b* values in all

clams, except for one gold clam (3110), decreased from the first to the second sample

period, indicating an increase in blue. This was clearly seen in the photographs of the

clams categorized as blue but not those of green and gold clams. The green and one gold

clams had minimal amounts of small blue portions of mantle tissue (clam 3110 days 30-

90, clam 311 day one). Between the third and the fourth sampling periods, the majority

of b* values increased, indicating a decrease in blue. This again was evident in the

photos of the blue clams but not in the green or the gold. Yellow color inside the mantle

tissue was usually shaded out by dark brown and was difficult for the spectrophotometer

to measure in b* values.

One clam had a dramatic color change from dark green to yellow (721). The high

spectral reflectance peak in the blue portion of spectrum may have been responsible for

the green found in its mantle tissue (Figure 3-5). Possibly, a mixture of brown

zooxanthellae and light blue tissue, most likely caused by iridocytes (Griffiths et al.

1992), created the greenish shade that was replaced by yellow tissue. Another possibility

is that different colored strains of zooxanthellae are selected for certain portions of

mantle tissue. Carlos et al. (2000), showed that multiple strains of zooxanthellae have









been found inside giant clams. If one of these strains contain less peridinin, an orange

based pigment, then it would appear green, due to the remaining chlorophyll a and c2

pigments. Other zooxanthellae strains containing more peridinin would appear brown as

a result of the combination of orange and green pigments. This "brown strain" of

zooxanthellae may have been what remained in the mantle of clam 721.

Only clams from the rollover treatments experienced tissue bleaching due to mass

zooxanthellae expulsion as indicated by photographs. This was likely due to loss of

brown zooxanthellae from the mantle tissue from increased temperatures (Buck et al.

2002). The remaining color left behind was a light cream/peach mantle with some light

blue tissue colored by iridocytes as seen in clam 8211. This was also found in clam 438;

the photograph showed minute amounts of green besides a very light brown. However,

bleaching did not occur in all clams from rollover tanks. For example, clam 412 showed

no signs of bleaching at the end of the study. This was unexpected since it was in the

treatment with the highest amount of light and heat (open x rollover). The zooxanthellae

in this clam may have acquired a tolerance to high light, preventing photoinhibition.

Only two clams with large blue mantles were recorded at the end of the study.

These were clams 715 from the open x recirculation treatment and 9210 from the shade x

recirculation treatment. Clam 715 had a higher blue spectral peak, while 9210 had a

lower blue spectral peak. This difference in blue was also evident in the photographs

taken of the clams 9210 and 715. Although clam 8211 from the shade x rollover

treatment had a large area of light blue during the fourth sample period, it was not as

vivid a blue as that found in both clams 715 and 9210.









Overall, L* and b* values showed more differences over time while a* values

stayed fairly constant. Two qualitative trends appeared in this study for both the open

and shade treatments according to L* and b* values (Table 4-1). The first qualitative

trend showed an increase in darkness from the first to the second sample period. The

second qualitative trend showed an increased blueness from the first to the third sample

period. This increase in dark pigment and blue color for the first three sample may

suggest that the clams from these two treatments were increasing the amount of

iridocytes and zooxanthellae density in response to environmental conditions (e.g.,

increase in sunlight). Only two of eight blue clams did not have photographs that agreed

with b* values. This was less problematic for the larger 5-cm clams. This may have

been due to larger, more uniform mantle tissue area of the 5-cm clams compared to the

smaller seed clams. Slight changes due to mantle tissue deformity would have greater

effects on readings with the smaller clams compared to the larger clams.

Although the measurements taken with the spectrophotometer were difficult to

perform and interpret for the seed size clams, color differences were obtainable when

mantle tissue did not vary in size or shape over time. Further investigation using the

spectrophotometer as an objective means to measure clam color is needed to optimize

environmental parameters for improved color in giant clams. This could be applied to

increase production of clams that exhibit brilliant blues and greens, allowing greater

economic success of ornamental giant clam producers.

Nutrients

Normal background levels of dissolved inorganic nitrogen as the sum of

ammonium nitrate and nitrite in non macroalgae dominated reefs is <15 tg/L (Lapointe

et al. 1993). Bivalves appear to have the capacity to tolerate high concentrations of









nitrogen. The bivalves Mercenaria mercenaria and Crassostrea virginica have 96-h

mean lethal tolerance limits of 110-880 mg/L, and 1081 to 2415 mg/L for ammonia and

nitrite, respectively (Epifanio and Srna 1975). One study of giant clams grown in

aquaculture effluent showed no adverse affects to T. maxima with exposures of nitrite up

to 1.3 mg/L (Sparsis et al. 2001). In the current study, clams in the recirculation system

showed no obvious detrimental effects of exposure to the increased levels of nitrite (1.5

mg/L) after week 9. Regular heavy dosing of ammonia did not occur until the

experiment began. This may explain why the establishment of a sufficient population of

Nitrobacter for biofiltration was delayed. Both ammonium and nitrite levels were spiked

using ammonium chloride and sodium nitrite in the months previous to the experiment,

but may have been insufficient to maintain a sufficiently large Nitrobacter colony

capable of keeping nitrite levels in check.

Total phosphate was kept below 20 tg/L in all tanks. Normal background levels of

soluble reactive phosphate found in non-macroalgae dominated coral reefs is less than 3

tg/L (Lapointe et al. 1993).

A study on T. maxima juveniles (Estacion et al. 1986), found that clams fed

Isochrysis and Tetraselmis once every other day had significantly higher growth than

unfed clams. Feeding live algae may improve growth rates enough for inland culture of

juvenile clams to be economically viable. However, this means added cost for the grower

to provide adequate amounts of algae. Nutrient buildup may be another concern with

algae feeding. Despite apparently high tolerance levels of giant clams to certain

nutrients, culture system reactions to increased amounts of nutrient loading need to be

examined more thoroughly.









Small clam species may be poor candidates for ocean growout and better suited for

inland production. Hart et al. (1998) studied the two smallest, but most colorful clam

species, T maxima and T crocea as well as the larger clam T derasa. He determined

that 35-mm standard length (SL) T crocea and 50-mm (SL) T maxima are in greatest

demand in the aquarium trade. However, due to their smaller and weaker shells, these

clams are at higher risk for predation than larger species such as T derasa. Hart et al.

(1998) found a mean growth rate in offshore cage culture of 2.9 (S.D.=0.6) mm/month

in juvenile T maxima over a 16-month period starting from 8-month-old seed ranging

from 10-30 mm SL. T maxima reached 50 mm in 9 months, while T derasa seed (10-30

mm) grew to a size of 80 mm in the same time period and under the same conditions.

Poor survival rates (-40%) were recorded for T maxima during the growout period in the

ocean cages compared to -90% survival in T derasa for the same study. The smaller T

maxima species is a poor candidate for ocean growout and better suited for inland

production where it is out of reach from such predators as rice snails and wrasses that can

find their way through clam cage openings. Greater protection from environmental

conditions such as storms and anthropological factors such as poachers and pollution can

more easily be maintained in inland culture systems.

In conclusion, the two most favorable treatments in this study were the shaded x

recirculation system and the open x recirculation system. Survival rate was significantly

(F 2,30 = 9.04, P = 0.0008) higher for these two lights (47.7% and 55.5% vs. 28.5%) than

for the halide. Also mean weight was significant (Fi,io = 10.31, P = 0.0093) for the

recirculation system (mean weight gain of 18.3%) compared to the rollover (mean weight

loss of -2.1%). No significant differences were found among treatments for shell growth,









but the open x recirculation system treatment had a slight advantage over the shade x

recirculation system treatment for the highest growth (9.5% vs. 7.1%). Lucas et al.

(1989) also found lower growth in the shade treatments, compared to sunlight treatments,

but survival was higher in the shade treatments. These two treatments produced the only

two healthy clams that exhibited blue mantle at the end of the experiment, while the rest

of the clams displayed a dull green or gold (yellow/brown) mantle tissue. These two

clams were in tanks that had a larger than average number of coral fragments inside the

shaded treatment racks. More coral fragments were introduced into these tanks because

their shade treatments were closer to the airlifts than other tanks and provided a higher

flow that corals need relative to clams. Clams have the ability to draw in water with their

gills, while corals have to rely on a current for food or expelling waste. Whether an extra

supply of zooxanthellae provided by the corals may have helped produce better color in

these two clams is not clear.

An unpublished study on coloration of juvenile T. maxima clams showed that light

intensity was not as much a factor as was keeping the clams in their natural oceanic

environment (personal communication, Lynette Kumar, University of the South Pacific,

unpublished, masters thesis). This study found that clams planted in different depths in a

longline nursery 50 m off the reef, had better coloration at most depths than clams grown

in onshore tanks that used varying levels of shadecloth to simulate the depths of the

experimental clams out on the sea.

More studies need to be conducted on inland culturing of giant clams. Despite the

problems found in this study, it could ease the overharvesting of wild stocks of marine

ornamentals throughout the world's coral reefs. Overharvesting has already eradicated






58


certain species of giant clams in the Philippines and is a growing problem with wild

stocks in Vietnam (Cheshire and Valeriano 2004). Cultivation technology for marine

ornamentals has been advancing and should be adopted by the industry before further

collapse in wild stocks can occur.











1600

1400


- 4


1200

h 1000

E 800


0
E


600

400

200

0


C) ION- CO OC 0 C- ( C0) LC) (OI- CO O\ 0 C -
*I \H


Hour- -
Hour


Tank 4 open treatment intensity Mean halide intensity

Figure 4-1. Daily solar profile taken on day 69 for tank 4 open sun treatment shown in
dashed line. Mean metal halide intensity, for all 4 sample periods shown in
solid line.






















0 %,. e
.4


"' ,. I,. \,."


F. *


o LO 0 *Dj-
M C cNJ CO
CO q- q- b-q


oM .- ;T co M M (ND (- LO oM CO I- LO o N (0D 0 CO
NT (0 r I M OC N M CO LO (0 I- Ol M C( CO M (0 I( OM 0
s- l -l LO LO LO LO LO LO LO (0 (0 (0 (0 (0 (0 I r
Wavelength (nm)


- -Day 1


Day 30 Day 60 Day 90


Figure 4-2. This graph displays spectral shift from day one to day 90 for one of the blue
clam seed chosen for color measurements (number 715 from the open x
recirculation treatment). Blue peaks around 440 nm increases in percent
reflection while green (500-560 nm) and brown (yellow 560-600, orange 600-
650) decrease in percent reflection. This was seen in photographic
documentation at those time periods with increased portions of the mantle
tissue turning blue over time.


6


- 5
.2o

4


3


2


1


0


.^***'







61


Table 4-1. Mean L*a*b* values for open and shade treatment clams measured for color.
S.E. included in parenthesis (n=6, n=4 for sample period 3 in shade clams).
Only clam 1237 survived from the halide clams measured for color (n=l).
Open Shade Halide
sample
period L* a* b* L* a* b* L* a* b*
0 -3.5(1.4) 0.7(0.5) 6.0(1.2) -2.4(1.3) 0.7(0.4) 5.1(1.7) -5.4 1.0 3.8
1 -9.1(2.5) 1.3(0.9) 5.3(1.7) -6.7(1.3) 1.3(0.7) 5.8(2.0) 4.3 -0.1 2.2
2 -2.5(0.9) 0.8(0.2) 0.5(1.4) -4.0(2.2) 0.6(0.7) 0.2(1.5) -8.1 -3.1 -0.3
3 -3.3(1.1) 0.3(0.3) 2.2(0.8) -3.7(1.8) 1.1(0.6) 1.1(0.9) -3.5 0.8 2.0







62



Table 4-2. Mean L*a*b* values for three categories of color measured clams: blue;
green; and gold clams. S.E. included in parenthesis (n = 6 for blue clams, n =
3 for green and gold clams).
blue clam mean green clam mean gold clam mean
sample
period L* a* b* L* a* b* L* a* b*
0 -3.5(1.3) 1.6(0.1) 4.2(1.3) -1.4(0.1) -0.5(0.1) 3.6(0.5) -5.2(2.8) 0.0(0.4) 9.1(0.1)
1 -8.5(2.5) 2.5(0.8) 3.2(1.4) -5.9(2.7) -0.5(0.5) 3.4(0.4) -7.9(2.2) 0.4(0.6) 11.5(0.6)
2 -4.3(1.4) 0.2(0.9) -2.3(1.2) -1.4(1.5) 0.3(0.4) 2.1(1.8) -6.1(3.2) 0.4(0.1) 2.5(0.4)
3 -4.2(0.8) 0.9(0.2) 1.4(0.7) -0.4(0.1) 0.2(0.1) 0.6(0.5) -3.9(2.7) 0.4(1.1) 3.3(1.4)














CHAPTER 5
MANAGEMENT APPLICATIONS

Based on this research, the two optimal treatments for growing out T. maxima seed

are the open x recirculation system and shade x recirculation system. The combination of

evaporative coolers with the rollover system was insufficient for controlling high

temperatures during the peak hours of the day leading to increased mortality. The halide

treatment had high mortality from harmful UV radiation and also proved difficult to

maintain in the high humidity conditions of a greenhouse.

Cost of clam seed was the main limiting factor of this project due to lack of

production and transport stress. For this study, clam seed was obtained at $1 per clam

including shipping. Currently, there are no sources of clam seed in Florida. Technology

to produce clam seed in Florida would need to be developed for a more consistent supply

of seed. This technology could be carried over from well-established bivalve hatcheries

such as those used to produce the hard clam Mercenaria mercenaria. A local source of

seed may allow increased survival since shipping is probably very stressful.

Care must be taken so that no potential diseases are transferred over to the hard

clam production facilities such as the Perkinsus strain found in some giant clams

(Williams and Bunkley-Williams 1990). The negative impacts of culturing exotic species

can be avoided by practicing the standard Best Management Practices (BMPs) of the

aquaculture industry.

Another aspect that needs to be addressed is selection of a suitable substrate for

growing out seed clams. The substrate should be more porous than ceramic tiles. Some









of the currently used substrates for giant clam production are concrete trays and basalt

chips. These may not be as practical as other types of substrate that can be acquired and

handled with more ease such as plastic or fiberboard.

Established marine ornamental producers, who already have inland production

tanks or systems in place, can add clams in for polyculture growout. This would lower

economic risk since there is less start up costs. Corals may be grown in conjunction with

these clams and may be beneficial for a source of differing strains of zooxanthellae.

Lowering the price of clam seed to the prices that are paid for Mercenaria seed

would allow for longer grow-out periods. More suitable substrate may also allow higher

survival. Only if these two challenges are addressed, can the culture techniques utilized

in this study become profitable for a small-scale giant clam production facility.
















APPENDIX A
CLAM COLOR READINGS

Table A-1. Individual color readings for Blue seed size clams. A* values are more
negative with more green color and more positive with more red. B* values
are more negative with more blue color and more positive with more yellow.
Spectral peaks are also listed for the blue (400-500 nm), green (500-560 nm),
and brown (yellow/orange) (560-650 nm) regions of the color spectrum.
Clam Color Readings

Blues clam ID # t(0) t(1) t(2) t(3)
Wavelength peak in blue region (nm) 112 440 415 442 437
Reflection (%) 112 2.9 1.4 8.3 3
Wavelength peak in green/brown region (nm) 112 573 605 573 573
Reflection (%) 112 3.2 0.8 7.4 1.8
B* 112 4.2 0.9 2 2.7
Wavelength peak in blue region (nm) 1111 433 441 435 425
Reflection (%) 1111 2.9 7.6 2.1 5.9
Wavelength peak in green/brown region (nm) 1111 605 605 605 573
Reflection (%) 1111 4.7 4.4 1.1 1.2
B* 1111 4.0 2 -1.2 0.5
Wavelength peak in blue region (nm) 715 442 443 437 439
Reflection (%) 715 4.6 5.2 5.4 5.8
Wavelength peak in green/brown region (nm) 715 573 573 573 573
Reflection (%) 715 5.9 4.8 1.1 2.1
B* 715 10.5 9.7 -4.9 3.7
Wavelength peak in blue region (nm) 522 443 442-469 439-500 dead
Reflection (%) 522 4 3.5 2-2.3 dead
Wavelength peak in green/brown region (nm) 522 573 605 573-605 dead
Reflection (%) 522 5 4.8 2.4-2.5 dead
B* 522 10.5 9.7 -4.9 dead
Wavelength peak in blue region (nm) 8211 432 438 415 427
Reflection (%) 8211 2.7 6.8 10.3 3.9
Wavelength peak in green/brown region (nm) 8211 573 605 605 605
Reflection (%) 8211 1 5.8 2.9 1.8
B* 8211 1.6 0.5 -5.9 -0.4
Wavelength peak in blue region (nm) 9210 417 442 412 406
Reflection (%) 9210 2 4.5 5.3 3.82
Wavelength peak in green/brown region (nm) 9210 573 608 573 573
Reflection (%) 9210 2 2.9 1.6 1
B* 9210 1.3 3.8 -3.4 -0.1
Wavelength peak in blue region (nm) 1237 443 463 433 410
Reflection (%) 1237 1.6 2.3 4.2 1.6
Wavelength peak in green/brown region (nm) 1237 573 605 573 653










Table A-1. Continued.


Clam Color Readings


Blues
Reflection (%)
B*


clam ID #
1237
1237


Wavelength peak in blue region (nm)
Reflection (%)

Wavelength peak in green/brown region (nm)
Reflection (%)
B*


dead dead
dead dead

dead dead
dead dead
dead dead











Table A-2. Individual color readings for Green seed size clams. A* values are more
negative with more green color and more positive with more red. B* values
are more negative with more blue color and more positive with more yellow.
Spectral peaks are also listed for the blue (400-500 nm), green (500-560 nm),
and brown (yellow/orange) (560-650 nm) regions of the color spectrum.

Clam Color Readings

Greens clam ID # t(0) t(1) t(2) t(3)
Wavelength peak in blue region (nm) 721 N/A N/A 455 400
Reflection (%) 721 N/A N/A 3.7 4
Wavelength peak in green/brown region (nm) 721 N/A N/A 573 573
Reflection (%) 721 N/A N/A 1.3 0.6
A* 721 N/A N/A -0.6 0
Wavelength peak in blue region (nm) 311 495 461 400 490
Reflection (%) 311 3.6 3.2 2.7 1.3
Wavelength peak in green/brown region (nm) 311 573 573 603 573
Reflection (%) 311 6.8 1.1 3 1
A* 311 -0.3 -0.9 1.1 0.3
Wavelength peak in blue region (nm) 7110 495 485 485 443
Reflection (%) 7110 4.1 3.2 2.6 1.3
Wavelength peak in green/brown region (nm) 7110 573 573 573 573
Reflection (%) 7110 5.5 3.9 4.3 1.6
A* 7110 -0.8 0.6 0.3 0
Wavelength peak in blue region (nm) 7212 490 495 475 dead
Reflection (%) 7212 2.7 3.1 2.3 dead
Wavelength peak in green/brown region (nm) 7212 573 573 500-600 dead
Reflection (%) 7212 4.1 4 2.1 dead
A* 7212 -0.5 -1.1 -0.4 dead










Table A-3. Individual color readings for Gold seed size clams. A* values are more
negative with more green color and more positive with more red. B* values
are more negative with more blue color and more positive with more yellow.
Spectral peaks are also listed for the blue (400-500 nm), green (500-560 nm),
and brown (yellow/orange) (560-650 nm) regions of the color spectrum.

Clam Color Readings

clam
Golds ID # t(0) t(1) t(2) t(3)
Wavelength peak in blue region (nm) 225 485 495 440 440
Reflection (%) 225 2.5 4.5 1.2 1.5
Wavelength peak in green/brown region (nm) 225 573 573 573 573
Reflection (%) 225 7.4 6.3 1.8 1.1
B* 225 9.3 10.4 2.3 0.7
Wavelength peak in blue region (nm) 1212 443 443 468 426
Reflection (%) 1212 2.5 8.3 4.5 2.5
Wavelength peak in green/brown region (nm) 1212 573 604 573 607
Reflection (%) 1212 3.5 9.7 6.3 0.9
B* 1212 8.8 12.5 3.3 4.1
Wavelength peak in blue region (nm) 3110 495 495 435 495
Reflection (%) 3110 1.3 2.5 2.1 1.5
Wavelength peak in green/brown region (nm) 3110 573 573 573 573
Reflection (%) 3110 5.2 3.7 0.9 2.7
B* 3110 9.1 11.6 1.8 5.2















APPENDIX B
WATER QUALITY


Table B-1. Water Quality for Recirculation System (mg/L except for TN


Week Ammonium
0 5
1 5
2 5
3 40
4 50
5 60
6 55
7 60
8 60
9 20
10 10
11 5
12 10
13 10


Date
04/01/2005
04/08/2005
04/15/2005
04/22/2005
04/29/2005
05/06/2005
05/13/2005
05/20/2005
05/27/2005
06/03/2005
06/10/2005
06/17/2005
06/24/2005
06/27/2005


NH3
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm


N02
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
1.5
0.75
0.6
0.45
0.3
<.15


N03
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10


ig/L).
TN
545





2645




2750




1690









Table B-2. Water Quality for Recirculation System (mg/L except for TP = ig/L).
Date Week CA test kits CA ICP Alkalinity TP salinity ppt
04/01/2005 0 438 370 171 13 35
04/08/2005 1 438 171 35
04/15/2005 2 412 171 35
04/22/2005 3 400 171 35
04/29/2005 4 420 171 35
05/06/2005 5 420 400 188 13 35
05/13/2005 6 420 205 35
05/20/2005 7 450 205 35
05/27/2005 8 475 222 35
06/03/2005 9 420 400 205 9 35
06/10/2005 10 400 171 35
06/17/2005 11 400 205 35
06/24/2005 12 420 188 35
06/27/2005 13 420 400 205 5 35










Table B-3. Mean Water Quality for Rollovers (mg/L except for TN


Date
04/01/2005
04/08/2005
04/15/2005
04/22/2005
04/29/2005
05/06/2005
05/13/2005
05/20/2005
05/27/2005
06/03/2005
06/10/2005
06/17/2005
06/24/2005
06/27/2005


Week
0
1
2
3
4
5
6
7
8
9
10
11
12
13


Ammonium
5
5
5
55
45
45
55
45
40
40
40
50
45
50


NH3
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm
<0.6 ppm


N02
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15


N03
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10


TN
559





1883




1387




1688






72


Table B-4. Mean Water Quality for Rollovers (mg/L except for TP = g/L).


Date
04/01/2005
04/08/2005
04/15/2005
04/22/2005
04/29/2005
05/06/2005
05/13/2005
05/20/2005
05/27/2005
06/03/2005
06/10/2005
06/17/2005
06/24/2005
06/27/2005


Week
0
1
2
3
4
5
6
7
8
9
10
11
12
13


CA test kits
423
417
422
417
407
407
413
412
407
412
413
412
423
422


CA ICP
370





400




410




420


Alkalinity
171
171
171
188
197
200
202
202
211
214
211
208
217
217


Salinity ppt
35
35
35
35
35
35
35
35
35
35
35
35
35
35






73


Table B-5. Mean, max, and min water temperatures for each recirculation treatment tank
for 90-day study.
Recirculation Temperatures (C)
Tank mean max min
1 26.3 29.4 23.5
2 26.2 29.5 23.5
3 26.3 29.6 23.6
6 26.3 30.4 23.6
7 26.2 30.2 23.6
9 26.0 29.3 23.4






74


Table B-6. Mean, max, and min water temperatures for each rollover treatment tank for
90-day study.
Rollover Temperatures (C)
Tank mean max min
4 28.3 34.7 23.5
5 27.1 33.2 22.8
8 28.1 34.3 23.5
10 27.3 33.2 22.8
11 27.3 33.1 22.6
12 27.6 33.7 22.6











32

31

030 -

29 -

S28
C.
27 v N. W I

2 6 A xf >#A -

25
1 2 3 4 5 6 7 8 9 10 11 12 13
Week

Figure B-1. Mean weekly temperatures for two culture systems. Rollovers are marked
by the solid line. Recirculation tanks are marked by the dashed line.
















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BIOGRAPHICAL SKETCH

Micah Alo was born in Manila, the capitol of the Philippines, in 1977. His family

soon immigrated to the United States in 1984 and settled in Plainsboro, New Jersey, for

his formative school years. Numerous family vacations to the Florida Keys had ignited a

passion for aquatic biology which drove Micah to receive his AA degree at the Florida

Keys Community College. From there he went on to finish his bachelor's degree in

marine biology at Florida Atlantic University in Boca Raton, Florida, earning his degree

cum laude and being initiated into Phi Theta Kappa in 2000. He then worked at the

University of Florida's Tropical Aquaculture Laboratory and the Whitney Laboratory for

Marine Bioscience for 4 years. Afterwards, he elected to pursue a Master of Science

degree in the University of Florida's Department of Fisheries and Aquatic Sciences,

which he hopes will help him to pursue a career in the field of aquaculture.