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Evaluation of the Calanoid Copepod Pseudodiaptomus pelagicus as a First Feed for Florida Pompano, Trachinotus carolinus,...

Permanent Link: http://ufdc.ufl.edu/UFE0025056/00001

Material Information

Title: Evaluation of the Calanoid Copepod Pseudodiaptomus pelagicus as a First Feed for Florida Pompano, Trachinotus carolinus, Larvae
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Cassiano, Eric
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: artemia, copepods, firstfeed, fish, larvae, marine, microalgae, pompano, rotifers
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Production of many marine fish species is currently impeded by poor performance during the larval phase. Traditional live feeds, such as rotifers, Brachionus spp., and brine shrimp, Artemia spp., can be nutritionally deficient, may not constitute the appropriate size range, and may not elicit a feeding response and therefore are inadequate for many marine fish species. An evaluation of copepods as a live feed for marine fish larvae is critical to the expansion and development of larval rearing techniques. Recently, advances in copepod culture have increased interest in their application as a live feed. In this thesis, the calanoid copepod Pseudodiaptomus pelagicus was evaluated as a first food for marine fish larvae. The development of culture techniques for P. pelagicus associated with marine fish larviculture, including batch cultures, mass-scale production, maintenance, nauplii collection, and microalgae cultures are discussed. Furthermore, five trials were conducted to evaluate the performance of Florida pompano, Trachinotus carolinus, larvae fed nauplii of P. pelagicus. The Florida pompano is a highly prized marine fish species whose larviculture protocol currently includes a live feeds regime of rotifers and brine shrimp nauplii. Copepods have not been evaluated as a live food for Florida pompano larvae. In this evaluation, Florida pompano larvae were fed five diets, which included P. pelagicus nauplii, and were compared to the standard reference diet (SRD), which consisted solely of rotifers. Dietary treatments were fed during the first 9 days post hatch (DPH) in both 13-L and 170-L tank systems. Phase feeding of copepods fed for the first day, the first three days, and mixed with rotifers for the entire trial were examined. Copepods fed exclusively during the entire trial and in a mesocosm were also examined. Significant increases in growth, survival, and resistance to prolonged durations of net stress resulted. Florida pompano larvae fed copepod nauplii for the first day of feeding, at a density of 2 3 nauplii/mL, consistently had advantageous results when compared to larvae fed only rotifers. Beneficial results were also documented when larvae were fed nauplii for the first three days of feeding. But increased quantities of nauplii were needed to provide sufficient nutrients for growth and survival beginning on approximately 3 DPH. This density dependence was reflected in the results of larvae fed copepods for the entire trial, where survival was significantly higher than the other dietary treatments, but growth was significantly reduced. Larvae fed the mixed diet had similar survival as larvae fed the SRD (approximately 40%) however greater growth and resistance to stress occurred with larvae fed copepods, possibly reflecting a nutritional advantage to copepods in the diet. Resistance to net stress was greater for those larvae fed copepods during the trials, and survival was directly related to the duration copepods were fed. Larvae fed copepods were consistently alive after 10 minutes of net stress. Poor results were obtained from trials examining the use of a mesocosm feeding strategy. Refinement of stocking techniques and copepod preparation is discussed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Eric Cassiano.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Ohs, Cortney.
Local: Co-adviser: Petty, Denise.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025056:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025056/00001

Material Information

Title: Evaluation of the Calanoid Copepod Pseudodiaptomus pelagicus as a First Feed for Florida Pompano, Trachinotus carolinus, Larvae
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Cassiano, Eric
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: artemia, copepods, firstfeed, fish, larvae, marine, microalgae, pompano, rotifers
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Production of many marine fish species is currently impeded by poor performance during the larval phase. Traditional live feeds, such as rotifers, Brachionus spp., and brine shrimp, Artemia spp., can be nutritionally deficient, may not constitute the appropriate size range, and may not elicit a feeding response and therefore are inadequate for many marine fish species. An evaluation of copepods as a live feed for marine fish larvae is critical to the expansion and development of larval rearing techniques. Recently, advances in copepod culture have increased interest in their application as a live feed. In this thesis, the calanoid copepod Pseudodiaptomus pelagicus was evaluated as a first food for marine fish larvae. The development of culture techniques for P. pelagicus associated with marine fish larviculture, including batch cultures, mass-scale production, maintenance, nauplii collection, and microalgae cultures are discussed. Furthermore, five trials were conducted to evaluate the performance of Florida pompano, Trachinotus carolinus, larvae fed nauplii of P. pelagicus. The Florida pompano is a highly prized marine fish species whose larviculture protocol currently includes a live feeds regime of rotifers and brine shrimp nauplii. Copepods have not been evaluated as a live food for Florida pompano larvae. In this evaluation, Florida pompano larvae were fed five diets, which included P. pelagicus nauplii, and were compared to the standard reference diet (SRD), which consisted solely of rotifers. Dietary treatments were fed during the first 9 days post hatch (DPH) in both 13-L and 170-L tank systems. Phase feeding of copepods fed for the first day, the first three days, and mixed with rotifers for the entire trial were examined. Copepods fed exclusively during the entire trial and in a mesocosm were also examined. Significant increases in growth, survival, and resistance to prolonged durations of net stress resulted. Florida pompano larvae fed copepod nauplii for the first day of feeding, at a density of 2 3 nauplii/mL, consistently had advantageous results when compared to larvae fed only rotifers. Beneficial results were also documented when larvae were fed nauplii for the first three days of feeding. But increased quantities of nauplii were needed to provide sufficient nutrients for growth and survival beginning on approximately 3 DPH. This density dependence was reflected in the results of larvae fed copepods for the entire trial, where survival was significantly higher than the other dietary treatments, but growth was significantly reduced. Larvae fed the mixed diet had similar survival as larvae fed the SRD (approximately 40%) however greater growth and resistance to stress occurred with larvae fed copepods, possibly reflecting a nutritional advantage to copepods in the diet. Resistance to net stress was greater for those larvae fed copepods during the trials, and survival was directly related to the duration copepods were fed. Larvae fed copepods were consistently alive after 10 minutes of net stress. Poor results were obtained from trials examining the use of a mesocosm feeding strategy. Refinement of stocking techniques and copepod preparation is discussed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Eric Cassiano.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Ohs, Cortney.
Local: Co-adviser: Petty, Denise.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025056:00001


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EVALUATION OF THE CALANOID COPEPOD PSEUDODIAPTOMUS PELAGICUS AS A FIRST FEED FOR FLORIDA POMPANO, TRACHINOTUS CAROLINUS LARVAE By ERIC JON CASSIANO 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 2009 1

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2009 Eric Jon Cassiano 2

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To my Family, for their enduring patience and unconditional love 3

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ACKNOWLEDGMENTS Above all, I would like to thank my wonderf ul wife, Mandy Cassiano, and son, Issac, for their love, patience, and inspirati on. Their presence in my life is a blessing and a true testament to the strength of family. I would also like to thank my parents, Coley Cassiano and Anna Ip, for their encouragement and support during periods of doubt and self delusion. Also thank you to their respective spouses, Meta Ca ssiano and Donald Ip, for providing the warmth and strength necessary for the completion of this project. To my brothers, Damon and Nathan Cassiano, thank you for not pretending to act interested in copepods and reminding me there is more to life than academia. I would like to acknowledge my major professor, Cortne y Ohs, for his education, guidance, and willingness to let me learn through mi stakes. I would also like to acknowledge my co-chair, Denise Petty, and committee member, Jeff Hill, for their shared expertise in their respective fields. I would like to acknowledge Chuck Weir ich for providing larvae and knowledge pertaining to the culture of Florida pompano, and Erik Stenn for providing copepods for the completion of larval trials. Lastly, I would like to thank the staff and students at the University of Floridas Indian River Resear ch and Education Cent er for their support, knowledge, and guidance during the comp letion of this thesis. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................10 ABSTRACT ...................................................................................................................................12 CHAPTER 1 INTRODUCTION................................................................................................................. .14 Traditional Live Feeds ............................................................................................................15 Rotifers ............................................................................................................................15 Brine Shrimp ...................................................................................................................17 Copepods as Live Feeds .........................................................................................................19 Poecilostomatoida ............................................................................................................19 Cyclopoida .......................................................................................................................20 Harpacticoida ...................................................................................................................20 Calanoida .........................................................................................................................21 Pseudodiaptomus pelagicus ............................................................................................24 Stress Resistance .....................................................................................................................26 Florida Pompano Larviculture ................................................................................................28 Objectives ...............................................................................................................................29 2 MICROALGAE AND CO PEPOD CULTURE.....................................................................30 Introduction .............................................................................................................................30 Water Treatment .....................................................................................................................30 Microalgae Culture Techniques ..............................................................................................31 Pseudodiaptomus pelagicus Culture Techniques ...................................................................33 Water Quality ..................................................................................................................33 Diet ..................................................................................................................................34 Equipment ........................................................................................................................34 Contamination .................................................................................................................36 Airlift Efficiency .............................................................................................................37 Pseudodiaptomus pelagicus Cultures .....................................................................................38 Stock Cultures .................................................................................................................39 200-L Cultures .................................................................................................................39 Mass-Scale Cultures ........................................................................................................41 Conclusion ..............................................................................................................................43 Observations ....................................................................................................................43 Future Studies ..................................................................................................................45 5

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3 TRIAL 1...................................................................................................................... ............46 Material and Methods .............................................................................................................46 Water Treatment ..............................................................................................................46 Spawning and Egg Incubation .........................................................................................46 Larval Rearing and Experimental Design .......................................................................47 Dietary Treatments ..........................................................................................................48 Rotifer Culture and Enrichment......................................................................................48 Copepod Culture..............................................................................................................49 Sample Collection and Morphometric Analysis ..............................................................49 Stress Resistance Analysis ..............................................................................................50 Net Stress .........................................................................................................................50 Salinity Stress..................................................................................................................51 Fatty Acid Analysis .........................................................................................................51 Statistical Analysis ..........................................................................................................51 Results .....................................................................................................................................52 Water Quality ..................................................................................................................52 Growth .............................................................................................................................52 Survival ............................................................................................................................53 Stress Resistance .............................................................................................................53 Net Stress .........................................................................................................................53 Salinity Stress..................................................................................................................53 Discussion ...............................................................................................................................54 4 TRIAL 2...................................................................................................................... ............62 Material and Methods .............................................................................................................62 Spawning and Egg Incubation .........................................................................................62 Larval Rearing and Experimental Design .......................................................................62 Dietary Treatments ..........................................................................................................62 Copepod Culture..............................................................................................................63 Stress Resistance Analysis ..............................................................................................63 Net Stress .........................................................................................................................63 Salinity Stress..................................................................................................................63 Results .....................................................................................................................................63 Water Quality ..................................................................................................................63 Growth .............................................................................................................................63 Survival ............................................................................................................................64 Stress Resistance .............................................................................................................65 Net Stress .........................................................................................................................65 Salinity Stress..................................................................................................................65 Discussion ...............................................................................................................................65 5 TRIAL 3...................................................................................................................... ............71 Material and Methods .............................................................................................................71 Spawning and Egg Incubation .........................................................................................71 6

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Larval Rearing and Experimental Design .......................................................................71 Dietary Treatments ..........................................................................................................71 Copepod Culture..............................................................................................................72 Sample Collection and Morphometric Analysis ..............................................................72 Results .....................................................................................................................................72 Water Quality ..................................................................................................................72 Growth .............................................................................................................................72 Survival ............................................................................................................................73 Discussion ...............................................................................................................................73 6 TRIAL 4...................................................................................................................... ............77 Material and Methods .............................................................................................................77 Spawning and Egg Incubation .........................................................................................77 Larval Rearing and Experimental Design .......................................................................77 Dietary Treatments ..........................................................................................................77 Rotifer Culture and Enrichment......................................................................................78 Copepod Culture..............................................................................................................78 Sample Collection and Morphometric Analysis ..............................................................79 Net Stress Resistance Analysis ........................................................................................79 Results .....................................................................................................................................79 Water Quality ..................................................................................................................79 Growth .............................................................................................................................79 Survival ............................................................................................................................81 Net Stress Resistance .......................................................................................................81 Discussion ...............................................................................................................................82 7 TRIAL 5...................................................................................................................... ............88 Material and Methods .............................................................................................................88 Spawning and Egg Incubation .........................................................................................88 Larval Rearing and Experimental Design .......................................................................88 Dietary Treatments ..........................................................................................................89 Rotifer Culture and Enrichment......................................................................................89 Copepod Culture..............................................................................................................89 Sample Collection and Morphometric Analysis ..............................................................90 Net Stress Resistance Analysis ........................................................................................90 Results .....................................................................................................................................90 Water Quality ..................................................................................................................90 Growth .............................................................................................................................90 Survival ............................................................................................................................91 Net Stress Resistance .......................................................................................................91 Discussion ...............................................................................................................................91 8 CONCLUSION................................................................................................................... ....97 LIST OF REFERENCES .............................................................................................................101 7

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BIOGRAPHICAL SKETCH .......................................................................................................108 8

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LIST OF TABLES Table page 3-1 Dietary treatments fed to Florida pompano larvae ( Trachinotus carolinus ) during the experimental trials. .............................................................................................................60 3-2 Water quality variables measured during the experimental trials. .........................................61 9

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LIST OF FIGURES Figure page 3-1 The mean standard length (m m) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary trea tments during trial 1. .........................................................................57 3-2 The mean body depth (mm) of Florida pompano larvae ( Trachinotus carolinus ) fed different dietary trea tments during trial 1. .........................................................................57 3-3 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 1. ......................................................................................58 3-4 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments exposed to net stress during trial 1. ......................................................58 3-5 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments after 2 hours of salinity stress during trial 1. ........................................59 3-6 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments after 18 hours of salinity stress during trial 1. ......................................59 4-1 The mean standard length (m m) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary trea tments during trial 2. .........................................................................68 4-2 The mean body depth (mm) of Florida pompano larvae ( Trachinotus carolinus ) fed different dietary trea tments during trial 2. .........................................................................68 4-3 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 2 .........................................................................................69 4-4 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments exposed to net stress during trial 2. ......................................................69 4-5 The mean time to death of Florida pompano ( Trachinotus carolinus ) larvae fed different dietary treatments exposed to 100 mg/L salinity seawater during trial 2. .........................70 5-1 Histology section (hemotoxy lin and eosin stain) displaying Vibrio sp. (V) within the gills of Florida pompano larvae ( Trachinotus carolinus ) fed copepods during trial 3. .....76 6-1 The mean standard length (m m) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary trea tments during trial 4. .........................................................................86 6-2 The mean body depth (mm) of Florida pompano larvae ( Trachinotus carolinus ) fed different dietary trea tments during trial 4. .........................................................................86 6-3 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 4. ........................................................................................87 10

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6-4 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments exposed to net stress during trial 4. ......................................................87 7-1 The mean standard length (m m) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary trea tments during trial 5. .........................................................................95 7-2 The mean body depth (mm) of Florida pompano larvae ( Trachinotus carolinus ) fed different dietary trea tments during trial 5. .........................................................................95 7-3 The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 5. ........................................................................................96 11

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF THE CALANOID COPEPOD PSEUDODIAPTOMUS PELAGICUS AS A FIRST FEED FOR FLORIDA POMPANO, TRACHINOTUS CAROLINUS LARVAE By Eric Jon Cassiano August 2009 Chair: Cortney L. Ohs Cochair: B. Denise Petty Major: Fisheries a nd Aquatic Sciences Production of many marine fish species is currently impeded by poor performance during the larval phase. Traditional live feeds, such as rotifers, Brachionus spp., and brine shrimp, Artemia spp., can be nutritionally deficient, may not constitute the appropriate size range, and may not elicit a feeding response and therefore are inadequate for ma ny marine fish species. An evaluation of copepods as a live f eed for marine fish larvae is critical to the expansion and development of larval rearing techniques. Recen tly, advances in copepod culture have increased interest in their ap plication as a live feed. In this thesis, the calanoid copepod Pseudodiaptomus pelagicus was evaluated as a first food for marine fish larvae. The development of culture techniques for P. pelagicus associated with marine fish larviculture, includi ng batch cultures, mass-scale production, maintenance, nauplii collection, and microa lgae cultures are discussed. Furthermore, five trials were conducted to evaluate the performa nce of Florida pompano, Trachinotus carolinus, larvae fed nauplii of P. pelagicus. The Florida pompano is a highly prized marine fish species whose larviculture protocol currently includes a live feeds re gime of rotifers and brine shrimp nauplii. Copepods have not been evaluated as a live food for Florida pompano larvae. In this evaluation, Florida pompano 12

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larvae were fed five diets, which included P. pelagicus nauplii, and were compared to the standard reference diet (SRD), wh ich consisted solely of rotifers. Dietary treatments were fed during the first 9 days post hatch (DPH) in both 13-L and 170-L tank systems. Phase feeding of copepods fed for the first day, the first three days, a nd mixed with rotifers for the entire trial were examined. Copepods fed exclusively during the entire trial and in a mesocosm were also examined. Significant increases in growth, survival, and resistan ce to prolonged durations of net stress resulted. Florida pompano larvae fed copepod nauplii for th e first day of feeding, at a density of 2 nauplii/mL, consistently had advantageous result s when compared to larvae fed only rotifers. Beneficial results were also doc umented when larvae were fed nauplii for the first three days of feeding. But increased quantities of nauplii we re needed to provide sufficient nutrients for growth and survival beginning on approximately 3 DPH. This density dependence was reflected in the results of larvae fed copepods for the enti re trial, where survival was significantly higher than the other dietary treatments, but growth was significantly reduced. Larvae fed the mixed diet had similar survival as larvae fed the SR D (approximately 40%) however greater growth and resistance to stress occurred with larvae fed copepods, possibly reflecting a nutritional advantage to copepods in the diet. Resistance to net stre ss was greater for those larvae fed copepods during the trials, and survival was directly related to the duration copepods were fed. Larvae fed copepods were consistently alive after 10 minutes of net stress. Poor results were obtained from trials examining the use of a me socosm feeding strategy. Refine ment of stocking techniques and copepod preparation is discussed. 13

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CHAPTER 1 INTRODUCTION Expansion of marine fish production methods is critical to the a dvancement of the food, bait, and ornamental aquaculture industries. Currently, only a limited number of marine fish species are being produced with variable suc cess. The major impediment to commercial production of currently grown species and success with candidate species is the utilization of an appropriate live feed during the fi rst feeding phase of the larval cy cle. This period is extremely crucial for marine fish larvae. A live feed w ith an adequate nutritiona l composition, constituting a suitable size range that stimulates a feeding response is necessary for the expansion of the number of species of marine fish cultured. Copepods have these characteristics and are the natural food of marine fish larvae. Therefor e, improvements in growth, survival, and stress resistance should be observed in cultu red marine fish larvae fed copepods. An important dietary requirement for the optim al development of marine fish larvae is sufficient levels of highly unsaturated fatty acids (HUFAs), particularly docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachi donic acid (AA) (Sargent et al., 1999a). Improvements in growth, survival, and stress re sistance are attained when suitable absolute amounts or ratios of these fatty acids are present in larval di ets (Sargent et al., 1999b). The optimal levels are species-specific, however, an approximate ratio for DHA:EPA:AA of 10:5:1 is believed to be appropriate (Bell and Sargent, 2003). Gape is al so a limiting factor in successful marine fish production. Many marine fish larvae have a small gape and are unable to consume traditional live feeds including rotifers, Branchionus spp., and brine shrimp nauplii, Artemia spp. (McKinnon et al., 2003; Chesney, 2005). Also, the ab ility of a live feed organism to elicit a feeding response is important to the successful development of aquaculture techniques for candidate species (Stottrup and Norsker, 1997). 14

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Traditional Live Feeds Traditionally, rotifers, Branchionus spp., and brine shrimp nauplii, Artemia spp., are fed to marine fish during the larval phase. Well documen ted culture protocols, recent improvements in nutritional supplementation, and readily available eggs make the use of these live feeds appealing to commercial producers (Rainuzzo et al., 1997; Lavens and Sorgeloos, 1999; Stottrup and McEvoy, 2003). Even though these live feeds ar e inadequate for many marine fish larvae, they are the best option available and some species can be successfully produced. Rotifers Rotifers (phylum Rotifera) are small metazoan s with over 2000 species described; most inhabit freshwater lakes and ponds (L ubzens and Zmora, 2003). Two species, Brachionus plicatilis and B. rotundiformis have been used to culture over 60 species of marine finfish larvae and 18 species of crustacean larvae (Dhert, 1996). First identified as a pest in pond cultures of eels in Japan during the 1950s, tests rev ealed there were advantages to feeding B. plicatilis to red sea bream, Pagrus major larvae. This led to the mass culture of B. plicatilis for use as a live feed for other marine fish larvae (Dhert, 1996; Lubzens and Zmora, 2003). Rotifers propagate quickly under suitable conditions, with popul ations doubling over a few days (Lubzens and Zmora, 2003). Cultures can become quite dense with over 1000 rotifers/mL commonly maintained (Dhert, 1996; Lubzens and Zmora, 2003). This is an advantage for fish hatcheries with a large demand for live feeds during the larval phase Bentley et al. (2008) estimated that 400 billion rotifers are needed to produce 10 million gilthead sea bream, Sparus aurata, fry. On average, 20,000 to 100,000 rotifers will be used per fish larva during a 2030 day larval period (Lubzens and Zmora, 2003). Typically, rotifers are grown as batch cu ltures at salinities from 15 to 20 g/L and temperatures of approximately 26C, although specific culture characteristics vary between 15

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species and strains (Dhert, 1996; Lu bzens and Zmora, 2003). Prior to rearing fish larvae, rotifer production is increased through the use of high nut rient diets (Dhert, 1996 ; Lubzens and Zmora, 2003). Rotifer populations or resting eggs can be purchased, but typically a new culture is started from a subsample of another culture. On ce a stock culture is es tablished, the need to obtain new rotifers is reduced. Although rotifers can be grown in sufficient numbers to satisf y a large hatchery, there are problems with their adequacy as a live feed. For example, they do not have the proper nutritional composition for marine fish larvae (Stottrup, 2000; Hamre et al., 2008). Rotifers do not have appropriate levels of DHA, EPA, or AA, nor do they have the ability to elongate shorter chain fatty acids, therefore they must be enri ched prior to feeding to marine fish larvae (Rainuzzo et al., 1997; Sargent et al., 1997; Stottrup, 2000). Current enrichment techniques allow them to retain potentially adequate levels of HUFAs for several hours, provided they are kept at 10C to reduce their metabolism (Rainu zzo et al., 1997; Sargent et al., 1997). Once rotifers are placed in the larval tank, metabolism resumes and those nutrients are only available to the fish larvae for a short period of time (Rainuzzo et al., 1997; Sargent et al., 1997). The quantity of rotifers within a larval system must be constantly monitored to ensure that appropriate densities are maintained (Dhert 1996; Lubzens and Zmora, 2003). If rotifers propagate beyond the grazing pressure of fish larvae, not only will the fish larvae consume a nutritionally inadequate pr ey item, but the water quality can quickly become detrimental to the survival of the fish larvae (Dhert, 1996; Lub zens and Zmora, 2003). Rotifers also constitute a small size range (90 microns) which may not be adequate for smaller fish larvae (Lubzens and Zmora, 2003; McKinnon et al, 2003). Evidence further suggests that rotifers are not easily 16

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digested (Schipp et al, 1999) and their slow movement may not elicit a feeding response by marine fish larvae (Chesney, 2005). Brine Shrimp Brine shrimp (phylum Arthropoda) are the most widely used form of live feed for marine fish larvae in the world with over 2000 metric to ns of cysts marketed annually (Van Stappen et al., 1996; Dhont and Van Stappen, 2003). Brine shrimp, Artemia spp., can be split into two different types, zygogenetic speci es and parthenogenetic specie s (Dhont and Van Stappen, 2003). Zygogenetic species reproduce by the fusion of ma le and female gametes and parthenogenetic species reproduce by development of an unfertilized female gamete. Currently seven species of zygogenetic types and many strains of a parthe nogenetic species are acknowledged (Dhont and Van Stappen, 2003). All are referred to as Artemia The production of dormant cysts is a key characteristic of all species, and this makes Artemia an appealing live feed for fish and crustacean larvae because of av ailability (Van Stappen et al., 1996; Dhont and Van Stappen, 2003). Artemia cysts are stored dry until needed; optim al hatching is initiated in 155 g/L salinity water (Dhont and Van Stappen, 2003). Nauplii of the instar I and II stages are the most common stages of Artemia fed to larval fish, and they develop within 24 and 36 hours after hatching, respectively (Van Stappen et al., 1996; Dhont and Van Stappen, 2003). The ability of cysts to be stored for long periods of time and relative predictability of hatching success makes Artemia a suitable live feed for many hatcheries a nd marine fish species (Van Stappen et al., 1996; Dhont and Van Stappen, 2003). Artemia however, are a nutritionally deficient live feed for mo st developing marine fish larvae (Rainuzzo et al., 1997; Sargent et al., 1997; Stottrup, 2000). Like rotifers, they have insufficient levels of DHA, EPA, and AA (Rainu zzo et al., 1997; Sargen t et al., 1997; Stottrup, 2000). The instar I, a non-feeding stage, is incap able of enrichment but all other life stages can 17

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be enriched prior to feeding to marine fish larvae (Rainuzzo et al., 1997; Dhont and Van Stappen, 2003). Although thes e enrichments temporarily am eliorate their nutritional composition, the improved fatty acid levels are of ten inconsistent (Stottrup, 2000; Palmtag et al., 2006). Newly hatched Artemia nauplii are usually too larg e (~450 microns) for most first feeding marine fish larvae, often only being fed during subsequent stages of larval development (Van Stappen et al., 1996; Dhont and Van Sta ppen, 2003; Lee, 2003). Typically they are fed after the rotifer feeding phase and up to the transition to an artific ial diet (Sorgeloos et al., 2001; Lee, 2003). Complications with the ha tching and growth ch aracteristics of Artemia can also be an impediment to the successful culture of larvae. Decapsulation, hatching, and molting all produce membranes and molts which if not dealt with properly, can quickly deteriorate water quality of the system (Van Stappen et al., 1996; Dhont and Van Stappen, 2003). With advancements in artificial larval diets and the reduced availability of Artemia cysts from wild populations (Van Stappen et al., 1996; Dhont and Van Stappen, 2003), the use of Artemia may decrease in the future. Rotifers and Artemia have advantages as live feeds for marine fish larvae. Both are relatively easy and predictable to culture in appropriate numbers and feeding protocols are well documented for many currently cultured marine fish species (Lavens and Sargeloos, 1999; Stottrup and McEvoy, 2003). Enrich ment techniques are constantly being improved to provide not only essential nutrients, but al so antibiotics and other forms of prophylactic treatments for fish larvae (Lavens and Sargeloos, 1999; Stottr up and McEvoy, 2003). However, their use does have limitations and they are not effective for the majority of marine fish species. The bottleneck to the production of most marine fish species is the early larval stage and the 18

PAGE 19

inadequacy of rotifers as a first food for larvae, therefore cult ure of these species will likely require the feeding of copepods. Copepods as Live Feeds Copepods (phylum Arthropoda) are one of the most ubiquitous groups of marine organisms, with over 21,000 species currently described, and are a major component of the marine zooplankton community (Smithsonian Ins titution, 2008). It is well documented that in the wild, copepods constitute a major link in the nutrient pathway from primary producers to fish larvae serving as a supplemental or primary diet for first feeding marine fish larvae (Hunter, 1981; Delbare et al., 1996; Stottrup, 2003). Their role in the marine trophic system is essential to the survival of many marine fi sh species. To date, a limited number of researchers have investigated the efficacy of using copepods to cult ure various fish species. Ten orders of the Copepoda are currently recognized, bu t only four have been explored as live feeds for fish and crustacean larvae. These include Poecilostomatoi da, Cyclopoida, Harpacticoida, and Calanoida (Lee et al., 2005). Poecilostomatoida The Poecilostomatoida have an unclear symbio tic relationship with marine bivalves and only one member, Pseudomyicola spinosus, has been evaluated for us e as a live feed (Ho, 2005). Populations of P. spinosus were maintained on strips of mussel, Mytillus sp., meat and eggs sacs were collected daily (Ho, 2005). Once hatched, nauplii were cultured to adulthood (Ho, 2005). Theoretically, copepod populations could be maintained on mussel, Mytilus sp., beds and pelagic nauplii harvested daily and fed to marine fish larvae. However, evaluations of the symbiotic relationship and the potential effects of copepod production on mussel beds should be understood before evaluating this copepod species as a marine fish larval food. Members from the other three orders have b een explored much more extensively as a live feed. 19

PAGE 20

Cyclopoida A few Cyclopoida have been grown for use as live feed, mainly belonging to the genera Apocyclops and Oithona (Stottrup, 2006). Farhadian et al. (2009) increased survival of black tiger prawn, Penaeus monodon, post larvae by feeding a mixed diet of Apocyclops dengizicus and Artemia Apocyclops panamensis was fed to red snapper, Lutjanus campechanus larvae and Apocyclops royi was fed to grouper, Epinephelus coioides larvae all with inconclusive results (Stottrup, 2006). Surviv al of striped patio, Eugerres brasilianus, larvae was doubled through the use of Oithona oculata (Hernandez Molejon and Al varez-Lajonchere, 2003). Oithona spp. has also been harvested in wild zoopla nkton communities and fed to grouper, E. coioides larvae and produced increased survival and larvae disp layed preferential feeding on copepod nauplii (Toledo et al., 1999). Members of this order show potential for mass-scale culture as they can be cultivated in high densities, have appropriate nutritional composition, accept a variety of diets, and reach maturation quickly (Shansudin et al., 1997; Stottrup, 2006). Harpacticoida Copepods of the order Harpacticoida are primar ily benthic or epiben thic (Stottrup, 2003). Tisbe, Tigriopus and Euterpina are the three most commonly pr oduced genera for feeding fish or crustacean larvae (Stottrup, 2003). Tisbe biminiensis was fed to larvae and post larvae of the white shrimp, Litopenaeus vannamei and improved feed consumption during the mysis 2 and 3 stages (de Lima and Souza-Santos, 2007). Tisbe holothuriae nauplii fed to first feeding turbot, Psetta maxima larvae improved growth and survival as both a supplemental and primary diet (Stottrup and Norsker, 1997). Stottrup and No rsker (1997) also noted an increased rotifer consumption rate for those larvae fed a mixed di et as opposed to larvae fed solely rotifers. Tisbe spp. were also successfully used as a s upplemental diet for yellowtail clownfish, Amphiprion clarkii larvae increasing growth, survival, and the le vel of insulin-like growth factors I and II, 20

PAGE 21

which promote cartilage and muscle growth (Olivotto et al., 2008). Tigriopus japonicus has been fed to numerous fish speci es and significantly improved grow th and survival in black sea bream, Mylio macrocephalus (Stottrup, 2003). Mahi mahi, Coryphaena hippurus larvae fed Euterpina acutifrons showed improved growth, survival, and resistance to stress and these results were associated with increased levels of DH A in the copepods (Kraul et al., 1993; Stottrup, 2003). Some harpacticoid copepods have potential for mass-scale production because they can be grown in dense cultures (100/mL), withstand a wide range of environmental parameters, accept a variety of diets, reach maturity quickly, and have available life stages for feeding marine fish larvae (Fleeger, 2005; Stottrup, 2003). In addition to the genera Tisbe, Tigriopus and Euterpina Amphiascoides atopus has recently been mass produced with daily yields of 2 million copepod nauplii for 4 months of continuous culture (F leeger, 2005; Sun and Fleeger, 1995). Sun and Fleeger (1995) documented that two speci es of crustacean, the white shrimp, L. vannamei and the grass shrimp, Palaemonetes pugio and the darter goby, Gobionellus boleosoma can be successfully cultured on A. atopus. Nitokra lacustris is another harpact icoid copepod that appears suitable for mass-scale production (R hodes, 2003). Although harp acticoid copepods can be grown in high densities, their nauplii are of ten benthically oriented and display negative phototactic behavior; this limits their presence in the water column as available prey for pelagic fish larvae (Payne and Rippingale, 2001a). Fo r these reasons, development of culture systems for copepods produced to feed marine fish larvae have primarily consisted of calanoid copepods (Payne and Rippingale, 2001a). Calanoida Copepods from the order Calanoida are pre dominantly pelagic f ilter feeders and are concentrated in the water column (Stottrup, 2003). Numerous genera of calanoid copepods have 21

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been investigated for mass-scale culture as a po tential food organism for marine fish larvae, these genera include Acartia Gladioferens, Pseudodiaptomus Eurytemora Temora, Centropages Bestiolina Parvocalanus Labidocera, and Calanus (Evjemo et al., 2003; McKinnon et al., 2003; Lee et al., 2005). One of the most researched species of copepod in terms of culture techniques is Acartia tonsa (Lee et al., 2005; Marcus and W ilcox, 2007). Parameters such as temperature, salinity, diet, and egg production have been investigated (Marcus an d Wilcox, 2007; Peck and Holste, 2006; Stottrup and Jensen, 1990). A. tonsa broadcasts eggs into the water column, which can be collected and stored either in freshwater for up to 35 days, or cold (2C) for up to a year (Drillet et al., 2006; Hojgaard et al., 2008). Ogle et al. (2005) developed a mass-scale production system for A. tonsa that has been used to feed red snapper, L. campechanus larvae since 1998, significantly increasing survival of larvae at day 7 post hatch from approximately 3.0% to 16.5%. Southern flounder, Paralichthys lethostigma larvae fed a mixed diet of A. tonsa nauplii and B. plicatilis had increased length and he ight and more than double the survival and weight gain then those larvae fed solely B. plicatilis or B. rotundiformis (Wilcox et al., 2006). A. tonsa has also been used as a live feed with spotted seatrout, Cynoscion nebulosus (Lemus et al., 2008), cod, Gadus morhua, ladyfish, Elops saurus and Fundulus spp. (Stottrup, 2003). Other Acartia spp. have also been cultured to serv e as a live feed for larval fish. Acartia clausi fed to fourteen day-old seabass, Lates calcarifer larvae significantly improved growth and survival (Rajkumar and Kumara guru vasagam, 2006). Grouper, E. coioides larvae fed zooplankton collected from the wild which consisted primarily of Acartia tsuensis and Pseudodiaptomus spp., had improved growth and survival (Toledo et al., 1999) and grouper larvae showed preference for Acartia tsuensis nauplii over rotifers (Doi et al., 1997). Acartia 22

PAGE 23

sinjiensis were fed to red snapper, Lutjanus argentimaculatus larvae with mixed results (Stottrup, 2003). Research with A. sinjiensis investigated temperature, salinity, and photoperiod effects on egg production, hatchi ng, and early life stage developm ent to elucidate mass-scale culture techniques (Camus and Ze ng, 2008; Milione and Zeng, 2008). The culture techniques and efficacy of Gladioferens imparipes as a live feed are also well documented (Payne and Rippingale, 2001a). In 500-L batch culture systems, 439,000 nauplii were produced per day (0.88 nauplii/mL) and 1000L semi-continuous populations yielded up to 520,000 nauplii per day for 184 days (0.5 nauplii/mL) (Payne and Rippingale, 2001a). The effects of diet, salinity, cold storag e, and enrichment techniques of G. imparipes have all been explored (Payne and Rippingale, 2000a; Payne and Rippingale, 2001b). Naup lii can be stored at 8C for 12 days with 99% survival allowing sufficient numbers to be collected prior to feeding to fish larvae (Payne and Rippingale, 2001b). Nauplii were enriched for 6 hours in a mixture of Tahitian strain Isochrysis galbana (T-ISO) and Nannochloropsis occulata to increase their HUFA content (Payne and Rippinga le, 2001b; Payne et al., 2001). G. imparipes nauplii were fed to juvenile pipefish, Stigmatopora argus, with improved growth and 99% survival (Payne et al., 1998). West Australian seahorse, Hippocampus subelongatus juveniles were fed copepod nauplii and enriched Artemia, and results showed length, weight and survival were all significantly greater for larvae fed copepods (Payne and Rippinga le, 2000b). When fed a mixed diet (1:1) of G. imparipes nauplii and rotifers, West Australian dhufish, Glaucosoma hebraicum larvae displayed significantly greater growth and survival during a 25 day experiment when compared to larvae fed only rotifers (Payne et al, 2001). Pink snapper, Pagrus auratus larvae were fed a mixed diet (4:1) of rotifers and c opepod nauplii, a diet cons isting of copepod nauplii for the first six days then transitioned to a diet of rotifers, and a diet of only rotifers. Results 23

PAGE 24

showed both treatments fed copepods yielded sign ificantly greater growth and survival when compared to larvae fed only rotifers (Payne et al., 2001). Pseudodiaptomus spp. also demonstrate characteristics that make them suitable for massscale culture and for use as a live feed (Chen et al., 2006; Ogle et al., 2005; Puello-Cruz et al., 2009; Rhyne et al., 2009; Toledo et al., 1999). Pseudodiaptomus annandalei exhibits a wide salinity tolerance, and the highest reproduction oc curs at salinities from 5 g/L (Chen et al., 2006). Sheng et al. (2006) evaluated the feedi ng behavior of larval three-spot seahorse, Hippocampus trimaculatus and found larvae preferred P. annandalei nauplii over rotifers during the first 3 days post hatch. Larval seahorse selectively grazed on copepodites from 4 to 10 days post hatch, then began to eat adu lt copepods (Sheng et al., 2006). P. annandalei, in a mixture of wild caught zooplankton, was also fed to grouper, E. coioides larvae and improved growth and survival and larvae displayed feeding preference for copepod nauplii even at low prey densities (Doi et al., 1997). Puello -Cruz et al. (2009) compared different algal diets for Pseudodiaptomus euryhalinus and the highest production occurr ed in those fed the diatom Chaetoceros muelleri Pseudodiaptomus pelagicus First described by Clarence L. Herrick in 1884, specimens of Pseudodiaptomus pelagicus collected from the Mississippi Sound were used to classify the genus Pseudodiaptomus (Walter, 1989). Early descriptions of P. coronatus and P. americanus are now believed to be descriptions of P. pelagicus (Walter, 1989). Taxonomic Information : Pseudodiaptomus pelagicus Herrick, 1884 Kingdom: Animalia Phylum: Arthropoda Subphylum: Crustacea Class: Maxillopoda 24

PAGE 25

Subclass: Copepoda Infraclass: Neocopepoda Superorder: Gymnoplea Order: Calanoida Family: Pseudodiaptomidae (ITIS, 2009) P. pelagicus is an estuarine calanoid copepod native to the eastern Atlantic and Gulf coasts of the United States, from Massachusetts to Mexico (Walter, 1989). Females (1.30.57 mm) and males (0.92.13 mm) often spend their mature li fe stage copulating as paired units (Jacobs, 1961; Walter, 1989). P. pelagicus are maternal egg brooders pr oducing asymmetrical ovisacs with the right ovisac c ontaining 2 eggs and the left containing 9 eggs (Rhyne et al., 2009; Walter, 1989). Upon hatching in the second na upliar stage, nauplii molt through five more naupliar stages reaching the firs t copepodite stage by approximately 6 days at a temperature of 20.3C (Jacobs, 1961; Grice, 1969). Growth conti nues through six copepodite stages and they reach adulthood by approximately 18 days at 20. 3C (Jacobs, 1961; Grice, 1969). Increases in temperature significantly increase mean development time with growth from nauplii to adult taking approximately 10 days at 28C (Rhyne et al ., 2009). Adults spend the majority of their time on substrate or the walls and bottom of culture tanks, while nauplii appear to lack the ability to cling to substratum and are found in the water column (Jacobs, 1961; Rhyne et al., 2009). Populations of P. pelagicus maintained at the University of Floridas Indian River Research and Education Center (IRREC) were is olated from South Florida waters in 2003 and have been kept in continuous culture for 6 year s (Rhyne et al., 2009). An optimal temperature range of 28C was determined; this range reduces mean development time, increases survival, and promotes a shortened brood interval (Rhyne et al., 2009). Sex ratio and brood size 25

PAGE 26

were not affected by temperature (Rhyne et al., 2009). P. pelagicus can survive in a wide range of environmental parameters, tolerate heavy aera tion, sedimentation and suspended solids, can be cultured on a variety of microalgae diets, a nd can reach densities of 5/mL; these make P. pelagicus an ideal candidate for mass production (Rhyne et al., 2009). Stress Resistance A key component to expanding the number of marine fish cultur ed through the larval stage is an increased resistance to stress. Diets w ith the appropriate levels of HUFAs, specifically DHA, EPA, and AA have been shown to increase stress resistance in marine fish larvae (Kanazawa, 1997; Bell and Sargent, 2003). Copepods constitute th e appropriate composition of HUFAs for marine fish larvae (Sargent et al., 1997), and can be manipulated by feeding different microalgaes (van der Meeren et al., 2008). Theref ore, the use of copepods as a live feed during the larval stage of production may increase stress resistance of marine fish larvae. Stress resistance in marine fish larvae has been evaluated via prolonged exposure to air (Kraul et al., 1993; Ako et al., 1994), responses to bacterial inoculation (Chair et al., 1994; Yang et al., 2008), handling and transf er (Koven et al., 2001), salinity stress (Brinkmeyer and Holt, 1998; Nhu et al., 2009), temperature stress, exposure to low levels of dissolved oxygen (Kanazawa, 1997), and low pH (Wasielesky et al., 1997). A larvaes ability to withstand these stressors is directly related to nutrition. HUFAs are extremely important in improving the stress resistan ce of marine fish larvae (Coutteau et al., 1997; Bell and Sargent, 2003; Tocher et al., 2008). Kanazawa (1997) fed larval red sea bream, Pagrus major and juvenile marbled sole, Limanda yokohamae a diet containing DHA at 1% of the diet, and phospholipids (soybean lecithin) at 5% of the diet. A series of stress tests was instituted in cluding exposure to air, increa sed water temperature, and low 26

PAGE 27

dissolved oxygen levels. This di et increased growth and survival of the larvae, and larvae fed this diet had improved resistance to stress in all tests (Kanazawa, 1997). Ako et al. (1994) displayed an increased resi stance to air exposure for striped mullet, Mugil cephalus, larvae that had been fed enriched Artemia nauplii, although growth and survival of the larvae were not significan tly different between dietary treatments. Enriched Artemia nauplii had higher levels of n-3 HUFAs includi ng DHA (Ako et al., 1994). Red drum, Sciaenops ocellatus, larvae fed a diet with a DHA:EPA ratio of 3.78 outperformed diets with lower DHA:EPA ratios in a hypersaline (70g/L) stress ch allenge (Brinkmeyer and Holt, 1998) A diet fortified with 3% EPA and DHA improved stress tolerance to air exposure in black sea bream, Acanthopagrus schlegeli, larvae compared to a commercial diet (Om et al., 2001). Increased tolerance of cobia, Rachycentron canadum larvae to hypersaline stress test s was significantly correlated with elevated levels of DHA present in the live feed a nd fish larvae at various periods of the larval cycle (Nhu et al., 2009). AA in the diet of gilthead seabream, Sparus aurata, larvae improved resistance to handling stress and increased surviv al during the transition period onto a diet of Artemia (Koven et al., 2001). Kraul et al. (1993) demonstrated the bene fits of feeding th e harpacticoid copepod Euterpina acutifrons to larval mahimahi, Coryphaena hippurus. Multiple tests were conducted comparing stress resistance of larvae fe d diets consisting of copepod nauplii and Artemia enriched with different concentrations of SuperS elco, a commercial live feed enrichment (Kraul et al., 1993). To determine resi stance to stress, larvae (15 da ys post hatch) were blotted dry and held out of water in netting for 60 or 120 se conds, after which they were returned to tank water and survival was ascertained after 25 minutes (Kraul et al., 1993). Larvae fed Artemia with increased levels of enrichment, and ther efore higher levels of DHA, showed a greater 27

PAGE 28

resistance to the stress tests. However, larvae fed copepod nauplii had significantly less mortality (3 times) than any of the Artemia fed treatments in all of the experiments, even though their DHA level was similar to that of the enriched Artemia (Kraul et al., 1993). It was postulated that DHA from the copepod nauplii as a phospholipid, is more effectively incorporated by larval mahimahi than DHA from enriched Artemia, as a triglyceride. It was also suggested that the higher amino acid levels found in the copepod nauplii might have led to higher incorporation of DHA by the fish larvae. Florida Pompano Larviculture The Florida pompano, Trachinotus carolinus is a highly prized commercial and recreational marine fish species with a distribution al ong the south Atlantic and Gulf coasts of the United States (Muller et al., 2002). Low ha rvests of wild populations coupled with rising market demand have increased interest in develo ping culture techniques fo r this species (Muller et al., 2002; Weirich et al., 2006). Like most marine fish species, 5% survival through the larval phase impeded the development of production techniques. However, recent advances in Florida pompano larviculture and rotifer enrichme nt techniques have in creased survival (~35%) of larvae during the first feeding pha se (Cavalin and Weirich, 2009). Since 2004, experiments evaluating the larval performance of Florida pompano have been conducted by the United States Department of Agriculture, Agricultura l Research Service, Center for Reproduction and Larviculture, located on the campus of Harbor Branch Oceanographic Institute at Florid a Atlantic University in Fo rt Pierce, FL (USDA-ARS). Researchers have defined a prot ocol for first feeding Florid a pompano larvae that includes rotifers and Artemia (Weirich and Riley, 2005). Enriched rotifers are fed to larvae beginning on 2 days post hatch (DPH) and transitioned onto Artemia instar I nauplii on 10 DPH. Larvae are then transitioned onto Artemia metanauplii on 15 DPH and finally transitioned onto a 28

PAGE 29

formulated diet by 22 DPH. Greenwater culture conditions are maintained only while feeding rotifers. In Cavalin and Weiric h (2009), various commercial enrichments and microalgae diets were evaluated to determine the most efficient and effective enrichment method for rotifers fed to Florida pompano larvae. Re sults showed larvae fed rotifers enriched with commercial enrichments had higher survival and gr owth than those fed rotifers enriched with microalgae diets. Furthermore, larvae fed rotifer s enriched with the commercial enrichment OriGreen (Skretting, Italy) had highe r survival and only slightly le ss growth when compared to other commercial enrichments. Since Ori-Green requires much less time for enrichment, it was determined to be the most cost effective and adopted as a standard rotifer enrichment protocol. Objectives The purpose of this thesis is to develop suitable culture techniques for P. pelagicus and evaluate its efficacy as a live feed for marine fi sh larvae. An examination of batch cultures, mass-scale culture, nauplii collection and micr oalgae culture was conducted and is thoroughly discussed. Furthermore, five experiments were conducted to evaluate various feeding regimes with P. pelagicus as a primary and supplemental diet fo r first feeding Florida pompano larvae, and were compared to the standard reference diet of rotifers devel oped by the USDA-ARS. Differences in survival, growth (notochord le ngth and body depth), a nd stress resistance (resistance to air exposure and salinity stress) were measured to determine the effect of feeding copepod nauplii to Florida pompano larvae. 29

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CHAPTER 2 MICROALGAE AND COPEPOD CULTURE Introduction The efficient transfer of nutri ents to higher trophic levels is extremely important to successful larval culture of marine fish species. Nutritional components including essential amino acids and fatty acids are critical to the de velopment and survival of marine fish larvae. Research must be conducted in order to understand the specific nutritional requirements of each fish species produced. Furthermore, developm ent of a live feed program, which includes suitable prey species at each trophic level, is impor tant to the successful r earing of most species of marine fish. The presence of microalgae in larval rear ing systems has improved the performance of over 40 species of marine fish larvae (Muller-Feuga et al., 2003), although the mechanisms behind these improvements are not yet fully underst ood. Hypotheses include direct and indirect supply of nutrition, water quality improvement, light contrast, stimulation of feeding behavior and physiological processes, regulation of opport unistic bacterial populations, and improvements in the quality of live prey including copepod na uplii and rotifers (Muller-Feuga et al., 2003). Combinations of these benefits likely contribute to the successful culture of marine fish larvae. Water Treatment Microalgae and copepods were cultured with a 2:1 mixture of natura l seawater (32 g/L salinity) and well water (0 g/L sa linity) at the University of Fl oridas Indian River Research and Education Center (IRREC). Prior to use, al l water was chlorinated (150 mg/L) with sodium hypochlorite for 24 hours, then aerated for 24 hours until 0 mg/L chlorine was detected. Water was then pumped into 20,000-L storage silos. From the storage silos, water was pumped into the hatchery facil ity after passing through both 50 and 5 micron mesh bag filters. Seawater 30

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was stored in a 2500-L polyethylene tank and fre shwater was stored in a 1350-L polyethylene tank. Sodium hypochlorite was added to both seaw ater and freshwater (4 mg/L chlorine) when stored in tanks for longer than one week. S eawater and freshwater were mixed in a 2500-L polyethylene tank (mixed tank) to a salinity of 22 g/L. The mixed tank water was then continuously circulated through an 80 Watt UV ster ilizer (Emperor Aquatics Inc., PA) at rate of 170 L/minute which provided a sterilizat ion intensity of 30,000,000 microWatts/cm 2 Prior to use, any chlorine detected in the wate r was neutralized with s odium thiosulfate. As water left the mixed tank, it passed through both 1.0 and 0.5 micron cartridge filters. Microalgae Culture Techniques Tahitian strain Isochrysis galbana (T-ISO) and Thalassiosira weissflogii (TW) were fed to copepods during these experiments. All microalg ae culture techniques used were adapted from Anderson (2005). Fritz f/2 nutrient media soluti ons A and B (Fritz Industries Inc., TX) were provided to both T-ISO and TW microalgae cultu res following manufacturer instructions, except twice the suggested amount was supp lied to TW to elicit sufficien t growth. Sodi um metasilicate (Fritz Industries Inc., TX) was also provided to fa cilitate the formation of the cell wall in TW, a diatom (Andersen, 2005). All aeration provided to microalgae was continuously mixed with CO 2 at a rate of approximately 70 mL/minut e and passed through a 0.20 micron in-line air filter. All water used to grow microalgae was autoclaved (Consolidated Stills and Sterilizers, MA) and maintained at 21C and a salinity of 22 g/L unless otherwise noted. Once water was autoclaved, sterile procedures were used to reduce the poten tial for contamination of the microalgae (Andersen, 2005). Small (1-L) flasks were used to maintain stoc k cultures of each microalgae species. Stock cultures were a sterile culture used to preserve th e microalgae species within the facility and to inoculate larger cultures. Stoppers equipped with a 0.20 micron filter were inserted in the flasks 31

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to allow exchange of air, but prevent entrance of contaminants. Cultures were exposed to light levels from 1000 to 2000 lux and no aeration was provided. Small glass bottles (4-L) were used to grow microalgae for small scale experiments and to inoculate larger cultures. A tw o-holed rubber stopper was inserted into the bottle; each hole had a 6-mm diameter glass tube inserted. One tube extended to the bottom of the bottle and was used for aeration. The second was short and extended from the bottom to the top of the stopper and allowed air to escape. Algae were exposed to light levels from 1000 to 2000 lux. Maximum densities for T-ISO (15 million cells/mL) a nd TW (1 million cells /mL) were achieved before being used to in oculate larger cultures. Glass carboys (20-L) were used to grow microalgae for experiments, maintenance of copepod and rotifer populations, and to inoculate larger cultures. A two-holed rubber stopper with glass tubes, in a similar de sign to those used for the 4-L cu ltures, provided aeration to the cultures. Cultures were exposed to light le vels from 3500 to 4500 lux. Maximum densities for T-ISO (15 million cells/mL) and TW (1 mill ion cells/mL) were achieved before being used for experiments, feeding, or to inoculate larger cultures. Bags (450-L) were used to grow large quantities of T-ISO for experiments and maintenance of copepod and rotifer cultures. The details of the system and bag design cannot be discussed due to a contractual agreement with an industry partner. T-ISO bags were exposed to gradually increasing light levels from 300 to 18000 lux and attained densities up to 30 million cells/mL. TW was not grown in volumes larger than 20-L. 32

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Pseudodiaptomus pelagicus Culture Techniques Since 2005, a population of Pseudodiaptomus pelagicus has been maintained at IRREC in Fort Pierce, FL. This population was isolated in 2003 from the Indian River Lagoon and is maintained at Algagen LLC (Vero Beach, FL). Water Quality Water for copepod cultures came from the mixe d water tank, and prior to use was heavily aerated for 24 hours. This was done to avoid a possible adverse response by the copepods to any chemical present, including plasticizers, or iginating from the polye thylene storage tanks. One chemical detected in water samples take n from the water treatment system was diphenyl sulfone. Diphenyl sulfone is an acaricid, a mite pesticide, also used in the production of some polyethylene tanks. Although an evaluation of c opepods exposed to this chemical and others present in the water treatment system was not conducted, deleterious effects on the performance of copepod cultures have been noted when using water directly from some polyethylene tanks. Rhyne et al. (2009) evaluated effects of temperature on vari ous culture parameters of P. pelagicus and defined an optimal range for survival and fecundity to be between 28C. In studies evaluating copepod performance at various salinities, an optimal range between 22 g/L was observed, although P. pelagicus can tolerate 10 g/L salinity (Ohs et al., submitted). The results of these experiments were used to ma intain copepod cultures. No studies have been conducted on the performance of P. pelagicus cultures in response to other water quality parameters. Dissolved oxygen was maintained be tween 5 mg/L, although occasionally levels of 3 mg/L were detected with no deleterious effects observed. The pH was maintained between 7.6 and 8.2. Total ammonia-nitrogen (TAN) was maintained below 0.7 mg/L and nitrite was maintained below 0.13 mg/L. P. pelagicus cultures were subjected to surface light levels of 90 240 lux and a photoperiod of 24 light and 0 dark. 33

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Diet P. pelagicus cultures were fed diets consisting of T-ISO, TW, or a 1:1 mixture of T-ISO and TW. Dietary effects on P. pelagicus fed the various microalgae diets has been evaluated (Ohs et al., unpublished data). Results showed adults fed monoalgal diets consisting of TW produced on average 3 times more nauplii than adults fed T-ISO, the algae which has been fed to P. pelagicus for over five years (Ohs pers. comm.). Therefore, a combination of T-ISO and TW was used to enhance nauplii production in trials 4 and 5. P. pelagicus cultures were fed TISO to a density of 200,000,000 cells/mL. The 1:1 mixture, consisting of equal volumes of T-ISO (10 million cells/mL) and TW (1 mill ion cells/mL), was fed to a density of 75,000 125,000 cells/mL. This algal density was sufficient for P. pelagicus cultures with a density of 0.25.00 individuals/mL and was adjusted accordingly. All new culture water was inoculated with an appropriate amount of microalgae prior to the addition of copepods. Equipment Aeration was provided to each P. pelagicus population with an ai r pump and airline tubing (4-mm inner diameter) weighted down with a porcelain electrical fence insulator. In tanks larger than 55-L, a coarse silica air stone was used to maintain dissolved oxygen concentrations. Airstones that create fine bubbles were avoided to prevent air from getting trapped under the carapace of the copepods. Weighted sponge filte rs (Aquarium Technology Inc., GA) were also used in each copepod culture tank la rger than 55-L to act as a mech anical and biological filter. By design, air gently bubbled and pulled water through the sponge which filtered suspended particulate matter from the water. Sponge filters have been observed to reduce the frequency and magnitude of bacterial blooms within copepod cu lture tanks. When cleaned, the sponge filters were taken out of the culture tanks, placed in s eawater, and the collected particulate matter was squeezed from them. At that point they were either placed back in the culture tanks or soaked in 34

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tap water and replaced with new ones. A 200-wa tt heater (Visi-therm, OH) was also placed in each culture tank to maintain temperature. A variety of sieves were used with the P. pelagicus cultures. A sieve was constructed from a PVC or acrylic pipe with a diameter of 10 to 20 cm cut into 15 to 20 cm lengths. A ring of the PVC or acrylic pipe of similar diameter and 2 to 3 cm high was also used. The ring was cut to fit snuggly inside the 15 to 20 cm section. Nylon scr een of the appropriate mesh size was then cut and fit tightly inside one end of the pipe with the ring holding it in pl ace. The ring held the screen tightly in place so the mesh size was not compromised and the screen was taut. The screen was then hot glued around the edge to av oid loss of copepods. A variety of nylon mesh screen diameters were utilized. A 240 micron nylon mesh screen (240 sieve) was used to retain paired (copulating) adults and larger females. A 200 micron nyl on mesh screen (200 sieve) was used to retain all adults. A 150 micron nylon mesh screen (150 sieve) was used to retain all adults and copepodites fed T-ISO. A 140 micron nylon mesh screen (140 sieve) was used to retain all adults and copepod ites fed a mixture of T-ISO and TW. A 50 micron nylon mesh screen (50 sieve) was used to retain the enti re population. Floating airlifts were constructed to passively separate nauplii from the rest of the P. pelagicus population. Airlifts consiste d of 19-L plastic buckets with two openings cut out which removed 2/3 of the area of the sides of the buc ket, the openings were covered with 50 micron nylon mesh screen and were hot glued in place. The bucket had a 5-cm hole removed from the bottom center and a bulkhead was attached. A PVC pipe extending from the bottom of the bucket was secured by inserting it into the bulkhe ad. A window was cut out of the PVC pipe and 150 or 140 micron nylon mesh screen covered the window and was secured with hot glue. A 150 micron nylon mesh screen was used for cope pod populations reared solely on T-ISO and a 35

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140 micron nylon mesh screen was used for cope pod populations reared on a mixture of T-ISO and TW. A styrofoam ring was cut to fit around the outer perimeter of the bucket, under the handles, which allowed the airlift to float freely in the copepod culture tank s. Inside the bucket a standpipe was inserted into the bulkhead and extended above the surface of the water by 2 cm when floating. A small ceramic airstone was placed inside the internal st andpipe which created a current that pulled copepod nauplii (<140 microns) through the screened pipe and concentrated them in the bucket. The copepodite and adult populations did not pass through the screens and were not disturbed by this nauplii harvest method. Contamination Copepods from the culture tanks were regularly inspected, under a microscope, for health issues or contamination. Cont amination within populations of P. pelagicus was either tolerated or immediately addressed, depending on the se verity and the species. A stalked ciliate, Vorticella sp., has been observed in the copepod culture tanks. Attempts to grow Vorticella -free cultures of P. pelagicus were not successful. However, there has been no observed negative effect of the Vorticella sp. on the copepod population. Sma ller ciliates are commonly observed in P. pelagicus populations but they are consumed by the copepods. Presence of some ciliates may improve the health of a copepod population by reducing the bacterial load. Rotifers, Brachionus plicatilis contaminated the copepod population on ce, requiring three attempts to eradicate them by physical separation. The entire P. pelagicus population was passed through a 240 sieve in an attempt to isolate a clean population of copepods because B. plicatilis is smaller than 240 microns. Although this method has worked for other copepod culturists, our attempt was successful for about two weeks before the rotif ers reappeared. Rotifer contamination has to be dealt with immediately as they will quick ly reproduce and outnumber the copepods, greatly reducing the performance of the population. Furthe rmore, rotifer producti on of cysts and asexual 36

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life stages increases the difficu lty in removing them entirely and long term from the copepod population. All equipment was routinely soaked in 20 mg /L chlorine for 24 hours. Sponge filters, sieves, filter bags, and airlifts were soaked in 20 mg/L chlorine for 1 hours and then rinsed with tap water and allowed to dry. Routin e cleaning will help reduce the potential of contamination, even though in culture tanks larg er than 19-L some contamination is likely. Airlift Efficiency Two sizes of airlifts were used at IRREC, a small (7.5-L collection bucket) and a large (19L collection bucket) size. Both had the same desi gn and differed only in the size of bucket used. A simple experiment was conducted to see which size airlift, small or large, harvested the greater percentage of copepod nauplii after one hour of airlift operation. Fo r this comparison, 200-L culture tanks were used. Three evaluations were conducted with the sm all airlift. For each evaluation, collected nauplii were maintained in a pproximately 10-L of culture water with mild aeration. The harvested nauplii were then homogenized with increased aeration. Af ter homogenization, 5, 2mL samples were taken from the 10-L of collected nauplii. These volumetric samples were used to estimate nauplii density and subsequent total number of nauplii harvested by the airlift. A 100 % water change was then performed on the culture tank. The copepod population was passed through a 150 sieve to collect the remaining nauplii that were not harvested by the airlift. The collected nauplii were then homogenized with aeration. After homogenization, 5, 2-mL samples were then taken from the collected nauplii and were used to estimate density and subsequent total number of nauplii not harvested by the airl ift. The estimated number of nauplii harvested was then divided by the estimated number of nauplii in the tank and multiplied by 100 to attain the percentage of nauplii that were collected with the airlift. 37

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When examining the large airlif t, four evaluations were c onducted. For each evaluation, collected nauplii were maintained in approximately 4-L of culture water with mild aeration. The harvested nauplii were then homogenized with increased aeration. Af ter homogenization, 3, 2mL samples were taken from the 10-L of collected nauplii. These volumetric samples were used to estimate nauplii density and subsequent total number of nauplii harvested by the airlift. A 100 % water change was then performed on the culture tank. The copepod population was then passed through a 150 sieve to collect the remaining na uplii that were not harv ested by the airlift. The collected nauplii were then homogenized with aeration. After homogenization, 3, 2-mL samples were then taken from the remaining naup lii and used to estimate density and subsequent total number of nauplii not harvested by the airlif t. The estimated number of nauplii harvested was then divided by the estimated number of nauplii in the tank and multiplied by 100 to attain the percentage of nauplii that were collected with the airlift. There was a significant difference between the percentage of nauplii harvested with the two sizes of airlifts (T-TEST, T 5 = 7.86; p = 0.0005). The mean percentage of nauplii harvested by the large airlift (90.3 4.7%) wa s significantly greater than the mean percentage of nauplii harvested by the small airlif t (60.6 2.5%). Although this re presents a small number of evaluations with each airlift size, the data shows the large airlif t was more efficient at nauplii collection and was the preferred method to harvest nauplii from a mixed age population. Pseudodiaptomus pelagicus Cultures Copepods were maintained in stock, 200-L, and mass-scale cultures. Each was designed for different types of production goals and rese arch purposes. Based on need, all or only one scale of copepod culture ma y be maintained. Techniques involv ed with each culture type apply to the culture techniques previously me ntioned unless otherwise noted. 38

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Stock Cultures Stock cultures of P. pelagicus were grown in small containers (1 L), in static conditions to maintain a long term population and to provide starter cultures for la rger populations. Water was maintained at 22C and a salinity of 20 ppt with light aeration. Low feeding rates, 50,000,000 cells/mL of T-ISO, were provided ev ery other day, and 100% weekly water changes were used to reduce growth and fecundi ty but maintain population vigor. Temperature was manipulated by placing the container in a wate r bath or temperature-controlled room. The stock population was kept young by occasionally removing adults to reduce crowding. To perform a water change, th e air supply was shut off and the air line was removed from the container. Approximately 10 minutes after removing aera tion, unwanted debris settled and the copepods came to the surface. The cope pods were decanted thro ugh a 50 sieve and all life stages were collected and placed into a steril e container with clean cu lture water to visually inspect the population. While collecting copepods, the sieve nylon mesh screen was always submerged in seawater under similar environmenta l conditions to reduce st ress. If the culture was too dense, adults could be separated fr om all younger stages by passing through a 240 sieve prior to collection on a 50 sieve. Also, some of the population could be decanted off prior to collection on a 50 sieve. The population collected on the 50 sieve was easily returned to new water by using a squirt bottle, containing culture water, to gently concentrate and transfer the population off of the screen. The copepods were th en returned to the water bath or temperature controlled room and gentle aeration was provide d with a new, clean air line. Separated populations may be used to establ ish other culture s or discarded. 200-L Cultures The 200-L, intermediate-size copepod populatio ns at IRREC were batch cultured at 28 30C and a salinity of 22 g/L in 200-L cylindr ical, flat-bottom polyethylene tanks. These 39

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served as an individual population (roughly 100,000,000 individual s) or to inoculate a massscale population. Copepod nauplii were harvested from tanks and used to inoculate new tanks. This allowed for greater control of the growth rate, feed efficiency, and nauplii production of each tanks population. Furthermore, multiple tanks at various stages of development could be maintained. At 28C, 100,000,000 nauplii fed T-IS O would reach adulthood in 9 to 10 days and produce 70,000,000 nauplii/day for approximately 10 days. Under the same conditions, nauplii fed a 1:1 mixture of T-ISO and TW would reach adulthood in 7 days and produce 150,000,000 nauplii/day for approximately 9 days. At 30C versus 28C, time to adulthood was accelerated by approximately 1 day, but no effect on nauplii production was observed. Adult P. pelagicus have a high escape response, remain attached to substratum, and do not homogenize in the culture tanks making estimations of adult density difficult. The 200-L tanks (97-cm high x 55-cm diamete r) had a 2.54-cm bulkhead inserted in the bottom approximately 8-cm from the edge. A 2.54-cm PVC ball valve was fitted to the bulkhead with a 2.54-cm PVC street 90 elbow fitted on the opposite end of th e ball valve. The rate of water flow leaving the 200-L tank could be controlled by the valve. Also, an internal standpipe was inserted in the tank to prevent water from entering the drain pipe and valve and creating an anaerobic area. A collection sump, consisting of a 19-L plastic bucket with a bulkhead inserted 15-cm from the top, was used during a water chan ge. An outlet pipe, attached to the 2.54-cm PVC street 90 elbow, led into the collection su mp. The end of the outlet pipe was positioned below the bulkhead of the collection sump so it would be submerged when the collection sump was full of water. A 40 micron mesh filter bag ( 80-cm long x 15-cm diameter) with a drawstring was tied to the end of the outlet pipe to collect the copepod population an d transfer them to a new tank. 40

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To perform a water change, the air supply to the sponge filter was shut off and it was removed from the tank. The air s upply to the air stone was decreased but it was not removed. This reduced the amount of debris suspended in the water while maintaining dissolved oxygen concentrations. The outlet pipe was attached to the valve on the bottom of the tank which led into the collection sump. The 40 micron mesh bag was tied to the end of the outlet pipe in the collection sump. The collection sump was filled w ith the appropriate salinity seawater until the mesh bag was submerged, alleviating damage to the copepods collecting in the mesh bag. The valve was first opened completely and then turned back to open, eliminating any residual air in the outlet pipe. The flow of the water moved th e copepods quickly but with limited injury from encountering the mesh bag. Once drained, the co pepods were concentrated in the mesh bag by bobbing the bag several times in the water and rolling the top as it was brought out of the water. The copepods were concentrated at the bottom of the bag. The population was emptied from the mesh bag into a new tank and the sponge filter and air stone, heater, and feed rate were adjusted accordingly. If needed, the separation of lif e stages can occur during the water change procedure, similar to the methods described in the stock culture se ction. If desired, the population would be emptied from the mesh bag into a 19-L plastic bucket containing clean culture water and then sieved for isola tion of the target life stage. Mass-Scale Cultures Mass-scale populations of P. pelagicus were attained in an 1800-L cylindrical, conicalbottom, polyethylene tank. The tank was provided mild aeration with two coarse airstones and three sponge filters were suspended in the tank. This population was grown to produce >1 million copepod nauplii per day to feed to marine fish larvae with reduced labor and use of water. At 30C, approximately 750,000 copepods of a mixed age population were fed a 1:1 mixture of T-ISO and TW to a densit y of 100,000,000 cells/mL, and this population 41

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produced approximately 1 million nauplii/day ov er an eight day period, gradually decreasing from 1.75 to 0.80 million nauplii/day during the eight days. The mass scale system consisted of an 1800-L tank and a 1600-L sump. The 1800-L tank (118-cm high x 173-cm diameter) had a 3.81-cm bulkh ead inserted into th e center of the bottom of the tank. A 3.81-cm PVC pipe was connected from the bottom of the tank to a 2.54-cm ball valve positioned over a 1600-L sump (90-cm x 152-cm diameter). A 2.54-cm ball valve was also present at the lowest point in the PVC pipe, for purging the pipe. The rate of water flow leaving the 1800-L tank could be controlled by the valve over the sump. Also, an internal standpipe was inserted into the bulkhead and prevented water from entering the pipe and creating an anaerobic environment. During a water change a collection device was used inside the sump to harvest copepod nauplii for a larval rearing expe riment. The collection device consisted of a 75-L polyethylene container placed inside anot her 110-L polyethylene container. The 75-L container had 4 sides cut out a nd 50 micron nylon mesh screen was hot glued to cover and seal the openings. The 75-L container fit loosely inside the 110-L cont ainer which had a hole drilled in the side, 20-cm from the top. The hole allowe d water to drain from the 110-L container into the sump and prevented the 75-L container inside it from over flowing, therefore concentrating the collected copepod nauplii. An outlet pipe was attached from the 2.54-cm ball valve over the sump leading into the collection device with the e nd of the outlet pipe submersed during harvest. An internal standpipe (60-cm high x 15-cm diameter) with windows cut out had 140 micron nylon mesh screen hot glued to cover the openi ngs (140 standpipe) and was designed to allow harvest of the copepod nauplii wh ile retaining the adult and cope podite populations within the 1800-L culture tank. Harvesting nauplii during a wate r change required the use of a small light suspended approximately 70-cm above the wate r surface illuminating the culture around the 42

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internal standpipe. P. pelagicus nauplii are positively phototactic and gathered near the light during harvest. To perform a water change, th e internal standpipe was repl aced with the 140 standpipe. The purge valve was opened and 15-L was removed and allowed to settle. The air flow to both the sponge filters and air stones were turned off and the light was suspended over the top of the 140 standpipe. After ten minutes, the valve ove r the sump was completely opened and then turned back to open, eliminati ng any residual air in the outlet pipe. The flow of the water moved the copepods quickly but limited injury from encounteri ng the collection device was observed. As the collection device harvested nauplii, culture water would overflow into the sump. By design, the 1800-L tank would not completely drain with the 140 standpipe in place; the copepod population was retained in approximately 200-L of culture water. Once drained, the nauplii were transferred to 19-L plastic buckets and maintained under mild aeration for feeding to fish larvae. During the marine fish larval feeding experiments, the 1800-L tank was filled with new 22 to 25 g/L salinity water and the sponge filter and air stone, heaters, and feed rate were adjusted accordingly. Conclusion Observations During 17 months of main taining populations of P. pelagicus at IRREC, many observations were made about copepod perfor mance. Once, when the copepod population was contaminated with rotifers, we sieved the population through a 240 sieve. This, undoubtedly, was a stressful event for the c opepod population. However, within 30 hours the remaining adults had produced enough copepod nauplii to conduct a marine fish larval feeding experiment. This same effect was observed in Rhyne et al. (2009) when nauplii producti on was greatest on day 1 of a 10 day experiment examining e ffects of temperature on 10 pairs of P. pelagicus adults. 43

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Again stocking the experiment was more than likely a stressful ev ent for the copepods. Increased nauplii production was repeatedly obser ved following exposure to seemingly stressful events. However, multiple continued stressful even ts do not have this effect and will reduce the health of the copepod population. A change in dens ity must also be considered when examining this response. A 200-L tank normally produced approximately 70,000,000 nauplii/day depending on algal diet and adult density. Two to three ti mes a year each tank produced approximately 1 million nauplii/day over a 2 day period. One hypothesis is a reaction to astronomical influences such as the moon or seasonal cycles. However, as exact dates of these events were not recorded it was impossible to compare production to these cy cles. Also, numerous factors affecting copepod performance were not controlled during this time, so these are only observations. Techniques to increase nauplii production of P. pelagicus were practiced in order to efficiently conduct marine fish larval rearing trials. In Rhyne et al. (2009), an optimal temperature of 28C was suggested, however, adva ntages were seen in nauplii production of copepods kept at <26C and increased to 30C over a 24 hour period. By doing so, the metabolic demand of the population was believed to be reduced prior to the increase in temperature which triggered incr eased nauplii producti on. It was also obser ved that low adult densities (<5 individual/mL) produced more c opepod nauplii than cultures with high adult densities (>5 individuals/mL). Increased nauplii production was also observed when the adult copepod density was maintained >5 individuals/mL and then reduced to < 5 individuals/mL over a 24 hour period. Counterintuitively, given that P. pelagicus are benthic orient ed, the addition of substrate in the form of plastic mesh within the 200-L tanks had no observed effect on copepod 44

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performance or nauplii production. However, th ese techniques and findings were based on anecdotal information and controlled experimental studies need to be co nducted to confirm these observations. Future Studies Little information is available pertaining to the maintenance of P. pelagicus populations. First an examination into the tolerances a nd optimal ranges of various water quality and environmental parameters should be conducted. Dissolved oxygen, pH, TAN, nitrite, light intensity, and photoperiod all shou ld be evaluated to determine optimal culture conditions for P. pelagicus. Although diet studies with live microalgae have been conducted at IRREC, studies examining commercial enrichments and mi croalgae pastes should be conducted. As contamination is a continual th reat especially in larger popu lations (>55 L), evaluation of copepod performance subjected to various chemical treatments will help elucidate efficient procedures for the eradication of those contamin ants. Furthermore, an evaluation of chemicals dissolved in the water originating from polyethyl ene tanks and the effects these chemicals have on P. pelagicus populations should be conducted. Rapid increases in temperature, decreases in salinity, decreases in density, and increases in feed density seem to increase levels of nauplii pr oduction; these should be evaluated in replicated studies. Observed increases in nauplii producti on as a response to stress also should be evaluated. Once evaluated, the syne rgistic effects of these parameters and changes can be used to induce increased nauplii production. 45

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CHAPTER 3 TRIAL 1 The goal of this study was to examine the benefits of feeding nauplii of the calanoid copepod Pseudodiaptomus pelagicus to larval Florida pompano, Trachinotus carolinus. Three treatment diets were evaluated to ascertain advantages in development and performance compared to larvae fed the standard reference diet (SRD). Two copepod diets, fed during the first three days of feeding, and a mesocosm feeding regime were provided to Florida pompano larvae spawned from F1 broodstock. Net stre ss and salinity stress e xperiments were also performed to observe advantages to Florida pompano larvae fed copepods. Material and Methods Water Treatment At the University of Florid as Indian River Research a nd Education Center (IRREC), larval fish were cultured in natural seawater ( 32 g/L salinity). Prio r to use, seawater was chlorinated (150 mg/L) with sodium hypochlorite for 24 hours, then aerated for 24 hours until 0 mg/L chlorine was detected. Seawater was then pumped into 20,000-L storage silos. From the storage silos seawater was pumped in to the hatchery facili ty and passed through both 50 and 5 micron mesh bag filters. Seawater was stored in a 3500-L polyethylene tank and continuously circulated through an 80 Watt UV ster ilizer (Emperor Aquatics Inc., PA) at rate of 170 L/minute which provided a sterilizat ion intensity of 30,000,000 microWatts/cm 2 Prior to use, if chlorine was detected in the wate r it was neutralized with sodium thiosulfate. As water left the tank, it would pass through both 1.0 and 0.5 micron cartridge filters. Spawning and Egg Incubation Florida pompano eggs were acquired from the United States Department of Agriculture, Agricultural Research Service, Center for Reproduction and Larvicultu re, located on the campus 46

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of Harbor Branch Oceanographic Institute at Flor ida Atlantic University in Fort Pierce, FL (USDA-ARS). Volitional spawning of F1 broods tock using a gonadotropin-releasing hormone analogue (GnRHa) was achieved following proce dures described by Weirich and Riley (2007). Eggs were transported to IRREC and incubated under static conditions in two 200-L circular, conical-bottom fiberglass tanks (trials 1, 2, and 5) or one 55-L circular, conical-bottom polyethylene tank (trials 3 and 4). Incubation occurred at a temperature of 26C and a salinity of 35.1 g/L with light aeration. Ha tching occurred 30 hours postfertilization. Larvae were volumetrically quantified following homogenizati on with increased aeration and were stocked into the experimental system within 8 hours post-hatch. Larval Rearing and Ex perimental Design A 7-day larval rearing trial was conducted in a flow thr ough system consisting of 28, 13-L cylindrical, flat-bottom fiberglass tanks with bl ack walls and a white bo ttom. An artificial photoperiod of 14 hours light and 10 hours dark was maintained dur ing the experiment. Newly hatched larvae, 0 days post hatch (DPH), were vol umetrically stocked into each tank at a density of 50 individuals/L. The initia l water flow was 27 mL/minute, gentle aeration was provided, and daytime surface light levels were maintained below 150 lux (Milwaukee Model SM700, NC) for the first two days. Beginning on 2 DPH, tanks were inoculated daily with T-ISO to a density of 100,000,000 cells/mL. At this time water flow increased to 37 mL/minute, daytime surface light levels ranged from 1000 l ux, and aeration was increased slightly. At 4 DPH, aeration was again increased slightly and water flow was ad justed to 55 mL/minute for the duration of the trial. In each replicate tank temperature and salinity (YSI Incorporat ed Model 30-10-FT, OH), dissolved oxygen (YSI Incorporated Model 550 A, OH), and pH (Hach Model sensION1, CO) were monitored daily. Total ammonia-nitrogen (TAN) and n itrite-nitrogen (NO 2 -N) were also measured daily with a Hach DR/4000U Spectrophotometer. 47

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Dietary Treatments Four dietary treatments were examined in this study, a standard reference diet (SRD) consisting solely of enriched rotifers, a one day diet, a three day diet, and a mesocosm treatment (Table 3-1). Each diet was replicated six times and four tanks were used to monitor unfed larvae. The one day experimental diet consisted of feeding larvae copepod nauplii on 2 DPH at a rate of 2.14 nauplii/mL/day and then sw itching to the SRD from 3 DPH. The three day experimental diet consis ted of feeding larvae copepod nauplii on 2 DPH at a rate of 2.14 nauplii/mL/day, on 3 DPH at a rate of 2.50 na uplii/mL/day, and on 4 DPH at a rate of 3.00 nauplii/mL/day and then switching to the SRD from 5 DPH. The mesocosm experimental diet consisted of a mixture of P. pelagicus adults (>240 m) stocked into each tank on 0 DPH at a density of 1.34 individuals/mL. Stocked adult copepods produced nauplii during the experimental trial and replicate populations we re only fed algae. The mesocosm treatment populations were fed T-ISO at a density of 100,000,000 cells/mL for the first two days until the entire system was inoculated with T-ISO. For all treatments, when copepod nauplii were fed, an internal standpipe with 50 micron nylon mesh screen was used; when rotifers were fed, an internal standpipe with 240 micron nylon mesh screen was used. From 2 DPH to 6 DPH larvae were fed diets consisting of enriched S-strain rotifers, B. plicatilis with a body width of 117.5 19.8 microns (Cavalin and Weirich, 2009) and P. pelagicus nauplii with a body width of 93.2 13.7 microns. Enriched rotifer s were fed to larvae four times daily (0900, 1300, 1700, and 2100) at a rate of 2.5 rotifers/mL/day (Cavalin and Weirch, 2009). Rotifer Culture and Enrichment Rotifers were cultured at 26C and a salinity of 20 g/L in a 120-L cylindrical, conicalbottom, fiberglass tank. Rotifers were fed 7.5 L of T-ISO (10 million cells/mL) and Culture Selco (INVE Aquaculture Inc., UT), a supplementa l yeast-based rotifer diet, twice daily during 48

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the trial according to manufactur er instructions. Approximately 5 million rotifers were harvested daily for enrichment prior to feeding to fish larvae. Rotifers were enriched with OriGreen (Skretting, Italy) in 10-L buckets following manufacturer instructions for 3 hours at 27 28C. After the enrichment period, the rotifers we re rinsed with 20 g/L seawater, placed in new 20 g/L salinity water (10-L) and the temperatur e was reduced to approximately 15C by placing frozen water bottles into the bucket. Once th e temperature was reduced, the bucket was placed in a refrigerator at 5C for cold storage. En riched rotifers were fed within 24 hours of cold storage. Copepod Culture Copepods were batch cultured at 28C and a salinity of 22 g/L in twelve 200-L cylindrical, flat-bottom polyethylene tanks. Aera tion was kept moderate and sponge filters were used within each tank. Each tank was fed 1 L of T-ISO (15 million cells/mL) daily during the trial. Nauplii were harvested daily by si eving populations through a 150 micron nylon mesh screen and the adults not passing through the sieve were placed in new culture water. Harvested nauplii were then placed in a graduated bucket containing 22 g/L salinity water, volumetrically quantified, and then volumetrica lly fed to fish larvae. Sample Collection and Morphometric Analysis Sample collection and morphometric anal ysis techniques described by Cavalin and Weirich (2009) were used for this study. On 0 DPH, 60 yolk-sac larvae were randomly selected from the incubation tank for morphometric an alysis. Thereafter, 10 larvae from each experimental tank were randomly sampled on 3 a nd 6 DPH for morphometric analysis. Larvae were euthanized via cold water immersion (~ 4C), placed on a glass slide, and photographed using a dissecting microscope at 40-X magnification with a high resolution digital camera (Sony Model DCRA-C171, CA). Photographs were stored as JPEG Images at 6.1 megapixels. A 0.0149

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mm micrometer was photographed prior to each larval series for calibration purposes. SigmaScan Pro 5.0 image analysis software (SPSS Science, IL) was used to measure photographed larvae. Standard length (SL; the dist ance parallel to the longitudinal axis of the body from the tip of the snout to the distal end of the hypural bone) and body depth (BD; the distance perpendicular to the long itudinal axis of the body from th e insertion of the first dorsal spine to the ventralmost point on the base of the body) were measured and recorded. When measuring yolk-sac larvae, BD was considered the distance perpendi cular to the longitudinal axis from the dorsal crest through the midpoint of the yolk-sac to the ventralmost point of the body. Larvae from each tank were counted to determine survival at the conclusion of the larval rearing trial. Stress Resistance Analysis On 7 DPH, a sample of larvae from all treatment replicates were combined and placed into separate 110-L aquaria, each representing a dietar y treatment. The aquaria contained water from the larval culture system and were maintained at 26C and a salinity of 35 g/L, with gentle aeration. Larvae were than randomly selected from the aquaria for net and salinity stress experiments. Larvae from the mesocosm diet ary treatment were excluded from the stress resistance analysis due to low survival. Net Stress To simulate net stress, units were construc ted consisting of a 0.5-L container which fit tightly into a 1.0-L container. The bottom of the 0.5-L container was removed and a 150 micron nylon mesh screen was hot glued to cover the opening. Five larvae were randomly selected from their respective aquaria with a 0.5-L screen-bottomed containe r and held out of water for 30, 60, 120, or 240 seconds. Each dietary treatment was replicated four times for each time interval. After their respective duration, larv ae were submerged into the 1-L containers containing culture 50

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system water (salinity of 35 g/L) and were mainta ined in a temperature c ontrolled water bath at 26C. Larval survival was recorded one hour af ter being submerged. Survival was defined as any movement detected within one minute of observation. Salinity Stress Prior to the salinity stress experiment, larvae were transported 1 mile from the IRREC hatchery facility to the IRREC aquaculture labora tory in buckets with gentle aeration. Five randomly selected larvae from their respective di etary treatment were then pipetted into a 1.0-L container containing 5, 15, 35, or 55 g/L salinity water with gentle aeration and held in water baths at 26C. Treatment diets were replicated four times for each salinity treatment. Larval survival was recorded 2 hours and 18 hours afte r exposure. Survival was defined as any movement detected within one minute of obs ervation. Salinities were acquired by mixing appropriate amounts of artificial sea salt with deionized water th ree days prior to the salinity stress experiments. Fatty Acid Analysis Larvae were collected at 0 DPH from the incu bation tank and at the c onclusion of the trial from each experimental treatment for fatty acid analysis. Copepod nauplii and enriched rotifers were also collected for fatty acid analysis at the conclusion of their use in the culture experiment. Larvae and live feed samples were rinsed with deionized water, plac ed in plastic vials, and stored in a freezer (-80C) until analyzed. Statistical Analysis All statistical analyses were performed w ith SAS version 8.02 software (Cary, NC). Percentage data were arc-sine-s quare-root transformed prior to analysis. Treatment means of all dependent variables were subjected to one-way analysis of variance according to the General Linear Model (PROC GLM) procedure of SAS. A least significant difference test (LSD) was 51

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used to compare treatment means for larval gr owth, survival, and stress resistance when the ANOVA was significant When two treatments were comp ared a Studentized T-test (PROC TTEST procedure of SAS) was conducted to detected differences in the m eans. All statistical tests were considered significant when p 0.05. Results Water Quality All water quality parameters were recorded within normal limits (Table 3-2). Growth At 0 DPH, larvae had a mean SL of 2.77 0.22 mm (mean SD) and a mean BD of 0.89 0.06 mm. At 3 DPH, mean SL of larvae fe d the mesocosm (2.96 0.19 mm), SRD (2.85 0.36 mm), one day (2.86 0.25 mm), and three day (2.87 0.18 mm) treatments were not significantly different (ANOVA, F 3,196 = 2.23; p = 0.0861) (Figure 3-1). The mean BD of larvae was significantly different betw een dietary treatments (ANOVA, F 3,196 = 3.39; p = 0.0192). Larvae fed the SRD had a significantly greater BD (0.77 0.08 mm) than the one day (0.73 0.06 mm) and mesocosm (0.74 0.05 mm) treatments (Figure 3-2). The BD of larvae fed the three day treatment (0.76 0.06 mm) was not significantly differe nt from any of the dietary treatments. At 6 DPH, mean SL of larvae was signifi cantly different between dietary treatments (ANOVA, F 3,196 = 12.94; p < 0.0001). The SL of larvae fed the three day treatment had a significantly greater SL (3.54 0.22 mm) than larvae from any of the treatment diets (Figure 31). SL of larvae fed the SRD (3.36 0.26 mm) was significantly greater than both the one day (3.22 0.52 mm) and mesocosm (3.21 0.12 mm) tr eatments. The mean BD of larvae was significantly different between dietary treatments (ANOVA, F 3,196 = 2.23; p < 0.0001). Larvae fed the one day treatment had a significantly greater BD (1.0 0 0.13 mm) than larvae from any 52

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of the treatment diets (Figure 3-2). BD of larvae fed the three day (0.93 0.08 mm) and SRD (0.91 0.11 mm) treatments were both significantly gr eater than the mesocosm treatment (0.78 0.06 mm), but were not different from each other. Survival At 7 DPH, there were significant differences in survival between treatments (ANOVA, F 3,196 = 12.94; p < 0.0001). Survival of larvae fe d the one day treatment (38.6 7.8%) was significantly greater th an survival from the SRD (20.8 10.5%) and mesocosm (6.5 2.8%) treatments (Figure 3-3). Survival of larvae fed the three day tr eatment (29.0 4.6%) was significantly greater than survival from the mesocosm treatment. No significant differences were detected in the survival from the three day tr eatment when compared to the one day and SRD treatments. Stress Resistance Net Stress No significant differences were detected in survival of larvae fed the various experimental diets following 30 (ANOVA, F 2,9 = 0.50; p = 0.6224), 60 (ANOVA, F 2,9 = 1.21; p = 0.3417), 120 (ANOVA, F 2,9 = 0.54; p = 0.6013), and 240 (ANOVA, F 2,9 = 3.66; p = 0.0687) seconds of exposure to net stress (Figure 3-4). Salinity Stress At the 2 hour time interval, no si gnificant differences were de tected in the survival of larvae from any of the experime ntal diets for the 15 g/L (ANOVA, F 2,9 = 2.48; p = 0.1389) and 35 g/L salinity treatments (ANOVA, F 2,9 = 0.40; p = 0.6798). There was a significant difference between treatments in the 5 g/ L salinity treatment (ANOVA, F 2,9 = 4.89; p = 0.0366). Survival of larvae fed the SRD (90.0 11.6%) and one day (85.0 19.1%) treatments exposed to the 5 53

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g/L salinity treatment were significantly greater than survival of larvae fed the three day treatment (50.0 25.8%) (Figure 3-5). At the 18 hour time interval, no significant diffe rences were detected in the survival of larvae from any of the experime ntal diets for the 15 g/L (ANOVA, F 2,9 = 1.44; p = 0.2857) and 35 g/L salinity treatments (ANOVA, F 2,9 = 0.91; p = 0.4355). There was a significant difference between treatments in the 5 g/ L salinity treatment (ANOVA, F 2,9 = 6.44; p = 0.0184). Survival of larvae fed the SRD (90.0 11.6%) and one day (85.0 19.1%) treatments exposed to the 5 g/L salinity treatment were significantly greater than survival of larvae fed the three day treatment (45.0 25.0%) (Figure 3-6). No larvae we re alive in the 55 g/L salinity treatment for any of the experimental diet s tested at the 2 hour or 18 hour time intervals. Discussion Improvements in growth, survival and resist ance to stress were noted for larvae fed copepods compared to those larvae fed the SRD. A one day di et, which consisted of feeding P. pelagicus nauplii to pompano larvae for the first day of exogenous feeding, resulted in nearly double the survival and increased growth in BD at 6 DPH when compared to larvae fed the SRD. The three day diet also result ed in higher survival and a sign ificant increase in the SL when compared to larvae fed the SRD. When examin ing the net stress resistance of pompano larvae, no significant differences were detected between the dietary treatments, though increases in sample sizes may have displaye d significant differences. At 6 DPH larvae from the mesocosm treatment had the lowest SL and BD, and recorded the lowest survival of larvae from any of the tr eatment diets (6.5 2.8%). At an assumed 1:1 sex ratio and an estimated nauplii production rate of 9.5 1.9 nauplii/femal e/day (Rhyne et al., 1999), tanks stocked at a density of 1.34 indi viduals/mL should have produced roughly 6.4 nauplii/mL/day. However, fewer nauplii (0.11/mL ) than expected were sampled from the 54

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mesocosm treatment tanks during the experimental trial. This observation coupled with the low growth and survival of larvae from the treatm ent tanks suggests that poor nauplii production occurred. One explanation may be that an insu fficient amount of the algae T-ISO was supplied to the adult copepods through greening the cu lture water in order for them to produce sufficient numbers of nauplii. In Rhyne et al. (2009), T-ISO was fed to adult P. pelagicus at densities of 200,000,000 cells/mL. During this experiment, T-ISO densities were maintained from 100,000,000 cells/mL. Another explanation is that a 1:1 sex ratio of copepods was not achieved at stocking. Only females were counted from samples of the populations used to stock the experimental sy stem, because a 1:1 sex ratio is typical for P. pelagicus under these culture conditio ns (Rhyne et al., 2009). Howe ver, during the experiment schools of male copepods were observed near th e tops of the mesocosm treatment tanks and antagonism was observed by unpaired males upon paired adults (t he reproductive state). An elevated number of males within the treatment tanks could have further reduced the amount of TISO available for paired adults and increased antagonism likel y reduced the amount of energy available for reproduction. Directly following this experiment, the condition of the copepod cultures quickly deteriorated yielding low survival and low na uplii production. The cultures became cloudy and food consumption was reduced. It was deduced that high levels of bacteria were present in the cultures, likely from injury and damage to the copepods during the repeated sieving of the populations. This method of nauplii collection should not be used for feeding larval marine fish or the health of copepod populations will decline. The results of this study provided information on Florida pompa no larviculture techniques. However, further studies feeding P. pelagicus nauplii to larval Florida pompano are warranted. 55

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Replicated feeding regimes of the one day and three day diets provided in this study examined on wild-caught broodstock are necessary to confirm advantages of feeding copepods to larval Florida pompano. Also, acquiring informa tion on the advantages of feeding copepods to pompano larvae up to the Artemia transitioning period (9 DPH) and using a larger system designed for commercial production is warranted. Further, refi ning a mesocosm technique for P. pelagicus is needed to examine the most cost effici ent methods of providing nauplii to larval marine fish species. 56

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2.25 2.75 3.25 3.75 4.25 4.75 3 DPH 6 DPHStandard Length (mm) Rotifer One Day Three Day Mesocosm A B C C A A A A Figure 3-1. The mean standard lengt h (mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 1. Values were recorded on 3 and 6 days post hatch (DPH). Standard error bars and LSD multiple comparisons test results are displayed. Bars with different lett ers are statistically different (p 0.05). 0.50 0.75 1.00 1.25 1.50 3 DPH 6 DPHBody Depth (mm) Rotifer One Day Three Day Mesocosm A B B C A B AB B Figure 3-2. The mean body depth (mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 1. Values were recorded on 3 and 6 days post hatch (DPH). Standard error bars and LSD multiple comparisons test results are displayed. Bars with different lett ers are statistically different (p 0.05). 57

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0 25 50 75Rotifer One DayThree DayMesocosmSurvival (%) A AB B C Figure 3-3. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 1. Standard error bars and LSD multiple comparisons test results are displayed. Bars with different letters are statistically different (p 0.05). 0 20 40 60 80 100 30 60 120 240Survival (%)Duration (seconds) Rotifer One Day Three Day A A A A A A A A A A A A Figure 3-4. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments after exposure to net stre ss during trial 1. Expos ure durations were 30, 60, 120 and 240 seconds. Standard error ba rs and LSD multiple comparisons test results are displayed. Bars with different letters are statisti cally different (p 0.05). 58

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59 Figure 3-6. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments after 18 hours of salinity st ress during trial 1. Salinities were 5, 15, 35, and 55 g/L. Standard error bars and LSD multiple comparisons test results are displayed. Bars with different lett ers are statistically different (p 0.05). 0 51 20 40 60 80 100 53Survival (%)Salinity (g/L) 55 5 Rotifers One Day Three Day Figure 3-5. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments after 2 hours of salinity stress during trial 1. Salinities were 5, 15, 35, and 55 g/L. Standard error bars and LSD multiple comparisons test results are displayed. Bars with different lett ers are statistically different (p 0.05). 0 20 40 60 80 100 51 53 55 5Survival (%)Salinity (g/L) A A A A B B A A A A A A A A A A A A Three Day One Day Rotifers

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Table 3-1. Dietary tr eatments fed to Florida pompano larvae ( Trachinotus carolinus ) during the experimental tr ials. Values are given as the number of live feeds (CN = copepod nauplii, R = rotifers) fed per milliliter per day. Th e day post hatch (DPH) that the feed was fed is also provided in pa renthesis. Stocking density for mesocosm treatments is also given (CA = copepod adults). Following provision of one day and three day treatments, larvae were fed the standard reference diet (SRD) for their respective trial. One Day Three Day Copepod Mix Mesocosm SRD Trial 1 2.14 CN (2) 2.14 CN (2) 1.34 CA /mL 2.50 R X 4 (2-6) 2.50 CN (3) 3.00 CN (4) Trial 2 2.43 CN (2) 2.43 CN (2) 2.50 R X 4 (2-6) 3.10 CN (3) 3.41 CN (4) Trial 3 1.25 CN (2) 3.50 CN (2) 1.25 CA /mL 2.50 R X 4 (2-7) 60 3.60 CN (3) 4.00 CN (4) 4.00 CN (5) 4.00 CN (6) 3.78 CN (7) Trial 4 2.50 CN (2) 2.50 CN (2) 2.00 R X 4 (2-8) 2.50 R X 4 (2-8) 3.00 CN (3) 3.00 CN (3) 0.50 CN X 4 (2-8) 4.50 CN (4) 4.50 CN (4) 6.25 CN (5) 7.80 CN (6) 8.00 CN (7) 7.80 CN (8) Trial 5 1.11 CN (2) 2.50 R X 4 (2-5) 3.00 R X 4 (6-9)

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61 Table 3-2. Water quality variables measur ed during the experimental trials. Vari ables including temperature, salinity, dissol ved oxygen (DO), pH, total ammonia-nitrogen (TAN), and nitrite-nitrogen (NO 2 -N) were measured. Values are given as the mean standard deviation and the range. The number of samples (n=) for each trial is given. Temperature (C) Salinity (g/L) DO (mg/L) pH TAN (mg/L) NO 2 -N (mg/L) Trial 1 n=168 26.1 0.8 24.2 27.2 35.1 0.3 34.4 35.6 5.77 0.27 5.02 6.25 8.03 0.05 7.94 8.13 0.10 0.02 0.03 0.18 0.0068 0.0045 0.0020 0.0493 Trial 2 n=161 24.9 0.9 22.7 26.8 35.3 0.3 34.8 35.6 6.04 0.22 5.42 6.54 8.07 0.05 7.94 8.26 0.14 0.13 0.00 0.74 0.0151 0.0008 0.0001 0.0407 Trial 3 n=196 25.8 0.6 24.7 26.8 36.0 0.2 35.2 36.3 5.32 0.28 4.17 5.79 8.07 0.04 7.87 8.16 0.04 0.09 0.00 0.61 0.0075 0.0032 0.0012 0.0231 Trial 4 n=224 27.6 0.6 26.2 28.5 33.9 1.3 31.6 36.0 6.11 0.18 5.46 6.55 8.22 0.05 8.02 8.30 0.03 0.06 0.00 0.40 0.0114 0.0107 0.0018 0.0099 Trial 5 n=96 30.7 0.7 28.9 32.5 35.9 1.0 34.6 37.2 5.49 0.48 3.83 6.63 8.14 0.08 8.00 8.13 0.11 0.10 0.00 0.35 0.0042 0.0221 0.0169 0.0892

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CHAPTER 4 TRIAL 2 The goal of this study was to examine the benefits of feeding nauplii of Pseudodiaptomus pelagicus to larval Florida pompano, Trachinotus carolinus during the first three days of feeding. Treatment diets similar to trial 1 were fed, however, an increased rate was used to ascertain advantages in development, performan ce and solidify the findings of trial 1. Dietary treatments were provided to Florida pompano larvae spawned from wild-caught broodstock. The duration of net stress exposure wa s extended and two salinity stress resistance experiments were conducted to determine the effects of diet on Florida pompano larvae. Material and Methods Material and Methods from trial 1 we re repeated unless otherwise noted. Spawning and Egg Incubation Volitional spawning of wild-caught broodsto ck supplied larvae for this trial. Larval Rearing and Ex perimental Design Experimental tanks were inoculat ed daily with Tahitian strain Isochrysis galbana (T-ISO) to a density of 120,000,000 cells/mL. Dietary Treatments Three dietary treatments were examined in this study, a standard reference diet (SRD) consisting solely of enriched rotifers, a one day diet, and a three day di et (Table 3-1). Each dietary treatment was replicated six times and four tanks monitore d unfed larvae. The one day experimental diet consisted of feeding larvae copepod nauplii on 2 days post hatch (DPH) at a rate of 2.43 nauplii/mL/day and then switching to the SRD from 3 DPH. The three day experimental diet consisted of feeding la rvae copepod nauplii on 2 DPH at a rate of 2.43 nauplii/mL/day, on 3 DPH at a rate of 3.10 na uplii/mL/day, and on 4 DPH at a rate of 3.41 62

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nauplii/mL/day and then switching to the SRD fr om 5 DPH. Six tanks monitored larvae fed copepod nauplii on 2 DPH at a rate of 2.43 nauplii/mL/day and not fed again for the entire trial. Copepod Culture Copepods were batch cultured in eight 200-L cylindrical, flat-bottom polyethylene tanks. Copepod nauplii were harvested twice daily from each tank via floating airlifts (Equipment section of Chapter 2) within each tank. Stress Resistance Analysis Net Stress Larvae were held out of water fo r 30, 120, 240, 360, or 600 seconds. Salinity Stress 1-L containers: Larvae were exposed to 1, 35, or 100 g/L salinity water. Larval survival was recorded 30 minutes after exposure. Salini ties were attained by mixing appropriate amounts of >100 g/L salinity solution with deionized water one day prior to the salinity stress experiment. A >100 g/L salinity solution was obtained by continuously boiling treated seawater. 30 mL containers: One randomly selected larva was removed from 35 g/L salinity and was transferred into a 30 mL container comprising 100 g/L se awater at 26C. 10 larvae were selected from each dietary treatment. Time to death for each larva was recorded in seconds. Results Water Quality All water quality parameters were recorded within normal limits (Table 3-2). Growth At 0 DPH, larvae had a mean standard leng th (SL) of 2.41 0.22 mm (mean SD) and a mean body depth (BD) of 0.81 0.07 mm. At 3 DPH, mean SL of larvae was significantly different between dietar y treatments (ANOVA, F 2,157 = 4.12; p = 0.0181). The SL of larvae from 63

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the one day treatment (2.49 0.14 mm) was signifi cantly greater than the SL of larvae fed the SRD (2.40 0.20 mm) (Figure 4-1). The thr ee day (2.44 0.14 mm) treatment was not significantly different from any of the treatment diets. The mean BD of larvae was significantly different between dietar y treatments (ANOVA, F 2,157 = 37.27; p < 0.0001). The BD of larvae fed the one day (0.79 0.06 mm) and three day (0.79 0.06 mm) treatments was significantly greater than the BD of larvae fed the SRD (0.71 0.05 mm) treatme nt, but were not significantly different from each other (Figure 4-2). At 6 DPH, mean SL of larvae was signifi cantly different between dietary treatments (ANOVA, F 2,157 = 14.35; p < 0.0001). The SL of larvae fe d the one day treatment (3.73 0.37 mm) was significantly greater than the SL of larvae fed the t hree day (3.48 0.45 mm) and SRD (3.29 0.44 mm) treatments (Figure 4-1). Larvae fed the three day treatment also had a significantly greater SL than larvae fed the SR D treatment. The mean BD of larvae was significantly different between dietary treatments (ANOVA, F 2,157 = 13.69; p < 0.0001). The BD of larvae fed the one day (0.98 0.11 mm) a nd three day (0.95 0.13 mm) treatments were significantly greater than larvae fed the SRD treatment (0.88 0.09 mm), but were not different from each other (Figure 4-2). Survival At 7 DPH, the mean survival of larvae was significantly different between dietary treatments (ANOVA, F 2,13 = 12.12; p = 0.0011). The survival of larvae fed the three day treatment (71.5 5.1%) was significantly greater th an survival from the one day (63.5 7.6%) and SRD (53.6 5.0%) treatments (Figure 4-3). Surv ival of larvae fed th e one day treatment was also significantly greater than survival from the SRD treatment. 64

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Stress Resistance Net Stress No significant differences we re detected in the mean survival of larvae fed the experimental diets following 30 (ANOVA, F 2,9 = 1.75; p = 0.2280), 120 (ANOVA, F 2,9 = 3.98; p = 0.0579), and 360 (ANOVA, F 2,9 = 1.80; p = 0.2200) seconds of exposure to net stress. At the 240 second duration, the mean survival of larvae was significantly diffe rent between dietary treatments (ANOVA, F 2,9 = 5.06; p = 0.0338). The mean survival of larvae fed the three day (65.0 34.2%) and one day (60.0 16.4%) treatments were significantly greater than larvae fed the SRD (15.0 19.2%) (Figure 4-4). At th e 600 second duration, mean survival of larvae was significantly different betw een dietary treatments (ANOVA, F 2,9 = 21.50; p = 0.0004). The mean survival of larvae fed the three day treatment (30.0 10.0%) wa s significantly greater than larvae fed the one day (0.00 0.00%) a nd SRD (5.0 10.0%) treatments, which were not significantly different from each other. Salinity Stress 1-L containers : No larvae were alive in the 1 and 100 g/L treatme nts. All larvae were alive in the 35 g/L treatment at the 30 minute time interval for all of the experimental diets tested. Therefore, no statistical analysis was performed. 30-mL containers: The mean time to death of larv ae was not significantly different between dietary treatments (ANOVA, F 2,27 = 1.71; p = 0.2003). The time to death of larvae fed the three day treatment was greater (764.5 330.2 seconds) than larvae fed either the one day (669.4 189.8 seconds) or SRD (577.8 89.7 seconds) treatments (Figure 4-5). Discussion Significantly greater growth, in SL and BD, a nd survival were detected for pompano larvae fed copepod treatment diets when compared to larv ae fed the SRD. Survival of larvae fed the 65

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three day treatment during tria l 2 was significantly higher than both the one day and the SRD treatments. In trial 1, however, no significant diffe rences were detected when survival from the three day treatment was compared to both the one day and SRD treatme nts. One explanation for the increased performance of larvae fed the t hree day treatment in tr ial 2 is the increased feeding rate from 2 DPH. During trial 2 la rvae were fed 0.30 nauplii/mL more on 2 DPH, 0.60 nauplii/mL more on 3 DPH, and 0.41 nauplii/mL more on 4 DPH. This increased feeding rate likely supplied more nutrients during the fi rst 3 feeding days and significantly increased survival in the three day treatment during tria l 2. Another explanation is the decreased water temperature recorded during trial 2, which was on average 1.2C cooler than water temperatures recorded during trial 1. A redu ced water temperature could have decreased the metabolism of the larvae, subsequently reduci ng the amount of feed organisms needed to maintain sufficient growth and survival. Also, copepod nauplii fed during trial 2 were supplied continuously throughout the day as airlifts were continually harvesting nauplii to meet the required feed densities. During trial 1, nauplii were fed two times, 0900 and 1600. Although internal standpipes with 50 micron nylon mesh kept copepod nauplii inside the experimental tanks, their ability to find refuge between feedings was increased during trial 1. In trial 2, nauplii were supplied to experimental tanks up to 8 times a day with little time for nauplii to seek refuge before being consumed. This continuous feedi ng strategy may have influenced the significantly greater results seen in all morphometric and survival data fo r both copepod fed treatments. Net stress resistance of pomp ano larvae fed copepod treatm ent diets was significantly higher than larvae fed the SRD at the 240 and 600 second durations. Remarkably, the three day treatment had at least one larva from ever y replicate still alive in the 600 second treatment duration after one hour, while only on e larva from combined replicat es of both the one day and 66

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SRD treatments was alive. The salinity stress e xperiments conducted in 1-L containers produced poor results. More than likely, the extreme salinities (1 and 100 g/L) examined and the 30 minute time interval were too great to detect di fferences in tolerance due to dietary nutrition. The salinity stress experiments conducted in 30 mL containers produced results with high variation. On 0 DPH a high level of rotifer contamina tion was noticed in the copepod populations. All tanks containing copepod popul ations were immediately siev ed through a series of 240 sieves. This allowed all rotif ers and younger life stages of co pepods to be flushed from the population, while copepod adults were retained on the sieves and placed into new culture tanks. Nauplii were harvested via airlift and fed to fish larvae roughly 36 hours after sieving the populations. Surprisingly, sufficient quantities of copepod nauplii were harvested to complete this larval trial, although a reduced number of c opepod adults were apparent The use of airlifts during this larval trial provided an efficient and passive method for the collection of nauplii that had no observed negative effect on the condi tion and health of the population. The results of this study confirm the increases in growth, survival, a nd resistance to stress that can be attributed to feeding copepods to first feeding Florida pompa no. However, acquiring information on the advantages of feeding copepods to pompano larvae up to the Artemia transitioning period (9 DPH) and developing appropriate feeding regimes for pompano larvae is warranted. 67

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2.25 2.75 3.25 3.75 4.25 4.75 3 DPH 6 DPHStandard Length (mm) Rotifer One Day Three Day A B C A B AB Figure 4-1. The mean standard lengt h (mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 2. Values were recorded on 3 and 6 days post hatch (DPH).Standard error bars and LSD multiple comparisons test results are displayed. Bars with different lett ers are statistically different (p 0.05). 0.50 0.75 1.00 1.25 1.50 3 DPH 6 DPHBody Depth (mm) Rotifer One Day Three Day A A B A A B Figure 4-2. The mean body depth ( mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 2. Values were recorded on 3 and 6 days post hatch (DPH). Standard error bars and LSD multiple comparison test results are displayed. Bars with different lett ers are statistically different (p 0.05). 68

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0 25 50 75 Rotifer One Day Three DaySurvival (%) A B C Figure 4-3. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 2. Standard error bars and LSD multiple comparisons test results are displayed. Bars with different letters are statistically different (p 0.05). 0 20 40 60 80 100 30 120 240 360 600Survival (%)Duration (seconds) Rotifer One Day Three Day A A A A A A A A A A A B A B B Figure 4-4. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments exposed to net stress during trial 2. Durations were 30, 120, 240, 360, and 600 seconds. Standard error bars and LSD multiple comparison test results are displayed. Bars with different letters are statistically different (p 0.05). 69

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500 600 700 800 900 Rotifer One Day Three DayTime (seconds) Figure 4-5. The mean time to death of Florida pompano ( Trachinotus carolinus ) larvae fed different dietary treatments exposed to 100 mg/L salinity seawater during trial 2. No significant differences in survival were det ected (p = 0.2003). Standard error bars are displayed. 70

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CHAPTER 5 TRIAL 3 The goal of this study was to examine the benefits of feeding nauplii of Pseudodiaptomus pelagicus to larval Florida pompano, Trachinotus carolinus up to the Artemia transitioning period, 9 days post hatch (DPH). A mesocosm tr eatment was also examined to develop feeding strategies for marine fish larvae and a one day dietary treatment was fed to determine the minimum duration of feeding copepod nauplii to larval Florida pompano necessary to have an effect. Material and Methods Material and Methods from trial 1 we re repeated unless otherwise noted. Spawning and Egg Incubation Volitional spawning of wild-caught broodsto ck supplied larvae for this trial. Larval Rearing and Ex perimental Design An 8-day larval rearing trial was conducted. Experimental tanks were inoculated daily with Tahitian strain Isochrysis galbana (T-ISO) at a density of 100,000,000 cells/mL. Dietary Treatments Four dietary treatments were examined in this study, a standard reference diet (SRD) consisting solely of enriched rotifers, a cope pod diet, a one day diet, and a mesocosm treatment (Table 3-1). The copepod experiment al diet consisted of feeding larvae copepod nauplii on 2 DPH at a rate of 3.50 nauplii/mL/da y, on 3 DPH at a rate of 3.60 nauplii/mL/day, on 4 DPH at a rate of 4.00 nauplii/mL/day, on 5 DPH at a rate of 4.00 nauplii/mL/day, on 6 DPH at rate of 4.00 nauplii/mL/day, and on 7 DPH at a rate of 3.78 nauplii/mL/day. The one day experimental diet consisted of feeding la rvae copepod nauplii on 2 DPH at a rate of 1.25 nauplii/mL/day and then switching to the SRD from 3 DPH. The mesocosm experimental diet 71

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consisted of 16,000 P. pelagicus copepodites (collected as nauplii 8 days prior) stocked into each experimental tank on -2 DPH. Copepodites were in itially stocked into the experimental tanks at half volume (6.5-L) and kept under static c onditions with gentle aeration. Tanks were maintained with ambient temperature and a salin ity of 22 g/L. 35 g/L salinity seawater was dripped into the mesocosm treatment tanks beginning on -1 DPH, to slowly acclimate P. pelagicus copepodites to larval rearin g conditions. By 0 DPH, meso cosm treatment tanks were filled with 35 g/L salinity seawater, and were suit able for larval stocking. Once filled (13-L), the copepod density within the mesocosm treatme nt tanks was 1.25 copepodites/mL. After inoculation, the mesocosm treatment wa s fed T-ISO daily to a density of 200,000,000 cells/mL until the entire system was inoculated w ith T-ISO. From 2 DPH to 7 DPH larvae were fed diets consisting of enriched S-strain rotifers, Brachionus plicatilis and P pelagicus nauplii. Copepod Culture Copepods were batch cultured in six 200-L cylindrical, flat-bottom polyethylene tanks with mild aeration and no sponge filters. Nauplii were harvested twice daily from each tank via floating airlifts (Equipment secti on of Chapter 2) within each tank. Sample Collection and Morphometric Analysis Ten larvae from each experimental tank were sampled on 3 and 7 DPH for morphometric analysis. Results Water Quality All water quality parameters were recorded within normal limits (Table 3-2). Growth Morphometric data were not analyzed. 72

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Survival Survival data were not analyzed. Discussion The survival of all copepod fed treatments be gan to decline on 4 DPH and was severely reduced by 8 DPH, when the trial was discontinued. The health of pompano larvae was evaluated and an examination of the copepod cult ure techniques and larv al feeding rates was warranted to explain the low survival experienced by larvae fed the copepod treatment diets. Also, a description of observations made regard ing the copepod performa nce in the mesocosm treatment is discussed. Upon visual inspection of the tr eatment tanks, a notably higher su rvival rate for larvae fed the SRD was observed when compared to the copepod fed treatments. Although these larvae were not counted, no deviation from the mean survival (30%) was apparent. Conversely, when larvae fed the copepod treatment were counted a survival of 3.75 3.97% (Mean SD) was recorded. Survival of the other copepod fed treatments, although not counted, appeared similar to the survival of larvae fed the copepod treatment. Therefore, at the conclusion of trial 3, samples of pompano larvae fed the copepod treatment, copepods, T-ISO, and water from the treat ment system were sent to the University of Floridas Fish Health Diagnostic Labora tory for analysis. Upon evaluation, a Vibrio sp. infection was detected in the gills of Florida pompano larvae fed copepods and likely caused increased mortality in those treatm ents (Figure 5-1). Since the conclusion of trial 2, some changes were made pe rtaining to the population of copepods maintained at the University of Floridas Indian River Research and Education Center (IRREC). During the first two trials, copepod popul ations became contaminated with rotifers. Afterwards, unsuccessful attempts were made to maintain rotifer-free populations of copepods. 73

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Therefore, the contaminated populations were bleached and a new population was acquired from Algagen LLC (Vero Beach, FL). Prior to th e acquisition of the ne w population, all copepod culture equipment was either thrown away or so aked for days in a 25 mg/L solution of sodium hypochlorite. From this time forward, a stock population of copepods was maintained in a biosecure room, while copepods grown for fish larval rearing were maintained inside the hatchery. Copepod culture conditions for trial 3 were identical to those of trials 1 and 2 except no sponge filters were used in any of the copepod culture tanks. Additionally, larvae in the copepod treatment were overfed during this trial in an attempt to identify an appropriate feeding level for Fl orida pompano larvae during the first 9 DPH. However, this quickly became a problem. As the su rvival in the experimental tanks declined, the concentration of copepod nauplii within each ta nk grew (>7 nauplii/mL). Larvae experienced frequent repeated physical intera ctions with copepod nauplii and this may have had deleterious effects. Also, elevated numbers of copepod nauplii drastically reduced the amount of algae within copepod treatment tanks, decreasing the contrast needed to visualize prey and likely affected the amount of indirect nutrition received. Continual provi sion of a low prey density (2 4 nauplii/mL) is recommended for future studies with Florida pom pano larvae fed copepod nauplii. Upon observation of the mesocosm treatment resu lts, a suitable ratio of males to females was achieved during this trial when compared to tria l 1. Most adults appeared to be paired (the reproductive state) and the presence of schooling males was reduced or absent in this trial. In trial 1, adults were taken from established populati ons and stocked into the experimental system. For this trial, copepod nauplii were harvested prior to the larval trial and reared together as a cohort. Once stocked into the experimental system, copepodites were acclimated from 22 g/L 74

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salinity to 35 g/L salinity over a 30 hour period. By 2 DPH, c opepodites had matured to adults and began to reproduce. However, samples of the culture water indicated the concentration of nauplii in the experimental tanks was still low (0.16 nauplii/mL). Copepod nauplii production may have, again, been reduced by insufficient am ounts of live T-ISO within the experimental tanks. Algal densities similar to those of trial 1 were initiated for this trial and fell below those suggested for optimal nauplii production by P. pelagicus (Rhyne et al., 2009). Future studies evaluating a mesocosm should include provision of sufficient algal densities and use copepods fully acclimated to larval rearing conditions. Although this trial was not completed, valuab le information was obtained regarding the condition of copepod cultures fed to marine fish larvae and co pepod feeding rates for Florida pompano larvae. Furthermore, useful insight in the developmen t of an appropriate mesocosm feeding regime for P. pelagicus was observed. 75

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V V Figure 5-1. Histology section (hemot oxylin and eosin stain) displaying Vibrio sp. (V) within the gills of Florida pompano larvae ( Trachinotus carolinus ) fed copepods during trial 3. (Photo and evaluation courtesy of B. De nise Petty, University of Florida) 76

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CHAPTER 6 TRIAL 4 The goal of this study was to examine the benefits of feeding nauplii of the calanoid copepod Pseudodiaptomus pelagicus to larval Florida pompano, Trachinotus carolinus, up to the Artemia transitioning period, 9 days post hatch (DPH). Florida pompano larvae were fed a copepod diet, consisting solely of copepod nauplii, and a mix diet, consisting of both rotifers and copepod nauplii, for the entire la rval trial. Additionally, larv ae were fed a diet of copepod nauplii for the first three days of feeding to c onfirm the results of trials 1 and 2. Net stress experiments were performed to detect differences in stress resistance of Florida pompano larvae fed dietary treatments. Material and Methods Material and Methods from trial 1 we re repeated unless otherwise noted. Spawning and Egg Incubation Volitional spawning of wild-caught broodsto ck supplied larvae for this trial. Larval Rearing and Ex perimental Design A 9-day larval rearing trial wa s conducted. Experimental tank s were inoculated daily with Tahitian strain Isochrysis galbana (T-ISO) at a density of 200,000,000 cells/mL. At 7 DPH, aeration was increased slightly and water flow was adjusted to 72 mL/minute for the duration of the trial. In each replicate tank, temperature, salinity, dissolved oxygen, and pH were monitored daily (YSI Incorporated Model 556 MPS, OH). Dietary Treatments Four dietary treatments were examined in this study, a standard reference diet (SRD) consisting solely of enriched rotifers, a copepod diet, a three day diet, and a mix diet (Table 3-1). Each dietary treatment was replicated seven times. The copepod experimental diet 77

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consisted of feeding larvae copepod nauplii on 2 DPH at a rate of 2.50 nauplii/mL/day, on 3 DPH at a rate of 3.00 nauplii/mL/day, on 4 DPH at a rate of 4.50 nauplii/mL/day, on 5 DPH at a rate of 6.25 nauplii/mL/day, on 6 DPH at rate of 7.80 nauplii/mL/day, on 7 DPH at a rate of 8.00 nauplii/mL/day, and on 8 DPH at a rate of 7.80 nauplii/mL/day. The three day experimental diet consisted of feeding larvae copepod nauplii on 2 DPH at a rate of 2.50 nauplii/mLday, on 3 DPH at a rate of 3.00 nauplii/mL/day, and on 4 DPH at a rate of 4.50 nauplii/mL/day and then switching to the SRD from 5 DPH. The mix diet consisted of feeding larvae a mixture of rotifers and copepods at a ratio of 4:1. Live feeds were fed to larvae four times daily (0900, 1300, 1700, and 2100), enriched rotifers at a rate of 2.0 rotifers/mL /day copepod nauplii at a rate of 0.5 nauplii/mL/day. From 2 DPH to 8 DPH la rvae were fed diets consisting of enriched Sstrain rotifers, Brachionus plicatilis and P. pelagicus nauplii. Rotifer Culture and Enrichment Rotifers were cultured in a 950-L cylindrical, fl at-bottom, fiberglass tank. Approximately, 20 million rotifers were harvested daily for enrichment prior to feeding to fish larvae. Rotifers were enriched in two 19-L buckets. Copepod Culture Copepods were batch cultured in five 200-L cylindrical, flat-bottom polyethylene tanks and one 1800-L cylindrical, conical-bottom tank. Copepod populations were fed a 1:1 mixture of T-ISO (15 million cells/mL) and TW (1 million cells/mL). Each 200-L population was fed 1 L of the algal mix and the 1800-L population was fed 18 L. Copepod nauplii were harvested twice daily from each tank via floating airlifts (Equipment section of Chapter 2) within each tank. 78

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Sample Collection and Morphometric Analysis Larvae from each experimental tank were randomly sampled on 3, 6, and 9 DPH for morphometric analysis. Larvae were euthan ized with buffered Tricaine-S (Tricaine Methanesulfonate; Western Chemical Inc., WA ) and photographed. For calibration purposes, larvae were photographed on a Sedgewick-Rafte r Cell S50 (PYSER-SGI Limited, Kent, UK) with a 1.0-mm square grid base. Net Stress Resistance Analysis On 9 DPH, ten larvae were randomly selected and held out of water for 180, 360, 540, or 720 seconds. Each dietary treatment was repli cated five times for each time interval. Results Water Quality All water quality parameters were recorded within normal limits (Table 3-2). Growth At 0 DPH, larvae had a mean standard leng th (SL) of 2.97 0.14 mm (mean SD) and a mean body depth (BD) of 0.90 0.08 mm At 3 DPH, the mean SL of larvae was significantly different between dietar y treatments (ANOVA, F 3,276 = 12.57; p < 0.0001). The mean SL of larvae fed the copepod (3.29 0.16 mm), mix (3.24 0.15 mm), and three day (3.26 0.22 mm) treatments were significantly greater than larvae fed the standard reference diet (SRD) treatment (3.10 0.21 mm), but were not significan tly different from each other (Figure 6-1). The mean BD of larvae was significantly di fferent between dietary treatments (ANOVA, F 3,276 = 34.91; p < 0.0001). The mean BD of larvae fed the mix (0.75 0.06 mm), three day (0.75 0.06 mm), and copepod (0.74 0.06 mm) treatments were significantly greater than larvae fed the SRD treatment (0.66 0.05 mm), but were not significantly different from each other (Figure 6-2). 79

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At 6 DPH, the mean SL of larvae was signifi cantly different between dietary treatments (ANOVA, F 3,276 = 3.03; p = 0.0300). The mean SL of larvae fed the SRD (3.92 0.35 mm) and mix (3.91 0.38 mm) treatments were significan tly greater than larvae fed the three day treatment (3.78 0.31 mm), but were not significan tly different from each other (Figure 6-1). The mean SL of larvae fed the copepod treatment (3.82 0.31 mm) was not significantly different from any of the other dietary treatmen ts. The mean BD of larvae was not significantly different for larvae fed the mix (0.99 0.14 mm), SRD (0.98 0.13 mm), copepod (0.95 0.12 mm), and three day (0.94 0.12 mm) treatments (ANOVA, F 3,276 = 1.97; p = 0.1180) (Figure 6-2). At 9 DPH, the mean SL of larvae was signifi cantly different between dietary treatments (ANOVA, F 3,276 = 16.65; p < 0.0001). The mean SL of larvae fed the mix (4.56 0.52 mm) treatment was significantly great er than larvae fed the SRD ( 4.40 0.46 mm), three day (4.25 0.29 mm), and copepod (4.08 0.37 mm) treatments (Figure 6-1). The mean SL of larvae fed the SRD treatment was significan tly greater than larvae fed the three day and copepod treatments and the mean SL of larvae fed the three day treatment was significantly greater than larvae fed the copepod treatment. The mean BD of larvae was significantly different between dietary treatments (ANOVA, F 3,276 = 18.20; p < 0.0001). The mean BD of larvae fed the mix (1.24 0.21 mm) treatment was significantly gr eater than larvae fed the SRD (1.18 0.21 mm), three day (1.11 0.14 mm), a nd copepod (1.03 0.14 mm) treatments (Figure 6-2). The mean BD of larvae fed the SRD treatment was significantly greater than larvae fed the three day and copepod treatments and the mean BD of larvae fed the three day treatment was significantly greater than larvae fed the copepod treatment. 80

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Survival At 9 DPH, the mean survival of larvae was significantly different between dietary treatments (ANOVA, F 3,24 = 7.58; p = 0.0010). The mean surv ival of larvae fed the copepod treatment (57.3 15.4%) was significantly greater than mean survival from the SRD (45.5 9.5%), mix (37.8 2.4%), and three day (34. 0 7.6%) treatments (Figure 6-3). The mean survival of larvae fed the SRD treatment was signi ficantly greater than larvae fed the three day treatment. The mean survival of larvae fed th e mix treatment was not significantly different from larvae fed the SRD or three day treatments. Net Stress Resistance After the 180 second duration of exposure to net stress, mean surviv al was significantly different between dietar y treatments (ANOVA, F 3,16 = 3.63; p = 0.0359). The mean survival of larvae fed the copepod treatm ent (80.0 12.2%) was significantly greater than mean survival of larvae fed the three day (56.0 19.5%) and SRD (54.0 11.4%) treatments (Figure 6-4). The mean survival of larvae fed the mix tr eatment (76.0 18.2%) was significantly greater than mean survival of larvae fed the SRD treatme nt, but no significant differences were detected when compared to the copepod or three day treatments. There were no significant differences between the three day and SRD treatments. After the 360 second duration of exposure to ne t stress, mean survival was not significantly different between larvae fed the mix (52.0 19.2%), copepod ( 46.0 15.2%), SRD (42.0 13.0%), and three day (38.0 14.8%) treatments (ANOVA, F 3,16 = 0.72; p = 0.5542). After the 540 second duration of exposure to ne t stress, mean survival was not significantly different between larvae fed the copepod (56.0 32.1%), three day (36.0 20.7%), mix (32.0 16.4%), and SRD (28.0 21.7%) treatments (ANOVA, F 3,16 = 1.41; p = 0.2775). 81

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After the 720 second duration of exposure to net stress, mean surviv al was significantly different between dietar y treatments (ANOVA, F 3,16 = 15.31; p < 0.0001). The mean survival of larvae fed the three day treatment (52.0 16.4%) was significantly greater than mean survival of larvae fed the copepod (28.0 16.4%), mix (12.0 11.0%), and SRD (0.0 0.0%) treatments (Figure 6-4). The mean surviv al of larvae fed the copepod treatment was significantly greater than survival of larvae fed the SRD treatment. There were no significant differences between the mean survival of larvae fed the mix treatment and mean survival of larvae fed the copepod or SRD treatments. Discussion Variable results were obtained from provi ding Florida pompano larvae with copepod diets for the entire 9 day trial. Improvements in growt h, survival and resistance to stress were detected for treatments fed copepods compared to those larvae fed the SRD. However, the benefits of those improvements were inconsistent between the copepod fed treatments. The copepod treatment had the greatest su rvival for any of the dietary treatments examined. At 9 DPH, however, the recorded growth of those larvae (SL and BD) was significantly lower than any of the other dietary treatments, al though reduced growth at both 3 and 6 DPH had not yet become apparent. One explanation for this decreased growth beyond 6 DPH for larvae fed the copepod treatment is an insu fficient feeding rate at the observed level of survival. One replicate in the copepod treatmen t recorded much lower survival (23.8%) than any other replicate in the treatmen t. The next lowest survival r ecorded by a replicate from the copepod treatment was 56.0%. When morphometric data, taken at 9 DPH, from larvae fed the copepod treatment with 23.8% surv ival (23 C) was compared to the replicate with the lowest survival from the SRD treatment (35.4%; 35 R) significant differences in both mean SL (TTEST, T 18 = 3.06; p = 0.0068) and mean BD (T-TEST, T 18 = 2.53; p = 0.0211) were detected. 82

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The SL of 23 C (4.58 0.37 mm) was significantly greater than the SL of 35 R (4.00 0.49 mm) and the BD of 23 C (1.22 0.15 mm) was significan tly greater than the BD of 35 R (1.03 0.19 mm). Further, when we examined replicates wi th similar survival rate s from both the copepod treatment (56.0%; 56 C) and the SRD treatment ( 57.0%; 57 R) no significant differences in SL (T-TEST, T 18 = 0.01; p = 0.9949) or BD (T-TEST, T 18 = -0.10; p = 0.9188) we re detected. Since most of the other replicates from the SRD treatment had a lower survival than 57.0% and morphometric means detected when compari ng 56 C and 57 R more closely resembled those recorded in the copepod treatment, it can be deduced that a higher feeding rate for the copepod treatment past 6 DPH could have resulted in greater growth in SL and BD. In addition, cannibalism was not observed in any of the replicate tanks for the entire trial. Similarly, upon examination of la rval growth fed the three day treatment a significant decrease when compared to both the mix and SRD treatments at 9 DPH is observed. At 6 DPH, a significant decrease in SL for larvae fed the three day treatment was detected when compared to the SL of larvae fed both the mix and SRD treatments. However, BD at 6 DPH and morphometric data at 3 DP H were not significantly differ ent among any of the dietary treatments. Again decreases in growth after 6 DPH for a dietary treatment fed copepods was observed. When comparing 9 DPH morphometric data from replicates with similar survival from the three day (37.0%; 37 T) and SRD (38. 0%; 38 R) treatments, no significant differences are detected for SL (T-TEST, T 31.9 = -1.97; p = 0.0572) or BD (T-TEST, T 30.7 = -1.85; p = 0.0742). However, the relatively low p-value for bot h SL and BD, infers that the differences are small and significant differences may be detected with an increased sample size. In addition, unlike the copepod treatment, th e three day treatment recorded the lowest survival at the conclusion of the trial. Therefore, a sudden mo rtality across all replicates would have had to 83

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occur just prior to ascertaining survival to see the low growth associated with an insufficient feeding rate for the three day treatment at 9 DPH. Nutritional disadvantages cannot be ruled out in the reduced growth of larvae fed copepods past 6 DPH. For the first time in any of the larval rearing trials, Thalassiosira weissflogii (TW) was used in the diet of P. pelagicus. Dietary effects on P. pelagicus fed microalgae diets comparing T-ISO, TW, Chaetoceros gracilis Rhodomonas lens and Tetraselmis suecica have been conducted (Ohs et al., unpublished data). Adults fed monoalgal diets consisting of TW produced on average 3 times more naupl ii than adults fed T-ISO, th e traditional diet fed to P. pelagicus Since trials 4 and 5 were simulta neously conducted, elevated number s of nauplii were required to meet the demand of the treatments for both trials However, published literature indicates an insufficient ratio of docosahex aenoic acid (DHA): ecosapentaenoic acid (EPA) : arachidonic acid (AA) for marine fish larviculture in Acartia tonsa a marine calanoid copepod, fed monoalgal diets of TW (Stottrup and Jensen, 1990; Stottrup et al ., 1999). Furthermore, cod, G. morhua, larvae fed Acartia tonsa nauplii reared on TW had the lowest growth of any of the treatment diets examined (St. John et al., 2001). With this in mind, our populations of P. pelagicus were fed a 1:1 mixture of T-ISO (15 million cells/mL), an algae high in DHA (Reitan et al., 1997), and TW (1 million cells/mL) in the hopes of bot h boosting nauplii production, but without the adverse effects on fish larvae from the low DHA found in a monoalgal TW diet. However, low levels of DHA may also account for the decreased growth seen in the copepod and three day treatments. Conversely, the mix treatment had the greatest growth for any of the dietary treatments examined during trial 4 with respect to SL a nd BD. Even though mean survival of the mix treatment was significantly lower than the copep od treatment, no significant differences were 84

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detected when compared to those larvae fed the SRD treatment. By feeding a mixture of enriched rotifers and copepod nauplii to pompano larvae for the entire trial, disadvantages in growth and survival from a potential insufficien t feeding rate and possible differences in the nutritional composition of liv e feeds were avoided. Pompano larvae fed copepod treatment diets showed significantly higher survival compared to larvae fed SRD treatments at the 180 and 720 second dur ation of net stress exposure. Remarkably, 14 of 15 replicates fed copepods had at least one larva alive in the 720 second treatment duration after one hour, but none of the larvae fed the SRD treatment were alive. The results of this study reflect the advantages in growth, su rvival, and resistance to stress that can be attributed to the a ddition of copepods in the diet of Florida pompano during the first 9 DPH. A better understanding of a feeding regime for pompano larvae that involves copepods has been achieved. Benefits are apparent when larvae are fed cope pods for the first 1 days of feeding and then are switched to the SRD. However, acquiring information on the advantages of feeding copepods to pompano in a system designe d for commercial production is warranted. 85

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2.25 2.75 3.25 3.75 4.25 4.75 3 DPH 6 DPH 9 DPHStandard Length (mm) Rotifer Copepod Three Day Mix A B C D A A A AB B A A A B Figure 6-1. The mean standard lengt h (mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 4. Values we re recorded at 3, 6, and 9 days post hatch (DPH). Standard error bars and LSD multiple comparisons test results are displayed. Bars with different letters are significantly different (p 0.05). 0.50 0.75 1.00 1.25 1.50 3 DPH 6 DPH 9 DPHBody Depth (mm) Rotifer Copepod Three Day Mix A B C D A A A A A A A B Figure 6-2. The mean body depth ( mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 4. Values were recorded at 3, 6, and 9 days post hatch (DPH). Standard error bars and LSD multiple comparisons test results are displayed. Bars with different lett ers are significantly different (p 0.05). 86

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0 25 50 75 Rotifer Copepod Three Day MixSurvival (%) A B BC C Figure 6-3. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 4. Standard error bars and LSD multiple comparisons test results are displayed. Bars with different letters are statistically different (p 0.05). 0 20 40 60 80 100 180 360 540 720Survival (%)Duration (seconds) Rotifer Copepod Three Day Mix A AB A BC A A C A A A A A A B BC C Figure 6-4. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments exposed to net stress during trial 4. Durations were 180, 360, 540, and 720 seconds. Standard error bars and LSD multiple comparisons test results are displayed. Bars with different lett ers are significantly different (p 0.05). 87

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CHAPTER 7 TRIAL 5 The goal of this study was to examine the benefits of feeding nauplii of Pseudodiaptomus pelagicus to larval Florida pompano, Trachinotus carolinus in a system designed to simulate commercial production. Commercial facilities often do not adhere to the strict guidelines required for scientific inquiry, and so an i nvestigation of pompano production in a greenhouse under ambient Florida weather conditions was co nducted. Florida pompano larvae were either fed the standard reference diet of rotifers (SRD ) or copepods for the first day of feeding. Net stress experiments were performed to detect differences in stress resistance of larvae fed the dietary treatments. Material and Methods Material and methods from Trial 1 were repeated unless otherwise noted. Spawning and Egg Incubation Volitional spawning of wild-caught broodstock s upplied larvae for this trial. Incubation occurred at ambient greenhouse temperatur es (27.8.1C) and a salinity of 34.7 g/L. Larval Rearing and Ex perimental Design A 10-day larval rearing trial was conducted in two recirculating a quaculture systems each consisting of 8, 170-L cylindrical, conical-bottom fiberglass tanks with black walls and bottom. A natural photoperiod of approximately 14 hours li ght and 10 hours dark with ambient daytime surface light levels below 6430 lux (Milwaukee Model SM700) recorded during the larval rearing trial. The initial wa ter flow was 354 mL/minute and gentle aeration was provided. Beginning on 2 DPH, tanks were inoculated daily with Nannochloropsis oculata paste (Algagen LLC, FL) to a density of 450,000,200,000 cells/mL and water flow was increased to 472 mL/minute. At 4 DPH, aerati on was increased slightly and wa ter flow was adjusted to 708 88

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mL/minute. At 7 DPH, aeration was again increa sed slightly and water flow was adjusted to 945 mL/minute for the duration of the trial. In each replicate tank, temperature, salinity, dissolved oxygen, and pH were monitored daily (YSI Inco rporated Model 556 MPS, OH). Temperature from one randomly selected replicate tank was al so recorded every 30 minutes during the entire trial with a StowAway Tidbit temperature logger (Onset Comput er Corporation, MA). Dietary Treatments Two dietary treatments were examined in th is study, a SRD consisting solely of enriched rotifers and a one day diet (Tab le 3-1). Each dietary treatment was replicated six times and two tanks from each system contained no larvae to reduce water quality problems associated with biomass. The SRD consisted of enriched ro tifers fed to larvae four times daily (0900, 1200, 1600, and 1900) at a rate of 2.5 rotifers/mL/day fr om 2 days post hatch (DPH) and a rate of 3.0 rotifers/mL/day from 6 DPH. The one day experimental diet consis ted of feeding larvae copepod nauplii on 2 DPH at a rate of 1.11 nauplii/mL/day and then switching to the SRD from 3 DPH. From 2 DPH to 9 DPH larvae were fed di ets consisting of enrich ed S-strain rotifers, Brachionus plicatilis, and P. pelagicus nauplii. Rotifer Culture and Enrichment Rotifers were cultured in a 950-L cylindrical, fl at-bottom, fiberglass tank. Approximately, 20 million rotifers were harvested daily and were enriched prior to feeding to fish larvae. Rotifers were enriched in two 19-L buckets. Copepod Culture Copepods were batch cultured in five 200-L cylindrical, flat-bottom polyethylene tanks and one 1800-L cylindrical, conical-bottom tank. Copepod populations were fed a 1:1 mixture of Tahitian strain Isochrysis galbana (T-ISO) (15 million cells/mL) and Thalassiosira weissflogii (TW) (1 million cells/mL). Each 200L population was fed 1 L of the algal mix 89

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and the 1800-L population was fed 18 L. Cope pod nauplii were harvested twice daily from each tank via floating airlifts (Equipment section of Chapter 2) within each tank. Sample Collection and Morphometric Analysis Larvae from each experimental tank were randomly sampled on 3, 6, and 9 DPH for morphometric analysis. Larvae were euthan ized with buffered Tricaine-S (Tricaine Methanesulfonate; Western Chemical Inc., WA ) and photographed. For calibration purposes, larvae were photographed on a Sedgewick-Rafte r Cell S50 (PYSER-SGI Limited, Kent, UK) with a 1.0-mm square grid base. Net Stress Resistance Analysis On 9 DPH, ten larvae were randomly selected from each replicate tank and gently poured into units (Material and Methods section of Chapter 3) contai ning culture system seawater maintained at ambient greenhouse temperature and a salinity of 34 g/L. Two replicates from each tank were tested. Larvae were then lifted out of 1-L containers with the screen bottomed 0.5-L containers and held out of wa ter for 75, 180, 360, or 720 seconds. Results Water Quality All water quality parameters were recorded within normal limits (Table 3-1). Growth At 0 DPH, larvae had a mean standard leng th (SL) of 2.91 0.20 mm (mean SD) and a mean body depth (BD) of 0.80 0.09 mm. At 3 DPH, mean SL of larvae was not significantly different for larvae fed the one day (3.07 0.20 mm) and SRD (3.02 0.24 mm) treatments (TTEST, T 98 = 0.97; p = 0.3344). The mean BD of larvae was not significantly different for larvae fed the one day (0.66 0.05 mm) and SRD (0.65 0.04 mm) treatments (T-TEST, T 98 = 1.08; p = 0.2816). 90

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At 6 DPH, mean SL of larvae was not signifi cantly different for larvae fed the one day (3.77 0.25 mm) and SRD (3.77 0.21 mm) treatments (T-TEST, T 98 = -0.14; p = 0.8871). The mean BD of larvae was not significantly diffe rent for larvae fed the one day (0.90 0.09 mm) and SRD (0.87 0.09 mm) treatments (T-TEST, T 98 = 1.41; p = 0.1631). At 9 DPH, mean SL (T-TEST, T 98 = 2.11; p = 0.0377) and BD (T-TEST, T 98 = 2.18; p = 0.0317) of larvae were significantly different between larvae fed th e dietary treatments (Figure 71). The mean SL of larvae fed the one da y treatment (4.11 0.30 mm) was significantly greater than the mean SL of larvae fed the SRD treatment (3.99 0.31 mm). The mean BD of larvae fed the one day treatment (1.05 0.10 mm ) was greater than the mean BD of larvae fed the SRD treatment (1.00 0.13 mm) (Figure 7-2). Survival At 9 DPH, mean survival of larvae fe d the SRD (29.3 9.4 %) and one day (24.6 6.0%) treatments were not significantly different (T-TEST, T 8 = -0.90; p = 0.3955) (Figure 7-3). Net Stress Resistance After the 75 second duration of exposure to net st ress, the mean survival of larvae fed the one day (36.0 25.0%) and SRD (25.0 27.2%) trea tments were not signi ficantly different (TTEST, T 18 = -1.06; p = 0.3013). No larvae were alive in any of the replicates from either dietary treatment at the 180, 360, and 720 second durations of exposure to net stress, so no statistical analysis was necessary. Discussion Improvements in growth were detected for larvae fed copepods compared to those larvae fed the SRD. At 9 DPH, larvae fed the one da y treatment had significan tly greater SL and BD. An appropriate feeding rate for larvae fed the one day diet on 2 DPH would have been 2 nauplii/mL. Unfortunately, not enough copepod na uplii were produced to meet that goal. 91

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Since larvae used in trial 4 and 5 came from the same spawn and the same SRD feeding regime was used in both trials, a comparison of those larvae fed the SRD treatments can be made between the two systems and their effect upon larval performance. However, larvae fed the SRD treatment in trial 5 (170-L syst em, Greenhouse = G) did have an increased feeding rate from 6 DPH and survival from that system was recorded one day later than survival recorded for trial 4 (13-L system, Hatchery = H). That may explai n why survival of larvae in H (45.5 9.5%) was significantly greater than survival of larvae in G (29.3 9.4%) (T-TEST, T 10 = 3.10; p = 0.0113). Reduced survival during that sp ecific 24 hour period may have been likely, as pompano larvae are routinely transitioned onto Artemia nauplii beginning on 9 DPH (C harles R. Weirich, pers. comm.). Since larvae were not fed Artemia nauplii during trial 5, a sufficient quantity of feed may not have been available. Furthermore, the elevated temperature (>26C) may have accelerated development of larvae and d ecreased the time when provision of Artemia should have been initiated. Another explanation is that surv ival was initially reduced prior to first feeding. Under the ambient conditions of the greenhous e, larvae were exposed to in cubation temperatures above those experienced in the hatchery (26C). This may have led to an accelerated depletion of yolksac reserves prior to first feed ing. When the ratio of body depth to standard length (BD:SL) for 0 DPH larvae from both incubation systems was compared, significant differences were detected (T-TEST, T 118 = -4.42; p < 0.0001). Larvae from the H incubation system (0.30 0.03) had a significantly higher BD:SL ratio than larvae from the G incubation system (0.27 0.04). Furthermore, the BD:SL of larvae from the in cubation systems in trials 1, 2, and 4 were significantly greater than the BD:SL of larvae from the incubation system in trial 5 (ANOVA, 92

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F 3,237 = 17.82; p < 0.0001). Upon examination of photogr aphs taken of larvae from both systems on 0 DPH, reduced yolk-sac reserves were seen in larvae from the G incubation system. No significant differences in growth at 3 DPH were detected for the SL or BD of larvae grown in either H or G. However, SL and BD at 6 and 9 DPH for larvae grown in H were significantly greater than SL and BD for larv ae grown in G. The increased feeding rate of 0.5 rotifers/mL/day for larvae in the G system may have had a negative effect on growth during this period. Interestingly, when comparing survival of larvae from the two systems, a reduction in survival from the G system is observed. The one day treatment in the G system may not have received sufficient nutrients due to a low feeding rate on 2 DPH. This would have subsequently led to a reduced performance in survival, gr owth, and stress resistance when compared to copepod fed treatments from the H system. Thes e differences, however, can not be applied to the SRD treatments from both systems, which r eceived identical diets during the larval trials. During this trial, 1.1 million nauplii were used to feed the one day treatment. This feeding rate (1.1 nauplii/mL/day) provided advant ages in growth for Florida pompano at that larval density. To reach a rate of 2 naupl ii/mL/day, approximately 2 million nauplii would have had to have been produced. Usi ng current methods, it is possible for P. pelagicus populations to produce this quantity of nauplii. However, the cons istency of that production is not known. Furthermore, as the feeding rate for Florida pompano increases, it would be difficult to produce appropriate amounts of copepod nauplii to satisfy thei r demand. We were able to feed copepod nauplii to Florida pompano larvae for one day, but continued f eeding at that rate would have been difficult. 93

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The results of this study showed the advantages in growth and resistance to stress attributed to the addition of copepods in the diet of Florida pompano on the first day of feeding compared to those larvae fed the SRD. Furtherm ore, these results do verify results obtained in the 13-L system and provide valuable reference to the number of copepods required to obtain beneficial effects. Although advantages in grow th were seen for Florida pompano larvae fed the one day treatment, an increased feeding rate may have further improved results. However, continuous copepod production at th e required scale has not been achieved and further research is warranted. 94

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2.25 2.75 3.25 3.75 4.25 4.75 3 DPH 6 DPH 9 DPHStandard Length (mm) Rotifer One Day A B A A A A Figure 7-1. The mean standard length (mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 5. Values we re recorded at 3, 6, and 9 days post hatch (DPH). Standard error bars and students t-test re sults are displayed. Bars with different letters are statistically different (p 0.05). 0.5 1.0 1.5 3 DPH 6 DPH 9 DPHBody Depth (mm) Rotifer One Day A B A A A A Figure 7-2. The mean body depth (mm) of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 5. Values were recorded at 3, 6, and 9 days post hatch (DPH). Standard error bars and st udents t-test results are displayed. Bars with different letters are statistically different (p 0.05). 95

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0 25 50 75 Rotifer One DaySurvival (%) A A Figure 7-3. The mean survival of Florida pompano larvae ( Trachinotus carolinus) fed different dietary treatments during trial 5. Standard e rror bars and students t-test results are displayed. Bars with different lett ers are statistically different (p 0.05). 96

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CHAPTER 8 CONCLUSION Five dietary treatments consisting of Pseudodiaptomus pelagicus fed to larval Florida pompano, Trachinotus carolinus for various durations, at various concentrations and combinations were examined. Subsequent stress resistance of those larvae was examined at the conclusion of each trial through either net or salinity stress experiments. Furthermore, microalgae and copepod culture methods are summarized. A feeding regime for Florida pompano larvae which includes P. pelagicus has been elucidated and methods for production and harvest of P. pelagicus are identified. Feeding copepod nauplii in addition to feeding rotifers, has traditionally proven beneficial for the production of marine fish larvae. Toledo et al. (1999) fed copepod nauplii (mainly Acartia tsuensis ) to grouper, Epinephelus coioides larvae from 2-6 days post hatch (DPH), switching to a rotifer diet from 7-18 DPH. When compared to larvae fed rotifers during the entire 18 day trial, improvements in feeding in cidence, survival, and growth were detected (Toledo et al., 1999). First feeding turbot, Psetta maxima larvae had higher growth and survival when fed Tisbe holothuriae nauplii for the first 3 DPH before switching to rotifers, when compared to rotifers for the en tire 8 DPH (Stottrup and Norsker, 1997). Payne et al. (2001) fed pink snapper, Pagrus auratus larvae nauplii of the copepod Gladioferens imparipes from 4 DPH and then switched to a rotifer diet for 15 more days. When compared to larvae fed rotifers for the entire 25 day period, increased growth from 6 DPH on was observed when copepods were fed (Payne et al., 2001). Survival and sw im bladder inflation were also greater for larvae fed copepods (Payne et al., 2001). Beneficial results were also seen during the course of the present study when copepod nauplii were fed to pompano larvae for either one or three days prior to transitioning to the 97

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standard reference diet of rotifers (SRD). Th ree different feeding rates for each period of copepod provision were evaluated and results co nsistently displayed advantages to pompano larvae in growth, survival, and stress resistance Pompano larvae fed the one day diet during the course of this evaluation consistently outpe rformed larvae fed the SRD. Pompano larvae fed the three day diet during trial 4 did not perform as well as larv ae fed the SRD. However, this may have been due to nutritional deficiencies of the microalgae, Thalassiosira weissflogii fed to the copepods during trial 4. Larvae fed the three day diet did not perform as well as larvae fed the one day diet during trial 1. But this ma y have been related to prey density and not providing adequate numbers of copepods. Re sults clearly indicate that providing copepod nauplii to Florida pompano larvae for at leas t the first day of feeding at a rate of 2 nauplli/mL/day improved growth, surv ival and stress resistance. A mixed diet, consisting of enriched rotifers and copepod nauplii fed to pompano larvae at a ratio of 4:1 increased growth a nd stress resistance compared to larvae fed the SRD. In Payne et al. (2001) significant increas es in growth were seen when pink snapper, Pagrus auratus larvae fed a 4:1 ratio of enriched rotifers, Branchionus spp., and copepod nauplii, Gladioferens imparipes compared to larvae fed only enri ched rotifers. Southern flounder, Paralichthys lethostigma larvae had significantly higher growth a nd survival when fed a 1:1 mixture of enriched rotifers, B. plicatilis and Acartia tonsa nauplii when compared to larvae fed rotifers exclusively (Wilcox et al., 2006). Finally, yellowtail clownfish, Amphiprion clarkii larvae fed a 1:1 mixture of rotifers, B. plicatilis and Tisbe spp. nauplii resulted in significantly higher growth and survival when compared to thos e larvae fed only rotifers or only Tisbe nauplii (Olivotto et al., 2008). During trial 4, pompa no larvae fed a mix diet had greater growth and stress resistance than larvae fed the SRD. By feeding a mix diet possible nutr itional deficiencies of 98

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enriched rotifers were avoided, and it is easier to maintain an appropriate density of live feeds within the larval rearing tanks when ro tifers are included in the daily ration. Larvae fed only copepods during th e entire larval trial record ed the highest survival but had the lowest growth from any of the dietary trea tments. This was likely due to an insufficient quantity of prey items available to the pompano larvae. Although some fish species, like red snapper, Lutjanus campechiensis require copepod nauplii for development during the larval phase (Lemus et al., 2008); pompano larvae do no t appear to require copepods. However, the clear benefits of providing cope pod nauplii to larval Florida po mpano have been identified. A mesocosm treatment was attempted two times and was not successful. However, valuable information was attained on the preparation of copepods for a mesocosm, including when and how many to stock into the larv al rearing system. The advant ages provided by this feeding strategy, especially gr eatly reduced labor, warrant a continued thorough examination. Significant increases in resistance to net stre ss were also observed for those larvae fed copepods during this evaluation. The increase in survival was dire ctly related to the duration of time larvae were fed copepods. The larvae fed th e mix diet also had significantly increased resistance when compared to larvae fed the SRD. Notably, 7 day old pompano larvae fed copepods and exposed to more than 600 seconds (10 minutes) of net stress were still alive one hour after being submerged in culture water. Si milar resistance to stress was reported by Kraul et al. (1993) when mahimahi, Coryphaena hippurus larvae were fed the harpacticoid copepod Euterpina acutifrons. In a similar net stress test, higher survival was recorded for larvae fed copepods at both the 60 and 120 second durations when compared to larvae fed enriched Artemia nauplii (Kraul et al., 1993). In that study, significantly higher levels of docosahexaenoic acid (DHA), provided by the copepods, were believed to have led to increas ed resistance to net stress. 99

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In Kanazawa (1997), increased levels of diet ary DHA were also repo rted to significantly improve net stress resistance in larval red sea bream, Pagrus major As larvae are often netted for transport within the hatchery facility or to another location, thei r ability to withstand exposure to net stress is important to in creased survival and subsequent production. Populations of P. pelagicus were successfully cultured and the use of airlifts for nauplii collection was examined. Airlifts should be the method used for harvesting nauplii to feed to marine fish larvae to reduce stress on the adul t and copepodite populations. Mass-scale culture of P. pelagicus was conducted and was shown to be very effective and labor efficient. Similar systems were developed by Pa yne and Rippingale (2001a) for Gladioferens imparipes Their systems incorporated biofiltration and were kept in continuous culture for approximately 14 months. In the current study, the system was onl y used for 8 days. However, results provided estimates of nauplii production from this scale of system and can be equated to the nauplii demanded by marine fish larvae. On a commercial scale, copepod nauplii, us ing current production methods, can only be fed to pompano larvae during the in itial feeding phase. As larval feeding rates increase with growth, the feasibility of feeding copepods d ecreases because of the production necessary. Based on results of this evaluation, a diet including copepod nauplii beyond 3 DPH appears unnecessary. Therefore, a feedi ng regime which includes copepods fed to pompano larvae on 2 3 DPH at a rate of 2 nauplii/mL/day is recommended. Most commercial marine fish production facilities do not have systems to grow copepod populations. However, given the short duration of copepod provision necessary, simply purchasing and feeding copepod nauplii for the initial 2 DPH is a viable option. 100

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LIST OF REFERENCES Andersen, R.A., 2005. Algal Culturing Techniques. Elsevier Academic Press, London, United Kingdom. 578 pp. Ako, H., Tamaru, C.S., Bass, P., Lee, C-S., 1994. Enhancing the resistance to physical stress in larvae of Mugil cephalus by the feeding of enriched Artemia nauplii. Aquaculture 122, 81. Bentley, C.D., Carroll, P.M., Watanabe, W.O., 2 008. Intensive rotifer produc tion in a pilot-scale continuous culture recirculating system using nonviable microalgae and an ammonia neutralizer. Journal of the World Aquacu lture Society 39, 625. Bell, J.G., Sargent, J., 2003. Arachidonic acid in aquaculture feeds: current status and future opportunities. Aquaculture 218, 491. Brinkmeyer, R.L., Holt, G.J., 1998. Highly unsat urated fatty acids in diets for red drum ( Sciaenops ocellatus ) larvae. Aquaculture 161, 253. Camus, T., Zeng, C., 2008. Effects of photoperiod on egg production and hatching success, naupliar and copepodite development, adult se x ratio and life expectancy of the tropical calanoid copepod Acartia sinjiensis Aquaculture 280, 220. Cavalin, F., Weirich, C.R. 2009. Larval perf ormance of aquacultured Florida pompano ( Trachinotus carolinus ) fed rotifers ( Brachionus plicatilis ) enriched with selected commercial diets. Aquaculture 292, 67. Chair, M., Dehasque, M., Van Poucke, S., Nelis H., Sorgeloos, P., De Leenheer, A.P., 1994. An oral challenge for turbot larvae with Vibrio anguillarum Aquaculture International 2, 270. Chen, Q., Sheng, J., Lin, Q., Gao, Y., Lv, J., 2006. Effect of salinity on re production and survival of the copepod Pseudodiaptomus annandalei Sewell, 1919. Aquaculture 258, 575. Chesney, E.J., 2005. Copepods as live prey: A revi ew of factors that influence the feeding success of marine fish larvae. In: Lee, C.S., OBryen, P.J., Marcus, N.H. (Eds.), Copepods in Aquaculture. Blackwell Publishing, Ames, Iowa. pp. 133. de Lima, L.C.M., Souza-Santos, L.P., 2007. The ingestion rate of Litopenaeus vannamei larvae as a function of Tisbe biminiensis copepod concentrati on. Aquaculture 271, 411. Delbare, D., Dhert, P., Lavens, P., 1996. Zooplankton. In: Lavens, P., Sorgeloos, P. (Eds.), Manual on the Production and Use of Live Food for Aquaculture. FAO Fisheries Technical Paper No. 361. FAO, Rome, Italy. pp. 252. Dhert, P., 1996. Rotifers. In: Lavens, P., Sorgeloo s, P. (Eds.), Manual on the Production and Use of Live Food for Aquaculture. FAO Fisher ies Technical Paper No. 361. FAO, Rome, Italy. pp. 49. 101

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Dhont, J., Van Stappen, G., 2003. Biology, ta nk production and nutritional value of Artemia In: Stottrup, J.G., McEvoy, L.A. (Eds.), Live Feeds in Marine Aquaculture. Blackwell Scientific Publications Ltd, Ox ford, United Kingdom. pp. 65. Doi, M., Toledo, J.D., Golez, M.S.N., de los Santos, M., Ohno, A., 1997. Preliminary investigation of feeding performance of larvae of early red-spotted grouper, Epinephelus coioides reared with mixed zooplankton. Hydrobiologia 358, 259. Drillet, G., Iversen, M.H., Sorensen, T.F., Raml ov, H., Lund, T., Hansen B.W., 2006. Effect of cold storage upon eggs of a calanoid copepod, Acartia tonsa (Dana) and their offspring. Aquaculture 254, 714. Evjemo, J.O., Reitan, K.I., Olsen, Y., 2003. Cope pods as live food organisms in the larval rearing of halibut larvae ( Hippoglossus hippoglossus L.) with special emphasis on the nutritional value. Aquaculture 227, 191. Farhadian, O., Yusoff, F.M., Mohamed, S., Saad, C.R., 2009. Use of cyclopoid copepod Apocyclops dengizicus as live feed for Penaeus monodon postlarvae. Journal of the World Aquaculture Society 40, 22. Fleeger, J.W., 2005. The potential to mass-culture harpacticoid copepods for use as food for larval fish. In: Lee, C.S., OBryen, P.J., Ma rcus, N.H. (Eds.), Copepods in Aquaculture. Blackwell Publishing, Ames, Iowa. pp. 11. Grice, G.D., 1969. The developmental stages of Pseudodiaptomus coronatus Williams (Copepoda, Calanoida). Crustaceana 17, 291. Hamre, K., Srivastava, A., Ronnestad, I., Mangor-Jensen, A., Stoss, J., 2008. Several micronutrients in the rotifer Brachionus sp. may not fulfill the nutritional requirement of marine fish larvae. A quaculture Nutrition 14, 51. Hernandez Molejon, O.G., Alvarez-Lajonc here, L., 2003. Culture experiments with Oithona oculata Farran, 1913 (Copepoda: Cyclopoi da), and its advantages as food for marine fish larvae. Aquaculture 219, 471. Ho, J.S., 2005. Symbiotic copepods as live feed in marine finfish rearing. In: Lee, C.S., OBryen, P.J., Marcus, N.H. (Eds.), Copepods in Aqu aculture. Blackwell Publishing, Ames, Iowa. pp. 25. Hojgaard, J.K., Jepsen, P.M., Hansen, B.W., 200 8. Salinity-induced quiescence in eggs of the calanoid copepod Acartia tonsa (Dana): a simple method for egg storage. Aquaculture Research 39, 828. Hunter, J.R., 1981. Feeding ecology and predation of marine fish larvae. In: Lasker, R. (Ed.), Marine Fish Larvae. Morphology, Ecology and Relation to Fisheries. University of Washington Press, Seattle, Washington. pp. 33. 102

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ITIS (Integrated Taxonomic Information System). 2009. Pseudodiaptomus pelagicus Herrick, 1884. ITIS-North America. Last accessed: June 2009. Available: http://www.itis.gov/servlet/SingleRpt/Singl eRpt?search_topic=TSN&search_value=8585 4 Jacobs, J., 1961. Laboratory cultiv ation of the marine copepod Pseudodiaptomus coronatus Williams. Limnology and Oceanography 6, 443. Kanazawa, A., 1997. Effects of docosahexaenoic acid and phospholipids on stress tolerance of fish. Aquaculture 155, 129. Koven, W., Barr, Y., Lutzky, S., Ben-Atia, I., Wei ss, R., Harel, M., Behrens, P., Tandler, A., 2001. The effect of dietary ar achidonic acid (20:4n-6) on grow th, survival and resistance to handling stress in gilthead seabream ( Sparus aurata ) larvae. Aquaculture 193, 107 122. Kraul, S., Brittain, K., Cantrell, R., Nagao, T., Ako, H., Ogasawara, A., Kitagawa, H., 1993. Nutritional factors affecting stress resistance in the larval mahimahi Coryphaena hippurus Journal of the World A quaculture Society 24, 186. Lavens, P., Sorgeloos, P., 1999. Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper No. 361. FAO, Rome, Italy. 305 pp. Lemus, J.T., Apeitos, A., Lee, M.S., de la Calzada, R., Snawder, J.M., 2008. Results of a demonstration of intensive culture of Acartia tonsa and use of copepod nauplii for fish culture at the Gulf Coast Research Labor atory. Aquaculture America 2008 Abstracts p. 201. Lee, C-S., 2003. Biotechnical advances in finfis h hatchery production: a review. Aquaculture 227, 439. Lee, C-S., OBryen, P.J., Marcus, N.H., 2005. Copepods in Aquaculture. Blackwell Publishing, Ames, Iowa. 269 pp. Lubzens, E., Zmora, O., 2003. Production and nutri tional value of rotifers. In: Stottrup, J.G., McEvoy, L.A. (Eds.), Live Feeds in Mari ne Aquaculture. Blackwell Scientific Publications Ltd, Oxford, United Kingdom. pp. 17. Marcus,N.H., Wilcox, J.A., 2007. A guide to the meso-scale production of the copepod Acartia tonsa National Sea Grant College Program. 26 pp. McKinnon, A.D., Duggan, S., Nichols, P.D., Ri mmer, M.A., Semmens, G., Robino, B., 2003. The potential of tropical par acalanid copepods as live feeds in aquaculture. Aquaculture 223, 89. Milione, M., Zeng, C., 2008. The effects of temperature and salinity on population growth and egg hatching success of th e tropical calanoid copepod, Acartia sinjiensis Aquaculture 275, 116. 103

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Muller, R.G., Tisdel, K., Murphy, M.D., 2002. The 2002 update of the stock assessment of Florida pompano (Trachinotus carolinus). Florida Fish and Wildlife Conservation Commission. Florida Marine Research Institute. 45pp. Muller-Feuga, A., Robert, R., Cahu, C., Robin, J ., Divanach, P., 2003. Uses of microalgae in aquaculture. In: Stottrup, J.G., McEvoy, L.A. (Eds.), Live Feeds in Marine Aquaculture. Blackwell Scientific Publications Lt d, Oxford, United Kingdom. pp. 253. Nhu, V.C., Dierckens, K., Nguyen, T.H., Tra n, M.T., Sorgeloos, P., 2009. Can umbrella-stage Artemia franciscana substitute enriched rotifers for cobia (Rachycentron canadum ) fish larvae? Aquaculture 289, 64. Ogle, J.T., Lemus, J.T., Nicholson, L.C., Barnes D.N., Lotz, J.M., 2005. Characterization of an extensive zooplankton culture system coupled w ith intensive larval rearing of red snapper Lutjanus campechanus In: Lee, C.S., OBryen, P.J., Marcus, N.H. (Eds.), Copepods in Aquaculture. Blackwell Publishing, Ames, Iowa. pp. 225. Olivotto, I., Capriotti, F., Buttino, I., Avella, A.M., Vitiello, V., Maradonna, F., Carnevali, O., 2008. The use of harpacticoid copepods as live prey for Amphiprion clarkii larviculture: effects of larval survival and growth. Aquaculture 274, 347. Om, A.D., Umino, T., Nakagawa, H., Sasaki, T., Okada, K., Asano, M., Nakagawa, A., 2001. The effects of dietary EPA and DHA fortifi cation on lipolysis activity and physiological function in juvenile black sea bream Acanthopagrus schlegeli (Bleeker). Aquaculture Research 32, 255. Palmtag, M.R., Faulk, C.K., Holt, G.J., 2006. Highly unsaturated fatty acid composition of rotifers (Brachionus plicatilis ) and Artemia fed various enrichments. Journal of the World Aquaculture Society 37, 126. Payne, M.F., Rippingale, R.J., Longmore, R.B., 1998. Growth and survival of juvenile pipefish ( Stigmatopora argus) fed live copepods with high and low HUFA content. Aquaculture 167, 237. Payne, M.F., Rippingale, R.J., 2000a. Evaluation of diets for culture of the calanoid copepod Gladioferens imparipes Aquaculture 187, 85. Payne, M.F., Rippingale, R.J., 2000b. Rearing West Australian seahorse, Hippocampus subelongatus juveniles on copepod nauplii and enriched Artemia. Aquaculture 188, 353 361. Payne, M.F., Rippingale, R.J., 2001a. Intensive cultivation of the calanoid copepod Gladioferens imparipes. Aquaculture 201, 329. Payne, M.F., Rippingale, R.J., 2001b. Effects of sa linity, cold storage, and enrichment on the calanoid copepod Gladioferens imparipes. Aquaculture 201, 251. 104

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BIOGRAPHICAL SKETCH Eric J. Cassiano was born in Buffalo, New York in 1975 and moved, at the age of 6 months old, to North Carolina. He spent his youth exploring the wilderness surrounding his house and developed an appreciation for wildlif e. After high school, Eric continued those explorations through the United States, spendi ng time in Oregon, Colorado, California, New York, and traversing the country numerous times before settling down to continue his academic career. Eric received his Bachelor of Science in Mari ne Biology from Hawaii Pacific University in the spring of 2002. After a brief period with the Oregon Department of Fish and Wildlife working in fisheries science, he began worki ng for the Oregon State University Molluscan Broodstock Program, where he was exposed to the culture of the Pacific Oyster, Crassostrea gigas After a few years there, he began a posi tion for the University of Florida where he focused on various aspects of the hard clam (Mercenaria mercenaria ) production industry. Eric was then awarded a graduate assistantship and began his thesis studies under Dr. Cortney Ohs and Dr. B. Denise Petty for the University of Florida. Eric has been blessed with a beautiful wi fe and a vivacious young boy, who both bring him joy and serenity. Furthermore, he wholehearted ly believes in the ideals of aquaculture as a means to preserve our natural aquatic systems wh ile continuing to meet the demands of the food, bait, and ornamental markets.