ASSESSMENT OF CANDIDATE MARINE SPECIES TO DIVERSIFY ORNAMENTAL FISH PRODUCTION IN FLORIDA: EVALUATIONS OF SPAWNING, HYPOSALINE TOLERANCE AND SELECT LARVAL PRODUCTION FACTORS

By

CARTER S. CYR

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

© 2017 Carter S. Cyr

To my grandfather, Gilbert Cyr

ACKNOWLEDGMENTS

I thank my parents, Scott and Phyllis, and sister, Danielle, for immeasurable material and moral support over the years. I want to thank Grace Thurston for her patience and compassion. I thank Colin Beron, John Downey, Isaac Lee, Michael Quinn and Charles Stanley for their friendship.

I would like to thank my advisor Cortney Ohs for his patience, guidance and commitment to my development. I thank my committee members Matthew DiMaggio and Craig Watson for all of their efforts. Thank you to Isaac Lee, Wesley Freitas, Jason

Broach, John Marcellus, Andrew Palau, Audrey Beany and Bryan Danson. Also, thank you to Dynasty Marine for their help in our research. I thank Judy St. Leger, Rising Tide

Conservation, and Sea World Busch Gardens Conservation Fund for funding my position and, finally, the University of Florida for accommodating me. Go Gators.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 11

LIST OF ABBREVIATIONS ...... 13

ABSTRACT ...... 15

CHAPTER

1 INTRODUCTION ...... 17

Marine ornamentals: The Role of Aquaculture in the Aquarium Trade ...... 17 Species Diversification in Marine Ornamental Aquaculture ...... 18 Carangidae ...... 19 Salinity ...... 21 Reproduction ...... 22 Gametogenesis and Embryology ...... 22 Embryology and Mating Strategy ...... 23 Larval Culture ...... 23 Copepods ...... 24 Chaetodontidae ...... 25

2 EARLY LIFE STAGE CULTURE OF Trachinotus goodei AND Gnathanodon speciosus ...... 26

Foreword ...... 26 Methods ...... 28 Live Feeds Cultures ...... 28 Experiment I. Larval Culture of Trachinotus goodei ...... 29 Evaluation: Growth and Morphometric Dynamics of T. goodei from Embryo to Juvenile Stage ...... 33 Experiment II, Effects of Copepod (Parvocalanus crassirostris) Supplementation on Larval Growth and Survival in Gnathonodon speciosus...... 34 Experiment III, The Effect of Stocking Density on Growth and Survival of Post-Larval Trachinotus goodei ...... 36 Results ...... 38 Experiment I. Larval Culture of Trachinotus goodei ...... 38 Evaluation: Growth and Morphometric Dynamics of T. goodei from Embryo to Juvenile Stage ...... 39

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Experiment II, Effects of Copepod (Parvocalanus crassirostris) Supplementation on Larval Growth and Survival in Gnathonodon speciosus...... 43 Experiment III, The Effect of Stocking Density on Growth and Survival of Post-Larval Trachinotus goodei ...... 43 Discussion ...... 53

3 OSMOREGULATORY RESPONSE OF T. goodei TO HYPOSALINITY...... 56

Foreword ...... 56 Methods ...... 58 Experiment IV.I, Acute Natural Sea Water Salinity Tolerance at a Temperature Gradient ...... 59 Experiment IV.II, Acute Natural Sea Water Salinity Tolerance without a Temperature Gradient ...... 60 Experiment V, Acute NaCl Water Salinity Tolerance ...... 61 Experiment VI, Stepwise Acclimation to 3 g/L Sea Water ...... 62 Results ...... 63 Experiment IV.I, Acute Natural Sea Water Salinity Tolerance at a Temperature Gradient ...... 63 Experiment V, Acute NaCl Water Salinity Tolerance ...... 64 Experiment VI, Stepwise Acclimation to 3 g/L Sea Water ...... 65 Discussion ...... 73

4 SPAWNING, INCUBATION AND LARVAL CULTURE OF Paracanthurus hepatus AND Chaetodon speciosus ...... 76

Foreword ...... 76 Methods ...... 79 Broodstock Acquisition and Maintenance ...... 79 Spawning and Fecundity ...... 80 Experiment VII, Hatching Success in P. hepatus ...... 81 Experiment VIII, Effect of Salinity on Hatch Success in P. hepatus ...... 82 Experiment IX, Temporal Variation in Hatch Success of P. hepatus at Various Temperatures ...... 83 Experiment X, Hatching Success in Chaetodon sedentarius...... 84 Experiment XI, Effect of Aeration on Hatching Success in Chaetodon sedentarius ...... 84 Larval Culture of Chaetodon sedentarius ...... 85 Results ...... 87 Spawning and Fecundity ...... 87 Experiments VII and X Hatch Success in Paracanthurus hepatus and Chaetodon sedentarius ...... 89 Experiment VIII, Effect of Salinity on Hatch Success in Paracanthurus hepatus ...... 89 Experiment IX, Temporal Variation in Hatch Success of P. hepatus at Various Temperatures ...... 90

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Larval Culture of Chaetodon sedentarius ...... 91 Discussion ...... 102

5 CONCLUSION ...... 107

LIST OF REFERENCES ...... 109

BIOGRAPHICAL SKETCH ...... 126

7

LIST OF TABLES

Table page

2-1 Allocation of live feeds (organisms/mL-culture volume) to two treatments (Rotifers, Rotifers +Copepods) employed during Experiment I...... 44

2-2 Notochord length (NL), eye diameter (ED), body depth (BD) and condition index (CI) of fish fed diets of solely rotifers and rotifers supplemented with copepods from 1-7 dph of Experiment I. All parameters except CI are in µm. .... 45

2-3 Total growth and specific growth rate (%/day) (mean ± SD) of notochord length (NL), eye diameter (ED), body depth (BD) and condition index (CI) of T. goodei fed diets of solely rotifers and rotifers supplemented with copepods between 1 and 7 dph of Experiment I. All parameters except CI are in µm...... 45

2-4 Water quality parameters [mean ± standard deviation (max-min)] measured in tanks holding T. goodei fed diets of solely rotifers and rotifers supplemented with copepods measured throughout Experiment I...... 46

2-5 Feeding regime used for larval culture of Trachinotus goodei. Live feeds values correspond to the density (organisms/mL) added to culture tanks. Dry diet values indicate frequency of feedings per day...... 46

2-6 Mean water quality parameters measured throughout larval culture of T. goodei (mean ± SD)...... 47

2-7 Allocation of Parvocalanus crassirostris copepods (C), Brachionus plicatilis rotifers (R) in four treatments throughout Experiment II (2-15 dph)...... 47

2-8 Fifteen dph survival (Surv.), notochord length (NL), body depth (BD) and gape height (GH) of G. speciosus receiving four different dietary regimes of including rotifers (R) and copepods (C). Values are expressed as mean ± SE...... 47

2-9 Water quality parameters measured in tanks holding G. speciosus fed one of four diets throughout Experiment II. Values are expressed as mean ± SE (minimum – maximum)...... 48

2-10 Values for wet weight, standard length (SL) body depth (BD) and feed conversion ratio (FCR) (mean ± SE) of T. goodei held at four stocking densities from 52-71 dph (Experiment III). Values are expressed as mean ± SE (minimum – maximum)...... 48

2-11 Total growth of standard length, body depth and wet weight (mean ± SE) of T. goodei held at four stocking densities from 52-71 dph (Experiment III)...... 49

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2-12 Water quality parameters expressed as mean ± SD (min – max) as measured throughout the course of Experiment III...... 49

3-1 Kaplan-Meier survival analysis for three acute transfer trials. Survival is expressed as the mean ± SE...... 66

3-2 Log rank comparisons of T. goodei survival following acute transfer to one of four sea water concentrations in Experiment IV.I...... 66

3-3 Log rank comparisons of T. goodei survival following acute transfer to one of six sea water concentrations in Experiment IV.II...... 67

3-4 Log rank comparisons of T. goodei survival following acute transfer to one of six NaCl concentrations Experiment V...... 67

3-5 Each pair Wilcoxon t-test comparisons of wet weight changes of T. goodei transferred to one of four natural sea water concentrations in Experiment IV.I. . 67

3-6 Tukey’s Multiple Comparisons of wet weight changes of T. goodei transferred to one of six natural sea water concentrations in Experiment IV.II...... 67

3-7 ANOVA (P-value) comparisons of wet weight changes of T. goodei transferred to one of six natural NaCl concentrations in Experiment V...... 68

3-8 Water quality measured during three acute salinity transfer trials, Experiments IV.I – V. Values are expressed as mean ± SD (minimum- maximum) ...... 68

3-9 Salinity levels and mean changes in salinity per hour during stepwise acclimation from 34.64 ± 0.69 g/L (33.6-35.56) to 2.89 ± 0.089 (2.76-3.16), Experiment VI. Values are reported as mean ± SD (min-max)...... 69

3-10 Water quality parameters measured in 48 hour observation tanks following stepwise acclimation to 3 g/L trials, Experiment VI. Values are reported as mean ± SD (min-max)...... 70

3-11 NSW concentrations (g/L) corresponding to treatments used in Experiment IV.I. Values are reported as mean ± SD (min-max)...... 70

3-12 NSW and NaCl concentrations (g/L) corresponding to treatments used in Experiments IV.II and V. Values are reported as mean ± SD (min-max)...... 70

3-13 Total hardness and total alkalinity of salinities corresponding to treatments in Experiment IV.I...... 70

3-14 Total Hardness and total alkalinity of salinities corresponding to treatments in Experiment IV.II...... 71

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3-15 Total Hardness and total alkalinity of salinities corresponding to treatments in Experiment V...... 71

4-1 Egg production models for P. hepatus and a pair of C. sedentarius. Statistics are derived only from spawns for which a full analysis or floating and sinking eggs was performed (see methods)...... 94

4-2 Water quality parameters measured from July 2015 – February 2016 in tanks holding a P. hepatus and C. sedentarius broodstock...... 94

4-3 Water quality parameters from bath used to incubate P. hepatus eggs in Experiments VII and X between 09/05/2015 and 09/30/2015 ...... 94

4-4 Water quality parameters measured in cups used to incubate P. hepatus eggs at one of four salinities of Experiment VIII...... 95

4-5 Water quality parameters measured in tanks used to incubate C. sedentarius eggs with and without aeration in Experiment XI and P-values relating the two treatments...... 95

4-6 Water quality parameters as measured throughout the larval culture of C. sedentarius trial...... 95

4-7 Embryo characteristics of C. sedentarius (n=8)...... 96

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LIST OF FIGURES

Figure page

2-1 Flow through larval culture system used in Experiment I...... 30

2-2 Survival (%) at 7 dph of T. goodei fed diets of solely rotifers and rotifers supplemented with copepods measured throughout Experiment I. Error bars represent standard error...... 49

2-3 Linear regression displaying reduction of T. goodei oil globule volume using larvae from the copepod supplemented treatment of Experiment I...... 50

2-4 Development of T. goodei……………………...... 50

2-5 Survival (%) at 15 dph of G. speciosus fed four different diets of rotifers (R) and copepods (C) during Experiment II...... 52

2-6 Mean wet weight of T. goodei held at four different stocking densities during Experiment III...... 52

3-1 Mean change in T. goodei body wet weight from the time of exposure to four concentrations of natural sea water until mortality or termination of Experiment IV.I...... 71

3-2 Mean change in T. goodei body wet weight from the time of exposure to six concentrations of natural sea water until mortality or termination of Experiment IV.II...... 72

3-3 Mean change in T. goodei body wet weight from the time of exposure to six concentrations of NaCl until mortality or termination of Experiment V...... 72

4-1 Schematic showing the broodstock husbandry tanks and egg collection units used to house and harvest clutches from Paracanthurus hepatus (Populations 1 and 2) and the Chaetodon sedentarius pair...... 80

4-2 Semi-continuous Parvocalanus crassirostris production system...... 87

4-3 Estimates of clutch size (number of floating eggs) as produced by P. hepatus from July 2015 – February 2016. Orange lines represent full moon days...... 96

4-4 Estimates of clutch size (number of floating eggs) as produced by a pair of C. sedentarius from July 2015 – February 2016. Orange lines represent full moon days...... 97

4-5 Hatching (% hatched) of P. hepatus at (26-28ºC) from 19-27 hour post- fertilization...... 97

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4-6 Hatching (%) of P. hepatus from 18 – 29 hours post-fertilization at four different temperatures (ºC)...... 98

4-7 Mean oil reserve volumes (mm3) measured during larval culture of C. sedentarius...... 98

4-8 Mean yolk volumes (mm3) measured during larval culture of C. sedentarius. .... 99

4-9 Mean length measured during larval culture of C. sedentarius...... 99

4-10 Mean mouth gape heights (µm) calculated during larval culture of C. sedentarius...... 100

4-11 Mean body depths (µm) measured during larval culture of C. sedentarius...... 100

4-12 Hydrated C. sedentarius eggs approximately 10-14 hours post-fertilization. .... 101

4-13 Larval development of Chaetodon sedentarius...... 101

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LIST OF ABBREVIATIONS

BD body depth cm centimeter

D dark d1 horizontal diameter d2 vertical diameter dph days post hatch

ED egg diameter

EyD eye diameter

EV egg volume

F female g gram h hour

Hy yolk height l light

L liter

Ly yolk length

M male

Max maximum min minimum mL milliliter mm millimeter m meter

NO2-N nitrite-nitrogen

NO3 nitrate

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OD oil globule diameter

OD:ED oil globule diameter to egg diameter

PS perivitelline space r radius

S surface area

SD standard deviation

SE standard error

SGR specific growth rate

NL notochord length sp. species

UV ultra violet

V volume

YD yolk diameter

YV yolk volume

YV:EV yolk volume to egg volume ratio

μm micrometer

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

ASSESSMENT OF CANDIDATE MARINE SPECIES TO DIVERSIFY ORNAMENTAL FISH PRODUCTION IN FLORIDA: EVALUATIONS OF SPAWNING, HYPOSALINE TOLERANCE AND SELECT LARVAL PRODUCTION FACTORS

By

Carter S. Cyr

May 2017

Chair: Cortney Ohs Major: Fisheries and Aquatic Sciences

Research was conducted to define methods to successfully culture several fin fish species which have not been cultured in captivity before. Four species were investigated in several experiments to improve their aquaculture potential. The purpose of each experiment was to evaluate methods conducive to commercial production, thereby demonstrating the aquaculture potential of the species. Results indicate feeding regimes which incorporated Parvocalanus crassirostris copepods enhance survival in the golden trevally (Gnathanodon speciosus) and palometa (Trachinotus goodei). T. goodei tolerated salinities as low as 3 g/L. Spawning of two highly valued species,

Paracanthurus hepatus and Chaetodon sedentarius occurred volitionally. C. sedentarius began spawning within three months of captivity. Year round spawning was achieved with constant temperature and photoperiod. Spawning occurred 1-3 times per week, occasionally interrupted by 1-4 weeks with no spawning. Spawning was continuous (1-3 spawns/week) in C. sedentarius from August 2015 - June 2016. Spawns were observed outside of previously described spawning seasons for other chaetodontids native to the western Atlantic Ocean. C. sedentarius larvae were capable of feeding on 3 dph (days

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post hatch) and survived to 23 dph during initial culture efforts. P. hepatus began spawning within a year of captivity and mated in harems. Severe male-male aggression was observed as part of the establishment of the social hierarchy in the harem.

Following the first observed spawn in a population, spawning events occurred 2-7 times per week with occasional gaps of no spawns over 7-28 days. P. hepatus spawning was continuous from July 2015 - June 2016.

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CHAPTER 1 INTRODUCTION

In the last fifty years, the majority of seafood supply has transitioned from wild harvest to aquaculture (Naylor 2009). Between 1984 and 2011, the percentage of total food fish from aquaculture grew from under 10% to over 40%. This figure has now surpassed 50%. The growth of capture yields has leveled over the past forty years

(FAO 2013). Continued growth of the world’s population keeps the rise in demand for fish steady. This is particularly prevalent considering the rapid development of parts of the world that rely heavily on fish as a high-quality protein source. As of 2000, fish constituted 26% of animal protein consumption in Africa, and 22% in Asia, compared to under 10% in the U.S. (FAO 2000).

Marine ornamental fish aquaculture has seen comparatively modest development over the past two decades, usually due to unknown methods for culturing one or more life stages. Establishment of culture methods for some marine ornamental species has resulted in commercial production. However, the inability to provide such methods for many highly valued species leaves the only supply to be wild collection.

Long term, the lack of diversity in the commercial production of marine ornamentals may leave the industry vulnerable. Increasing environmental awareness presents a moral obligation and economic opportunity to potentially offset wild harvest with diversified aquaculture production.

Marine Ornamentals: The Role of Aquaculture in the Aquarium Trade

Florida is responsible for the majority of ornamental fish exports in the United

States (Chapman 1997). Ornamental aquaculture constitutes nearly 50% of Florida’s aquaculture production and 7% of U.S. aquaculture production by value. Between 1985

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and 1997, 80% of U.S. ornamental aquaculture yield originated from Florida farms. This was during a timespan in which ornamental fish production more than doubled nationally (FASS 1999; Tlusty 2002; Watson and Hill 2006). Aquaculture produced marine ornamental fish make up less than 1% of Florida’s aquaculture output, and less than 2% of the marine ornamental trade worldwide (Hoff 1996; Wabinitz 2003; Watson and Hill 2006). As of 2002, 100 of 800 commonly marketed marine ornamental fish species had been aquaculture produced, but only 21 of these 100 had demonstrated commercial scale production and marketing (Tlusty 2002). The past 10 years have been a time of substantial development in marine ornamental aquaculture, as economic potential and ecological necessity was realized. Diversification of live feeds and development of breeding and culture techniques have put many species in production which were previously available only through wild collection (Olivotto et al. 2006;

Watson and Hill 2006; Olivotto et al. 2008a; Olivotto et al. 2008b; Olivotto et al. 2010;

Ohs et al. 2010; Olivotto et al. 2011; Barden et al. 2014).

Species Diversification in Marine Ornamental Aquaculture

The ability to diversify marine ornamental aquaculture production depends on the development of commercially viable culture methods for new species. Identification of appropriate live feed organisms, development of live feed culture methods, and subsequent establishment of an appropriate feeding regime. All are vital to culturing any fish species. Inability to produce commercial quantities of live feeds, particularly copepods, is a major obstacle for producers trying to culture new marine ornamental species. Copepods are the preferred live feed organism for culturing larval marine fish, due to their small size, movement patterns and natural nutritional composition (Delbare et al. 1996; Laidley et al. 2009). Advancements in copepod culture techniques have 18

facilitated the successful culture of fish species previously unachieved. More recent reports have identified other zooplankton species, such as ciliates, as potentially effective first feeding supplements (Olivotto 2005; Laidley et al. 2008). Other studies have documented new copepod production techniques that can yield commercial volumes (Kline and Laidley 2015). These techniques were used to produce the copepods which were fed to the first cultured yellow and blue tangs (Zebrasoma flavescens and Paracanthurus hepatus). Mass scale copepod production was an integral component of this breakthrough. Commercial production of blue and yellow tangs will likely require the integration of mass scale copepod production into commercial hatcheries.

In the past few years, many highly valued marine ornamental species have been cultured. For these species to be commercialized, copepod production methods need to be used. To date, lack of effective culture methods and the costs and labor associated with producing mass quantities of copepods has hindered their widespread usage.

When establishing new species for marine ornamental production, the feasibility with which the industry can incorporate the culture of the species is critical. The vast majority of the ornamental aquaculture infrastructure in Florida is geared towards freshwater species. Identifying species that, exhibit rapid growth, do not rely on copepods, and tolerate a broad range of abiotic conditions should be prioritized when developing methods for culturing marine ornamental fish.

Carangidae

Carangids (Carangidae) include jacks, pompanos, jack mackerels, runners, and scads. They are characteristically aggressive feeders and many species are commercially important food and game fish. Feasibility of larval culture and aquaculture 19

compatibility have been demonstrated in many Carangids (Watarai 1973; Riley et al.

2009; Stuart et al. 2012; Ma et al. 2014: Broach et al. 2015). The use of copepods in larval feeding has not been required in culturing most carangids, however, the benefits of their use have also been reported (Cassiano et al. 2011; Roo et al. 2014).

In addition to closing the lifecycles of traditionally sought after ornamental species, the introduction of new species would expand and stabilize the industry.

Recent commercialization of golden trevally (Gnathanodon speciosus) production is a good example. Juvenile G. speciosus (2-3 cm) are sold at USD $40-50 in retail pet stores (Broach et al. 2015). Also a targeted game and food fish in the Indo-Pacific, G. speciosus can reach 1 m in length, but unique coloration makes the juveniles an attractive candidate for public and private aquaria. Like other carangids, G. speciosus was proven to be relatively easy to culture (Broach et al. 2015). The multi-market potential (food fish, game fish, and ornamental fish) of G. speciosus adds to the viability of this species as an aquaculture candidate and is a desirable characteristic in commercializing production of new species.

The Florida pompano (Trachinotus carolinus) has likely been the most researched Carangid over the past three decades. T. carolinus is valued at USD $1.8-

3.6/kg (ex-vessel). Retail prices generally range from $18-28/kg (McMaster 2014).

Profitable production of T. carolinus in the Dominican Republic and pilot scale domestic operations are a result of thorough documentation of culture techniques for this species

(Main et al. 2007).

Palometa (Trachinotus goodei) are a close relative of T. carolinus. They can be found throughout coastal waters of Florida, the Caribbean, and occasionally as far north

20

as Massachusetts (Randall 1983; Fishbase.org). T. goodei has shown potential for aquaculture (Thouard et al. 1990; Cole et al. 1997). Dramatically extended dorsal and anal fins (Figure 2-4) and relatively small size make it a potentially attractive species for public and private aquaria. T. goodei have been produced commercially, but there is no documentation of larval development or general aquaculture protocols. Refinement of culture techniques and determination of T. goodei’s resilience to various conditions could increase commercial interest in this species. Determining the potential benefits of feeding copepods to larval G. speciocus and T. goodei at first feeding is also of interest.

The ability to culture larvae without the use of copepods would make these species less expensive to culture and more attractive to producers not set up for copepod production.

Salinity

A species’ tolerance to hyposalinity adds to its aquaculture candidacy by reducing costs associated with sea water aquisition. Certain marine species are very tolerant of hyposaline conditions, sometimes growing faster or even exhibiting increased survival at low salinities (Craig et al. 1995; Woo and Kelly 1999; Boeuf and Payan 2001;

Imsland et al. 2003; Prashanth et al. 2012). The effective use of hyposalinity as therapeutic or preventative treatment of ectoparasites is well-documented. Minor reductions in salinity are commonly used to reduce energy used for osmoregulation and to combat parasites (Dunphy et al. 2003; Fajer-Ávila 2008). Furthermore, hyposaline tolerance may facilitate pond production.

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Reproduction

Gametogenesis and Embryology

Survival through larval metamorphosis is the greatest bottleneck to culturing many marine species, and this limits commercial production. It is important to have a general understanding of how gametes are produced by meiosis (gametogenesis), and its stages in order to establish reproductive capability and readiness of brood animals

(Coward et al. 2002).

The major stages of gametogenesis are primary growth, secondary growth, and maturation (Senthilkumaran 2013). There are various methods for determining stage of gonadal development in fishes (Rhody et al. 2013). Evaluation of oocytes provides information on the reproductive potential of brood. Cannulation and wet mount of oocytes or sperm is used to determine sex and/or reproductive stage. This technique is relatively cost effective and often minimally invasive. However, in the context of marine ornamental fish aquaculture, the small size, cost, and fragile nature of broodstock are reasons for concern in using cannulation. However, when applicable it is a valuable method to establish brood sex and oocyte stage. Concerns surrounding cannulation of marine ornamentals also apply to hormonal injection. The ability to condition marine ornamental broodstock to spawn with without these procedures is at the least advantageous, but may be a necessity. Furthermore, maintenance of frequent, volitional, year round spawning is important for commercializing the production of pelagic spawning species that have low rates of survival to metamorphosis. Such spawning activity would also be important to research efforts in culturing these species.

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Embryology and Mating Strategy

Marine species can be segregated into the two primary egg dispersal strategies, demersal and pelagic spawners. Demersal eggs, such as those of clownfish and many

Pomacentrids are often adhesive (Olivotto 2016). Amongst demersal spawners, clutches are cared for in various ways and to various extents by parents. Upon hatching, larvae of demersal spawning species are generally more developed than those of pelagic species. Parental care is not observed in pelagic spawning species and these larvae usually hatch in an altricial state without eyes or developed mouths.

Often demersal larvae are easier to culture than their pelagic counterparts, although there are exceptions. Culture of many pelagic spawning carangids has been feasible.

Conversely, some Pomacentrids (Pomacentridae) such as the green and blue chromis

(Chomis viridis, Chromis cyaneus) hatch from demersal eggs without mouths or pigmented eyes.

Embryological development and incubation techniques vary from species to species (Ellis et al. 1997; Ignatius et al. 2001; Komar et al. 2004; Jantrakajorn and

Wongtavatchai 2015; Puvanendran et al. 2015; Siddique et al. 2015).

Establishment of successful incubation techniques is critical to successful hatching. The effects of abiotic conditions and the potential for fouling needs to be considered when establishing incubation protocols.

Larval Culture

The highest mortality is common during larval culture. Metamorphosis, or the morphological transition of fish from larvae to juvenile, involves key developments such as the formation of a functional mouth, digestive system, eyes, fin appendages, as well as ossification. These events all require a substantial amount of energy and a means of

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supplying this energy in a very small package. Much research has been conducted to identify and culture nutritionally suitable zooplankton and develop feeding regimes for marine ornamental species (Delbare et al. 1996; Stottrup and Norsker 1997; Marcus and Murray 2001; Olivotto et al. 2003; Olivotto et al. 2006a; Olivotto et al. 2008a;

Olivotto et al. 2008b; Olivotto et al. 2008c; Olivotto 2016; Gopakumar and Santhosi

2009; Laidley et al. 2009).

Copepods

Feeding larvae copepods has enabled the metamorphosis of marine ornamental species that were previously not succesful using only enriched rotifers. Historically, difficulties culturing copepods and undefined culture methods have prevented widespread commercial usage of copepods (Payne and Rippingdale 2001; Støttrup and

McEvoy 2003; Peck and Holste 2006; Souza-Santos et al. 2006; Stottrup 2006; Jepsen et al. 2007; Rhyne et al. 2009; Ohs et al. 2010). Labor and material costs of culturing copepods are assumed to higher than those associated with rotifer culture. However, the superiority of copepods as first feed items are generally accepted, and the benefits are many (Kraul et al. 1992; Stottrup 2000, 2006; Schipp 2006; Camus and Zeng 2009;

Cassiano et al. 2011; Experiment 1 and 2 herein). Benefits of copepods have been attributed to characteristics including nutritional composition, small size, larval preference, forage inducing locomotion, wide, distribution and a natural prey (Shields et al. 1999; Hamre et al. 2002; McKinnon et al. 2003; Buskey 2005; Marcus 2005; Schipp

2006; Zavala-Leal et al. 2013; Degidio 2014). Over the past 10 years, documentation and refinement of copepod culture techniques has facilitated commercial production of copepods (Drillet et al. 2011; Kline and Laidley 2015).

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P. crassirostris have beneficial characteristics including small size, ability to proliferate to high densities, coastal abundance, and global distribution (Turner 1982;

Hopcroft et al.1998; Gómez-Erache et al. 2000; Rong et al. 2002; McKinnon et al. 2003;

Hsieh and Chiu 2004; Alajmi and Zeng 2015).

Recent development of intensive, high density culture methods for Parvocalanus crassirostris and other copepod species have demonstrated increased productivity and viability of copepods (Drillet et al. 2011; Abate et al. 2015; Alajmi et al. 2015; Arndt et al.

2015; Kline and Laidley 2015; Rayner et al. 2016). Alajmi et al. (2015) showed domestication (> 2 years) of P. crassirostris can improve reproductive performance, resilience in culture, and may increase fatty acid content compared to wild strains.

Chaetodontidae

Chaetodontids (Chaetodontidae), which include butterflyfish and banner fish are well represented in the aquarium trade. The attractive coloration and peaceful behavior of butterflyfish makes them desired by hobbyists. Degidio (2014) provided a thorough evaluation of the relationship between sex ratios and fecundity as well as larval culture methods for the milletseed butterflyfish (Chaetodon milliaris). In that study, C. milliaris survived until 43 dph. Prior investigations documented spawning behavior, estimates of fecundity, and early larval culture methods for various butterflyfish (Suzuki et al. 1980;

Colin 1988; Lobel 1989; Tricas and Hiramoto 1989; Londraville 1990; Hioki 1997;

Yabuta 1997; Yabuta and Kawashima 1997; Tanaka et al. 2001). In 2015, C. klieni and

H. diphreutes became the first successfully cultured Chaetodontids (Frank Baensch,

Reef Culture Technologies, LLC). The popularity of Chaetodontids warrants further research to commercialize their production. High fecundity, ease of conditioning, and volitional spawning would promote the commercial potential of butterflyfishes. 25

CHAPTER 2 EARLY LIFE STAGE CULTURE OF Trachinotus goodei AND Gnathanodon speciosus

Foreword

Establishment of spawning induction and larval culture protocols for

Gnathanodon speciosus (golden trevally) stimulated domestic production and distribution of this species into ornamental markets (Broach et al. 2015). Like golden trevally, Trachinotus goodei (palometa) have potential as a recreational game, and ornamental fish.

Early larval mortality is a major bottleneck in commercializing a marine aquaculture candidate (Sifa and Mathias 1987). Heavy losses during this stage have been attributed to sensitivity of larvae and failure to provide adequate nutrition to fuel fast development (Shields et al. 1999). The use of rotifers as a primary and often sole first feed was common practice. Resilience to adverse water quality conditions, slow movement, ability to control their nutritional composition, and their proliferation to high densities in culture contribute to rotifer’s popularity (Watanabe et al. 1983; Theilacker and Kimball 1984; Schlüter and Groeneweg 1985; Abu-Rezq et al. 1997; Fu et al. 1997;

Yoshimura et al. 1997; Suantika et al. 2000). Many studies have demonstrated the nutritional superiority of copepods compared to rotifers. Some species previously cultured without copepods have exhibited various improvements when provided copepods as a first feed item. Such effects included improved survival, growth, and stress resistance in larvae (Kraul 1983; 2006; Schnipp 2006; Cassiano et al. 2011; de

Melo-Costa et al. 2015). Noted benefits included HUFA content and bioavailability, digestibility, size, and movement theorized to elicit a feeding response (Watanabe et al.

1983; Pedersen 1984; Stottrup and Jensen 1990; McKinnon et al. 2003; Kraul 2006;

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Schipp 2006). First feeding larvae of various species have demonstrated preferential feeding on copepod nauplii over rotifers (Zavala-Leal et al. 2013). Determining the necessity and/or specific benefits of copepods for first feeding larval stages of a given species is needed.

Stocking density is important for efficiency of aquaculture. Higher stocking densities help maximize efficiency of labor and resource allocation. High stocking density has been shown to cause reductions in yield, growth, survival, and/or increases in feed conversion for many species of finfish including halibut [Hipoglossus hippoglossus (Bjornsson 1994)], brook trout [Salvelinus fontinalis (Vijayan and

Leatherland 1988)], Nile tilapia [Oreochromis niloticus (Yi et al. 1996)] and African catfish [Clarias gariepinus (Hengsawat et al. 1997)]. Irwin et al. (1999) indicated that lower stocking densities may produce more homogenous growth. Maintenance of homogenous growth rates are of concern for Carangid species as they are prone to cannibalism which can lead to losses (Broach 2015 pers. comm.).

Information on larval and early juvenile development and best culture methods are lacking and need further research. Thus, three experiments were conducted to address three sub-objectives: (I) prove the feasibility of larval culture methods for T. goodei with and without copepod (P. crassirostris) supplementation; (II) assess various feeding regimes, and evaluate the utility of copepods as a dietary component for first feeding G. speciosus; and (III) determine the effects of stocking density on the growth and survival of post larval T. goodei.

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Methods

Live Feeds Cultures

Parvocalanus crassirostris culture

Live feeds were cultured and maintained using continuous methods. P. crassirostris (copepods) were fed the Tahitian strain of Isochrysis galbana (T-ISO).

Copepod populations were cultured in 25 g/L salinity, sterilized, Atlantic Ocean water.

Culture vessels were 150 L plastic barrels. Daily, nauplii were harvested from copepod cultures using a bucket air lift (Cassiano et al. 2015).

Microalgae culture

T-ISO and Chaetoceros sp. stocks were cultured in 3.8 L and 18.9 L containers with sterilized Atlantic Ocean water with F/2 media (Fritz Aquatics, Mesquite, TX) added according to manufacturer’s recommendations. Additionally, Chaetoceros sp. water had silicates added prior to inoculation. Smaller containers of T-ISO were used as inoculants for 18.9 L containers and cultured for approximately 7 days before harvesting.

Inoculated containers were exposed to constant light supplied by T8 fixtures. Containers were continuously aerated and enriched with CO2 (10 L/min).

Brachionus plicatilis culture

Brachionus plicatilis (rotifers) were maintained in semi-continuous culture on a concentrated Nannochlorpsis sp. algae (Reed Mariculture, Campbell, CA). Prior to experimental feeding, rotifers were harvested and screened (30-150 µm) manually and enriched with Ori-green® (Nutreco, Amersfoort, Netherlands) enrichment according to manufacturer’s instructions.

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Experiment I. Larval Culture of Trachinotus goodei

Two dietary treatments were compared to assess feasibility of intensive propagation of T. goodei, and identify potential benefits of copepods as a supplement in their first feeding. Treatment 1 (T1) consisted solely of rotifers (5-7.5 individuals/mL).

Treatment 2 (T2) consisted of a mix of copepods and rotifers (0.8-1:4-6.6

[copepods/mL:rotifers/mL]). The experiment lasted from 1-7 dph. Feed was administered from 3-7 dph. The total number of live feed organisms added to each tank was consistent across treatments on every day of the experiment.

Experimental system and design

Eight identical, 93 L cylindrical, white-bottomed fiberglass tanks were filled with sterilized Atlantic Ocean water. Prior to experimental usage, water was bleached, dechlorinated with sodium thiosulfate, and then continuously circulated through an 80W

UV sterilizer (Emperor Aquatics, Pottstown, PA). Moderate aeration was maintained using a single ceramic air stone placed centrally within each culture tank. Each tank was equipped with a 250 µm Nitex covered standpipe inserted into a central outflow.

This enabled the containment of eggs and larvae with continuous flow through of water.

Prior to first feeding (3 dph), water was exchanged through culture tanks at a rate of

2.0-3.1 mL/s. Flow rate was increased to 3.7-4.6 mL/s at first feeding. This flow rate was used for the remainder of the experiment. Water exchange was stopped daily on days

3-7 between 06:00 and 17:30 for 6.5-11.5 hours to ensure retention of live feed organisms and microalgae. Photoperiod was maintained at 12L:12D. Water quality parameters (temperature, salinity, pH and dissolved oxygen) were monitored in two tanks per treatment daily throughout the experiment with a YSI 556 MPS (YSI Inc.,

- Yellow Springs, OH). Total ammonia nitrogen (TAN) and nitrite-nitrogen (NO2 N) levels 29

were monitored in each tank throughout the course of the experiment via a HACH

DR/4000V spectrophotometer (HACH Company, Loveland, Colorado). Water samples collected for testing nitrogenous waste accumulation were collected from two tanks per treatment on 1, 2 and 4-7 dph.

Figure 2-1. Flow through larval culture system used in Experiment I. Actual system consisted of 14 105 L tanks. Tanks were operated at a volume of 93 L during this experiment.

Embryo acquisition, stocking and experimental Design

Fertilized eggs were collected by a commercial producer (Proaquatix; Vero

Beach, Florida) and transported to the University of Florida Indian River Research and

Education Center (IRREC) Aquaculture Facility on October 12, 2015. Eggs were staged on a 1 mm graded Sedgewick Rafter cell and photographed using a dissecting microscope (Amscope, Irvine, CA). Egg and oil diameters were measured using ImageJ software ([ImageJ, USA] Faulk et al. 2007). Eggs were volumetrically enumerated and apportioned to culture tanks by sieving then funneling them into a graduated cylinder.

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The positively buoyant eggs floated within the graduated cylinder. An adjustable volumetric pipette (Eppendorf Inc. Hamburg, Germany) was then used to remove subsamples of the floating eggs to enumerate and calculate the total number of eggs.

Eggs were then distributed into culture tanks at a density of 21 eggs/L of culture water using a pipette (Eppendorf; Hamburg, Germany).

Feeding regimes

Live feeds were first fed on 3 dph. Rotifers were fed once daily to T1 at densities ranging from 5-7.5 rotifers/mL. Rotifers and copepods were added to T2 once daily at densities ranging from 4-6.5 rotifers/mL and 0.8-1 nauplii/mL. Daily live feed densities varied slightly throughout the experiment depending on the productivity of copepod cultures (TI= 5-7.5 /mL [rotifers]; T2= 0.8-1:4-6.6 [copepods:rotifers]) (Table 2-1). Total concentration of live feed organisms (rotifers + copepods) was maintained constant between treatments so that the same number of food organisms were added to each tank daily. Live T-ISO algae was added to each tank once daily to achieve a concentration of 62,500 cells/mL. Algae cells were counted using a hemocytometer.

Larval sampling and microphotograph documentation

A sub-sample of two larvae were randomly removed and photographed from each culture tank on 1-7 dph. Larvae were photographed (Canon, PowerShot S5IS) on a Sedgewick rafter slide with a 1 mm grid (Sigma-Aldrich, St. Louis, Missouri). Larvae were euthanized upon removal from respective culture tanks via overdose of MS-222

(500 mg/L) (Western Chemical, Ferndale, Washington). Upon termination of the experiment (7 dph) all larvae were manually enumerated from each culture tank. At this

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time, 10-100% of surviving larvae were subsampled from each culture tank, euthanized and photographed using the same methods as the daily subsampling.

Size and growth parameter measurement

Notochord length (NL), body depth (BD) and eye diameter (EyD) measurements were made upon completion of Experiment I using ImageJ software. A mean condition index (CI) was calculated for sampled larvae using the equation CI=BD/to quanitfy general condition (Koslow et al. 1985; Temple et al. 2004). Specific growth rates (SGR) from 1-7 dph were calculated for all morphometric parameters (NL, EyD, BD and CI) via

-1 the equation SGR (% day ) = ([ln(Mt) – ln(M0)]/t) * 100. In this equation Mt was the respective parameter measurement at 7 dph. M0 was the respective parameter measurement at 1 dph, and t was the length of time (6 days) (Temple et al. 2004).

Statistical analysis

Survival to 7 dph, four morphometric parameters (NL, BD, EyD, CI) and specific growth rates for each morphometric parameter were compared between the two treatments. Survival data were arcsine square root transformed prior to analysis.

Comparisons of all growth parameters between treatments were made for each day of subsampling and for total growth of the parameters from 2-7 dph. All data were checked for normality (Shapiro-Wilkes test) and homogeneity of variance. If these assumptions were satisfied then a Student’s T-test was used for. Data for the following comparisons violated the normality assumption and transformations failed to correct this violation: 6 dph NL, 5 and 6 dph CI, as well as 4 and 7 dph EyD and EyD SGR. These relationships were compared statistically using a Wilcoxon’s test. Six dph BD data violated the assumption of normality. A log transformation was applied to these data resulting in a

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normal distribution. A Student’s T-test was then used on the log transformed data. A

Welch’s T-test was used in the following cases where unequal variances could not be fixed by transformation of data: CI SGR and 1 dph BD. Water quality parameters measured on each day of the experiment were compared using a Student’s T-test.

Differences were considered significantly different at P≥0.05. Statistical tests were performed using JMP 13 (Cary, NC).

Evaluation: Growth and Morphometric Dynamics of T. goodei from Embryo to Juvenile Stage

Embryo morphology of T. goodei

Egg diameter (ED), oil globule diameter (OD), and yolk diameter (YD) were measured on eight T. goodei eggs (ImageJ software). These measurements were used to calculate egg volume (EV), oil droplet volume (OV), yolk volume (YV), perivitelline space (PS), egg surface area (ES), and oil droplet surface area (OS). Ratios of OV:EV, and YV:EV, ES:EV, and OS:OV were also calculated. Calculations were made with the

3 2 following equations: r = (d1 + d2)/4, V = 4/3 πr , YV = π/6 Ly Hy , PS = (ED-YD)/2 and S

= 4πr2 (Bonislawska et al. 2001; Imanpoor and Bagheri 2010; Bagarinao 1986; Markle and Frost 1985). In these equations r was radius, d1 was horizontal diameter, d2 was vertical diameter, V was volume, Ly was yolk length, Hy was yolk height, PS was perivitelline space, ED was egg diameter, YD was yolk diameter and S was surface area.

Morphology of T. goodei

Larvae from T2 of Experiment I were utilized. Residual yolk and oil globules were measured using the equations previously listed. From 1-13 dph, lengths were measured from the most posterior point of the head to the anterior terminal of the notochord (NL).

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Upon flexion and formation of a hypural plate, lengths were measured from the most anterior point of the head to the posterior edge of the hypural plate. Upon formation of the caudal peduncle, lengths were measured from the most anterior point of the head to the most posterior terminus of the caudal peduncle (standard length). The terminus of the caudal peduncle was defined as the most posterior point of flesh adjacent to the caudal fin ray. Gape heights were measured using the equation GH2 =UJ2 + LJ2 [UJ = upper jaw length, LJ = lower jaw length (Wittenrich et al. 2007)]. Body depth (BD) was measured as the length from the dorsal edge of the body to the ventral edge of the body as a vertical line adjacent to the posterior edge of the eye.

Culture methods of T. goodei

Surviving larvae from T2 of Experiment I were utilized. These larvae were consolidated into 93 L tanks upon completion of Experiment I.

From 7 dph to 13 dph, larvae were fed a combination of cultured copepod nauplii

(P. crassirostris and P. pelagicus), wild zooplankton (40-100 µm) enriched rotifers (B. plicatilis) and live Artemia nauplii. Weaning onto live Artemia nauplii started at 7 dph. By

13 dph fish were fed exclusively Artemia nauplii. Weaning onto Otohime dry feed

(Sakura Nisshin Feed Company, Tokyo, Japan) started at 14 dph. By 18 dph fish were fed exclusively B1 size Otohime dry diet.

Water quality parameters were monitored using equipment and methods listed for Experiment I methods.

Experiment II, Effects of Copepod (Parvocalanus crassirostris) Supplementation on Larval Growth and Survival in Gnathonodon speciosus.

To assess benefits of P. crassirostris (copepods) in the larval culture of

Gnathodon speciosus, four dietary treatments were evaluated. Live feed items were

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provided daily. Treatment 1 (T1, n=5) received solely B. plicatilis rotifers at concentrations between 3 and 20 rotifers/mL of culture water. Treatment 2 (T2, n=7) received equal amounts of copepods and rotifers daily totaling 3-20 feed items/mL.

Treatment 3 (T3, n=7) received solely copepods on the first day of feeding (2.7/mL) and solely rotifers from the second day of feeding until completion of the experiment on 15 dph. Treatment 4 (T4, n=7) received equal amounts of copepods and rotifers daily totaling 3-5 feed organisms/mL for the first 8 days of this experiment. For the remainder of the experiment T4 received solely rotifers (15-20 /mL). All treatments were fed once daily between 10:00 and 13:00.

A spawn was collected on 09/04/2014. On 09/05/2014, upon hatching larvae were stocked into 14.75 L culture tanks at a density of 25 larvae/L. Water was exchanged at a rate of 0.85 mL/s in all experimental replicates. Central drains were outfitted with a 240 µm nitex screen. A 12:12 light:dark photoperiod was maintained throughout the experiment. Dietary treatments were applied via daily allotments of live feed organisms respective to treatment (Table 2-7). Replicates were fed daily from 2-15 dph. Enumeration of residual live feed organisms was conducted in one tank per treatment from 6-12 dph in order to determine appropriate feeding densities throughout the course of the experiment. Water was greened with 250,000 cells/mL T-ISO algae each day of feeding. Water quality parameters (temperature, salinity, pH and dissolved oxygen) were monitored in each tank throughout the experiment. Total ammonia nitrogen (TAN) and nitrite-nitrogen (NO2-N) levels were measured in one tank per treatment on 5, 7 and, 11 dph.

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Fish (5-10) were subsampled from one tank in each treatment on 7 and 11 dph and photographed on a Sedgwick rafter cell using similar procedures outlined for

Experiment 2-1. Upon completion of the experiment, survival in each tank was measured by enumerating live larvae. Larvae (5-25) from each tank were again photographed on a Sedgwick rafter cell.

Size and growth parameter measurement

Notochord length (NL), body depth (BD) and gape height (GH) measurements were conducted with ImageJ software. GH was calculated using the equation GH2 =UJ2

+ LJ2 (UJ = upper jaw length, LJ = lower jaw length). Notochord length was measured from the most anterior point on the upper jaw to the most posterior point on the notochord or hypural plate. BD was measured in a line tangent to the most posterior edge of the eye from the dorsal to the ventral extremities.

Statistical analysis

Survival to 15 dph and all measured growth and water quality parameters were compared. Survival data was arcsin square root transformed and analyzed via ANOVA and Tukey’s Multiple Comparison’s test. Data transformations failed to correct violations of normality in NL, BD and GH data. These parameters were compared using a Kruskal-

Wallis test. Differences were considered significantly different at P≥0.05. Statistical tests were performed using JMP 13 (Cary, NC).

Experiment III, The Effect of Stocking Density on Growth and Survival of Post- Larval Trachinotus goodei

Experimental design and sampling

To assess the effects of stocking density on the growth and survival of recently settled T. goodei, four treatments were used: 3, 6, 12, and 24 fish/culture tank

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(Treatment 3, 6, 12, 24, n=3). These treatments corresponded to densities of 0.1, 0.2,

0.4, 0.7 g/L, respectively. Standard lengths, body depths and wet weights of fish at stocking were 29.3 ± 2.0 mm, 7.3 ± 2.0 mm and 1.0 ± 0.1 g, respectively. Fish were sedated via immersion in a 45-60 mg/L solution of MS-222 (Western Chemical,

Ferndale, WA) for preliminary measurements of wet weight, standard length, and body depth. Lengths and body depths were measured using ImageJ software. Fish were stocked into 12 identical 34 L fiberglass, cylindrical, black-walled, white bottomed culture tanks according to treatment density. Flow through rates of 2.8-8 tank volumes/24 hours were applied to all treatments to control potential accumulation of nitrogenous wastes. Water quality parameters (temperature, salinity pH, DO, TAN, NO2-

N) were monitored in each tank throughout the course of the experiment using the previously stated methods and instruments.

All culture tanks were fed Otohime C1 daily. Feed was allocated proportionally across all culture tanks relative to biomass. On 51 dph each tank received 0.75% of total tank biomass (total BM) in feed. Between 52 and 54 dph culture tanks received 5% total BM in feed. Culture tanks subsequently received 6-7% total BM in feed daily until the termination of the experiment. Culture tanks were subsampled for wet weight and length measurements on 57 and 64 dph. Upon completion of the experiment (71 dph), all fish were measured for wet weight and length.

Statistical analysis

Mean measurements of standard length, body depth, wet wet weight, and feed conversion ratios were compared among treatments. The following comparisons were made via ANOVA without the need for data transformations: mass at 57 and 64 dph, BD

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at each sampling period, and length at 64 dph. Mass at 64 dph and SL data at 57 dph initially violated assumption of normality. A log transformation was applied to these data sets, resulting in acceptable normality, and an ANOVA was subsequently performed.

The following data sets violated the assumption of homogeneity of variance and were compared via Welch’s ANOVA when transformations failed to satisfy this assumption:

FCR and length at 71 dph. Differences were considered significantly different at P≥0.05.

Statistical tests were performed using JMP 13 (Cary, NC).

Results

Experiment I. Larval Culture of Trachinotus goodei

A student’s t-test indicated survival of T1 (Rotifers) and T2 (Rotifers +

Copepods) was statistically significant (P=0.0255). T2 survival was 0.068 ± 0.036%

(mean ± SD). T1 survival was 0.011 ± 0.008% on day 7 (Figure 2-2). A Wilcoxon test indicated notochord length was significantly greater in T2 on 6 dph (P=0.0433). A student’s T-test indicated significantly greater condition index in T2 on 7 dph

[(P=0.0433) Table 2-2]. No other significant differences between T1 and T2 were detected for any of the measured growth parameters on any day of the experiment

(Table 2-2). No significant differences between T1 and T2 in total growth or specific growth rate with respect to any of the measured growth parameters were detected

(Table 2-7). All measured water quality parameters were held at consistent and acceptable levels throughout the course of the experiment. There were no significant differences between treatments for any of the monitored water quality parameters

(Table 2-4)

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T. goodei eggs were pelagic, non-adhesive, and positively buoyant, and measured 990-1029 µm (mean ± SD=1010 ± 11, n=8) in diameter. The chorion was transparent and contained a single oil globule. A homogenous yolk and perivitelline space were distinguishable in each specimen (Figure 2-4). Oil globule diameters measured 260-244 µm (mean ± SD=252 ± 5 µm). Perivitelline space measured 29.82 -

71.140 (mean ± SD=56 ± 13 µm). Yolk volumes were calculated to be 0.394 - 0.446 mm3 (mean ± SD=0.420 ± 0.022 mm3). Oil globule volumes were calculated to be 0.008

- 0.009 mm3 (mean ± SD=0.008 ± 0.001 mm3). Ratios of oil globule volume to egg volume were 0.014 - 0.017 mm3 (mean ± SD=0.015 ± 0.001 mm3). Ratios of yolk volume to egg volume were 0.710 - 0.851 mm3 (mean ± SD=0.763 ± 0.046 mm3).

Ratios of egg surface area to egg volume were 5.831 - 6.062 (mean ± SD=5.910 ±

0.080). Ratios of oil globule surface area to oil globule volume were 23.090 - 24.570

(mean ± SD=23.780 ± 0.502).

Evaluation: Growth and Morphometric Dynamics of T. goodei from Embryo to Juvenile Stage

On 1 dph (10/13/2015) mean larval notochord length (NL) was 2913 – 3061 µm

(mean ± SD=2983 ± 69 µm, n=8). Ventral ellipse shaped yolk sacs were approximately

10-20 % of the volume of those observed in embryos (mean ± SD=0.091 ± 0.036 mm3).

Oil globules were persistent and situated at the posterior terminus of the yolk sac

(Figure 2-4). Eyes were unpigmented and no evidence of gut or mouth development was noticeable.

On 2 dph notochord length increased (mean ± SD=3480 ± 123 µm.) Yolk and oil globule volumes reduced by roughly 90% and 55% respectively (mean ± SD=0.0124 ±

0.001 mm3, 0.001 ± 0.001 mm3). Extension of dorsal and ventral fin folds was evident

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across observed specimens. This development resulted in larvae with increased depth, particularly at the central axis. The gut tract was open at the anus in all observed specimens, however, eyes were still unpigmented. Pigmentation was present along the outer edges of dorsal and ventral fin folds and surrounding the notochord, gastrointestinal tract and residual yolk reserve (Figure 2-4).

At 3 dph mouths were fully formed, eyes pigmented and larvae were capable of feeding with a mouth gape height of 581 µm. Increase in notochord length from 2-3 dph was negligible (< 1 %, n=8). Observed increases in body depth were dramatic (mean ±

SD=36.39 ± 32.79 %). Yolk reserves were no longer apparent and oil globules had decreased to a mean of 23% of their original volume (mean ± SD=0.002 ± 0.001 mm3, n=8).

At 4 dph no significant notochord growth or body depth growth was observed.

Relative to 3 dph measurements, mouth gapes increased by 12.6% (mean ± SD=654 ±

67 µm). Residual oil globules were 2.9-5.7% of the original volume (mean ± SD=0.001 ±

0.000 mm3, n=4).

At 5 dph oil globules were no longer present.

At 7 dph notochord lengths were 34534-3724 µm (mean ± SD=3619.1 ± 119.6

µm, n=4). Mouth gape heights were 678-856 µm (mean ± SD=761 ± 69 µm, n=6). Body depths were 563-1010 µm (mean ± SD=791 ± 144 µm, n=21).

Development after 7 dph was rapid and by 9 dph larvae achieved notochord lengths, body depths and gape heights of 4235, 1018, 984 µm, respectively. Freshly hatched Artemia were measured (maximum posterior to anterior body length) and found to be smaller than 60% of this mouth gape (mean ± SD=577 ± 40 µm, n=4).

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At 11 dph dorsal and ventral fin folds were beginning to segment, however, a fusiform body shape persisted. Notochord length was 4905 µm, body depth was 1284

µm and gape height was 1106 µm.

At 13 dph the first sign of flexion was observed with the notochord tip migrating dorsally. The distance from the most posterior point of the head to the edge of the hypural plate measured 4470 µm. A body depth and gape height of 1600 and 1086 µm were observed. Curbed lengthening and persistent increase in depth resulted in an oblong body shape. A distinct, ovoid caudal fin had formed. Dorsal and ventral fin folds were further segmented. The ventral fin fold was completely divided forming two separate fins (anal and pelvic). The dorsal fin fold was continuous but consisted of two distinct, round lobes: a larger anterior lobe and a smaller posterior lobe. A silvery pigmentation had appeared on the body.

At 15 dph no notochord was visible. The hypural plate was clearer, and had a more angular shape. Two dorsal and ventral fins had formed larger, triangular, unpigmented anterior and shorter, pigmented, posterior fins with fully formed spines.

The posterior dorsal fin consisted of 4-5 spines and rays. The posterior ventral fin

(pelvic) consisted of 2-3 spines and as many rays. Ovoid, transparent pectoral fins were present. The caudal fin was emarginated. Length was 7250 µm, body depth was 2780

µm and gape height was 1800 µm. The silvery pigmentation was now more prominent.

At 17 dph, contrary to 15 dph, a notochord was still visible. However, dorsal migration of the notochord was more advanced than that seen on 13 dph. A length, body depth and gape height of 5380, 1730, 1210 µm (respectively) were observed. Fin

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formations were consistent with those observed on 15 dph, however, rays and spines were less clear.

At 19 dph pigmented cells covered the hypural plate forming a caudal peduncle with a rounded edge. From this time forward length was measured from the tip of the snout to the most anterior point of the caudal peduncle (standard length). Primary dorsal and pelvic fins were more pronounced. The formation of myomeres was observable.

Standard length was 9330 µm, body depth was 3000 µm and gape height was 1910

µm.

At 21 dph a standard length, body depth, and gape height of 8730, 2830 and

1570 µm (respectively) were observed.

At 23 dph a standard length, body depth, and gape height of 12210, 3730 and

2210 µm, were observed. Elongation of the body and overall increase in the size of the caudal fin had occurred. The primary dorsal and pelvic fins were further elongated.

Body pigmentation was entirely silver.

At 28 dph a standard length, body depth and gape height of 12150, 4040 and

2570 µm, respectively were observed.

At 30 dph a standard length, body depth, and gape height of 15810, 4410 and

3360 µm, respectively were observed.

At 34 dph a standard length, body depth, and gape height of 20880, 6060 and

3600 µm, respectively were observed. Characteristic of T. goodei, the most anterior fin segments were continuing to extend.

At 36 dph a standard length, body depth, and gape height of 17330, 4440 and

3010 were observed.

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At 40 dph a standard length, body depth, and gape height of 20460, 5750 and

3900 µm were observed.

At 48 dph standard lengths were measured from 28,380-32,710 µm (mean ±

SD=29270 ± 2010 µm, n=86). Body depths were 28,380-32,710 µm (mean ± SD=7320

± 2010 µm, n=67). Fish weighed 0.80-1.22 g (mean ± SD=0.997 ± 0.137 g, n=11).

Water quality parameters were maintained within acceptable parameters throughout the course of larval culture (Table 2-6).

Experiment II, Effects of Copepod (Parvocalanus crassirostris) Supplementation on Larval Growth and Survival in Gnathonodon speciosus.

Survival was higher in treatments which incorporated copepods. Mean (± SE) survivals for T1-T4 were 19 ± 5, 43 ± 7, 36 ± 4, and 40 ± 4% respectively. ANOVA indicated significant differences in survival among the four treatments (P=0.0483). A

Tukey’s HSD test indicated the only significant relationship was between T1 (no copepods) and T2. No significant difference was detected among treatments with respect to any of the measured growth parameters (Table 2-8).

Experiment III, The Effect of Stocking Density on Growth and Survival of Post- Larval Trachinotus goodei

Survival was 100% in all treatments. No significant differences were detected among treatments for any of the growth parameters measured (Table 2-11). Feeding behavior appeared more vigorous in higher density treatments. During the two weeks of the experimental period, Treatment 3 consistently required more feeding events to consume the daily allotment. Despite the fact that the food/biomass was held consistent between treatments, tanks with only three fish appeared to feed less vigorously, reaching apparent satiation faster than fish in other treatments. No significant differences among treatments were detected for any water quality parameters

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measured throughout the experiment (Table 2-12, Figure 2-6). No mortalities were observed in any of the treatments.

Table 2-1. Allocation of live feeds (organisms/mL-culture volume) to two treatments (Rotifers, Rotifers +Copepods) employed during Experiment I. Rotifers + Rotifers Copepods C 0.0 0.0 1 dph R 0.0 0.0 C 0.0 0.0 2 dph R 0.0 0.0 C 0.0 1.0 3 dph R 5.0 4.0 C 0.0 1.0 4 dph R 7.5 6.5 C 0.0 1.0 5 dph R 5.0 4.0 C 0.0 0.8 6 dph R 5.0 4.2

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Table 2-2. Notochord length (NL), eye diameter (ED), body depth (BD) and condition index (CI) of fish fed diets of solely rotifers and rotifers supplemented with copepods from 1-7 dph of Experiment I. All parameters except CI are in µm. Rotifers Rotifers + Copepods P NL 2866 ± 101 2983 ± 69 0.110 ED 207 ± 52 222 ± 20 0.654 1 dph BD 632 ± 18 570 ± 75 0.667 CI 0.2245 ± 0.011 0.1921 ± 0.021 0.094 NL 3481 ± 138 3480 ± 123 0.994 ED 327 ± 44 311 ± 16 0.526 2 dph BD 634 ± 150 592 ± 48 0.623 CI 0.1814 ± 0.0371 0.1705 ± 0.0176 0.615 NL 3410 ± 91 3487 ± 30 0.155 ED 318 ± 7 321 ± 11 0.700 3 dph BD 669 ± 96 797 ± 153 0.203 CI 0.1956 ± 0.0228 0.2287 ± 0.0437 0.228 NL 3427 ± 47 3444 ± 59 0.670 ED 318 ± 17 329 ± 10 0.567 4 dph BD 662 ± 52 656 ± 81 0.892 CI 0.1935 ± 0.0156 0.1906 ± 0.0241 0.847 NL 3402 ± 110 3314 ± 34 0.176 ED 319 ± 25 307 ± 24 0.542 5 dph BD 601 ± 68 552 ± 36 0.247 CI 0.1765 ± 0.0152 0.1665 ± 0.0116 0.149 NL 3199 ± 83 3450 ± 190 0.0433 ED 301 ± 12 326 ± 30 0.170 6 dph BD 574 ± 46 631 ± 90 0.304 CI 0.1795 ± 0.0131 0.1841 ± 0.0344 0.885 NL 3523 ± 229 3620 ± 120 0.487 ED 345 ± 28 363 ± 9 0.248 7 dph BD 641 ± 109 788 ± 66 0.061 CI 0.1820 ± 0.0279 0.2181 ± 0.0215 0.0434

Table 2-3. Total growth and specific growth rate (%/day) (mean ± SD) of notochord length (NL), eye diameter (ED), body depth (BD) and condition index (CI) of T. goodei fed diets of solely rotifers and rotifers supplemented with copepods between 1 and 7 dph of Experiment I. All parameters except CI are in µm. Total Growth Specific Growth Rate Rotifer + Rotifer + Rotifer Copepod P Rotifer Copepod P NL 658 ± 218 636 ± 138 0.871 3.4 ± 1.1 3.2 ± 0.7 0.874 EyD 190 ± 129 197 ± 108 0.211 1.0 ± 2.3 2.6 ± 1.0 0.724 BD 7 ± 219 196 ± 109 0.174 4.8 ± 2.6 0.4 ± 5.5 0.973 CI 0.0006 ± 0.0595 0.0475 ± 0.0378 0.608 0.1803 ± 5.3825 4.1060 ± 3.2397 0.231

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Table 2-4 Water quality parameters [mean ± standard deviation (max-min)] measured in tanks holding T. goodei fed diets of solely rotifers and rotifers supplemented with copepods measured throughout Experiment I. Rotifer Rotifer + Copepod T 26.02 ± 0.56 (25.29 - 26.87) 26.08 ± 0.47 (25.4 - 26.75) Salinity 33.94 ± 0.48 (33.27 - 34.8) 33.93 ± 0.48 (33.28 - 34.85) pH 7.82 ± 0.1 (7.63 - 7.98) 7.83 ± 0.1 (7.64 - 7.97) DO 5.09 ± 1.02 (2.96 - 6.85) 4.98 ± 0.99 (3.63 - 6.39) TAN 0.01 ± 0.02 (0 - 0.05) 0.03 ± 0.05 (0 - 0.13) NO2-N 0.015 ± 0.009 (0.003 - 0.036) 0.015 ± 0.010 (0.003 - 0.039)

Table 2-5. Feeding regime used for larval culture of Trachinotus goodei. Live feeds values correspond to the density (organisms/mL) added to culture tanks. Dry diet values indicate frequency of feedings per day. T-ISO P. Wild P. Dry dph (cells Rotifers Artemia crassirostris Plankton pelagicus Diet mL-1) 13-Oct 1 6.0-7.0 14-Oct 2 6.0-7.0 15-Oct 3 6.0-7.0 1 4 16-Oct 4 6.0-7.0 1 6.5

17-Oct 5 6.0-7.0 1 4

18-Oct 6 6.0-7.0 0.8 4.2 19-Oct 7 6.0-7.0 0.71-1 6.5-6.79 20-Oct 8 6.0-7.0 0.5 7.5 1 1 21-Oct 9 6.0-7.0 1 7.5 1 0 22-Oct 10 6.0-7.0 10 0.05 24-Oct 12 8 2 -3.5 0.02 0.25-0.5 25-Oct 13 6 0.25 26-Oct 14 0.75 27-Oct 15 0.5-1.75 3 28-Oct 16 0.6-1.4 5 29-Oct 17 0.3-2.3 3 30-Oct 18 2

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Table 2-6. Mean water quality parameters measured throughout larval culture of T. goodei (mean ± SD). T (ºC) 26.08 ± 0.47 Salinity (g/L) 33.93 ± 0.48 pH m (g/L) 7.83 ± 0.10 DO (mg/L) 4.98 ± 0.99 TAN (mg/L) 0.03 ± 0.05 NO2-N (mg/L) 0.020 ± 0.011

Table 2-7. Allocation of Parvocalanus crassirostris copepods (C), Brachionus plicatilis rotifers (R) in four treatments throughout Experiment II (2-15 dph). C 2 dph R:C 2-8 dph R 2-15 dph R:C 2-15 dph R 3-15 dph R 9-15 dph C 0 1.5 2.7 1.5 2 dph R 3 1.5 0 1.5 C 0 2.0 0 2.0 3 dph R 4 2.0 4.0 2.0 C 0 4.5 0 4.5 4 dph R 7 4.5 7.0 4.5 C 0 5.0 0 5.0 5-9 dph R 10 5.0 10.0 5.0 C 0 7.5 0 0 10-11 dph R 15 7.5 15.0 15.0 C 0 10.0 0 0 12-15 dph R 20 10.0 20.0 20.0

Table 2-8. Fifteen dph survival (Surv.), notochord length (NL), body depth (BD) and gape height (GH) of G. speciosus receiving four different dietary regimes of including rotifers (R) and copepods (C). Values are expressed as mean ± SE. C 2 dph R:C 2-8 dph R 2-15 dph R:C 2-15 dph R 3-15 dph R 9-15 dph P Surv. (%) 19.4 ± 4.6 42.9 ± 7.4 36.3 ± 4.2 39.9 ± 3.7 0.0483 NL (µm) 4039 ± 187 4636 ± 311 4380 ± 148 4406 ± 134 0.360 BD (µm) 1323 ± 63 1521 ± 115 1447 ± 67 1536 ± 46 0.151 GH (µm) 1111 ± 45 1266 ± 68 1198 ± 36 1239 ± 39 0.160

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Table 2-9. Water quality parameters measured in tanks holding G. speciosus fed one of four diets throughout Experiment II. Values are expressed as mean ± SE (minimum – maximum). C 2 dph R:C 2-8 dph R 2-15 dph R:C 2-15 dph R 3-15 dph R 9-15 dph Temp 26.77 ± 0.25 26.75 ± 0.19 26.76 ± 0.22 26.64 ± 0.16 (ºC) (26.27-27.13) (26.48-27.14) (26.37-27.16) (26.4-26.92)

Salinity 34.11 ± 0.62 33.94 ± 0.54 33.94 ± 0.53 33.88 ± 0.62 (g/L) (33.14-34.86) (32.99-34.79) (33.01-34.64) (32.96-34.81)

pH----- 7.80 ± 0.03 7.81 ± 0.05 7.80 ± 0.04 7.79 ± 0.06 - (7.76-7.86) (7.69-7.91) (7.74-7.87) (7.7-7.86)

DO 6.21 ± 0.17 6.07 ± 0.30 6.13 ± 0.30 6.16 ± 0.39 (mg/L) (6.00- 6.52) (5.48-6.53) (5.68-6.53) (5.49-6.67)

TAN 0.16 ± 0.10 0.13 ± 0.170 0.19 ± 0.162 0.16 ± 0.06 (mg/L) (0-0.31) (0.01-0.25) (0.1-0.38) (0.09-0.21)

NO2-N 0.016 ± 0.011 0.033 ± 0.026 0.031 ± 0.033 0.032 ± 0.026 (mg/L) (0.008-0.039) (0.015-0.052) (0.007-0.069) (0.015-0.062)

Table 2-10. Values for wet weight, standard length (SL) body depth (BD) and feed conversion ratio (FCR) (mean ± SE) of T. goodei held at four stocking densities from 52-71 dph (Experiment III). Values are expressed as mean ± SE (minimum – maximum). Treat (g/L) Wet Weight (g) SL (mm) BD (mm) FCR 0.1 0.9 ± 0.3 32.1 ± 4.2 7.3 ± 0.7 1.2 ± 0.4 32.3 ± 3.7 7.8 ± 0.8 57 dph 0.2 0.4 1.2 ± 0.4 36.0 ± 2.6 7.7 ± 0.7 0.7 1.1 ± 0.3 33.1 ± 3.0 7.5 ± 0.8 P 0.752 0.713 0.926 0.1 1.4 ± 0.2 32.1 ± 3.5 9.4 ± 0.7 1.5 ± 0.3 38.9 ± 3.7 9.7 ± 0.6 64 dph 0.2 0.4 1.3 ± 0.2 37.2 ± 1.50 9.2 ± 0.3 0.7 1.8 ± 0.2 41.5 ± 1.9 10.3 ± 0.5 P 0.751 0.734 0.736 0.1 1.9 ± 0.2 43.3 ± 1.4 9.6 ± 0.6 1.9 ± 0.8 2.0 ± 0.2 41.6 ± 1.5 9.5 ± 0.6 1.2 ± 0.8 71 dph 0.2 0.4 2.0 ± 0.2 42.0 ± 9.0 9.1 ± 0.6 1.2 ± 0.8 0.7 1.8 ± 0.1 40.8 ± 8.6 9.3 ± 0.7 1.5 ± 0.1 P 0.817 0.439 0.947 0.347

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Table 2-11. Total growth of standard length, body depth and wet weight (mean ± SE) of T. goodei held at four stocking densities from 52-71 dph (Experiment III). 0.1 g/L 0.2 g/L 0.4 g/L 0.7 g/L SL (mm) 1.4 ± 0.2 1.2 ± 0.1 1.3 ± 0.0 1.2 ± 0.1 BD (mm) 0.2 ± 0.1 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 Wet Weight (g) 0.9 ± 0.3 1.0 ± 0.1 1.0 ± 0.1 0.8 ± 0.1

Table 2-12. Water quality parameters expressed as mean ± SD (min – max) as measured throughout the course of Experiment III. 0.1 g/L 0.2 g/L 0.4 g/L 0.7 g/L

T (ºC) 25.69 ± 0.79 25.67 ± 0.75 25.60 ± 0.93 25.71 ± 0.82 (24.52-27.27) (24.51-27.00) (23.11-27.25) (24.4-27.18) Sal 34.02 ± 1.00 34 ± 0.99 33.96 ± 1.02 34.08 ± 1.00 (g/L) (32.94-35.68) (32.86-35.69) (32.57-35.64) (33.06-35.72) pH 7.81 ± 0.07 7.80 ± 0.08 7.79 ± 0.07 7.74 ± 0.21 (7.71-8.01) (7.65-7.97) (7.7-7.91) (7.17-8.08) DO 5.86 ± 0.39 5.84 ± 0.39 5.65 ± 0.52 5.64 ± 0.71 (mg/L) (5.27-6.93) (5.03-6.95) (4.97-7.07) (4.07-7.70) TAN 0.16 ± 0.21 0.10 ± 0.10 0.15 ± 0.19 0.13 ± 0.09 (mg/L) (0-0.60) (0-0.22) (0-0.60) (0.01-0.26) NO2-N 0 ± 0 0.010 ± 0 0.020 ± 0.020 0.010 ± 0.010 (mg/L) (0-0.008) (0.004-0.018) (0.001-0.043) (0.001-0.025)

b 9%

8%

7%

6%

5%

4%

3%

2% Percent Survival Percent a 1%

0% Rotifers Rotifers + Copepods Treatment Figure 2-2. Survival (%) at 7 dph of T. goodei fed diets of solely rotifers and rotifers supplemented with copepods measured throughout Experiment I. Error bars represent standard error.

49

0.014

0.012 ) 3 0.01

0.008

0.006

0.004 y = -0.0029x + 0.011 R² = 0.8611 Oil Volume (mm Volume Oil 0.002

0 0 1 2 3 4 5 dph

Figure 2-3. Linear regression displaying reduction of T. goodei oil globule volume using larvae from the copepod supplemented treatment of Experiment I.

Figure 2-4. Development of T. goodei. A) embryos. B) 1 dph. C) 2 dph. D) 3 dph larvae; eyes are pigmented, mouth is opened, and larvae are capable of feeding. E) 6 dph with absorbed yolk sac. F) 9 dph. G) 13 dph with flexion and partially formed hypural plate. H) 21 dph. 50

Figure 2-4 continued. I) 34 dph. J) 128 dph. K) 238 dph.

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60% b

50% ab ab 40%

30% a

20% Percent Survival Percent 10%

0% R 2-15 dph R:C 2-15 dph C 2 dph, R 3-15 dph R:C 2-8 dph, R 9-15 dph

Treatment Figure 2-5. Survival (%) at 15 dph of G. speciosus fed four different diets of rotifers (R) and copepods (C) during Experiment II.

2.5 3 fish 6 fish 12 fish 24 fish

2

1.5

Mass (g) Mass 1

0.5

0 49 57 64 71 Age (dph)

Figure 2-6. Mean wet weight of T. goodei held at four different stocking densities during Experiment III.

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Discussion

This was the first documentation of the larval culture of T. goodei. Spawning was volitional. Intensive culture of T. goodei is possible and may be commercially viable.

Supplemental administration of P. crassirostris copepods significantly increased survival of T. goodei. However, copepods are not ultimately necessary for the survival of

T. goodei. This is consistent with larval culture studies on Seriola rivoliana which do not require copepods to reach metamorphosis (Roo et al. 2014). Significant differences in notochord length and condition index on 6 and 7 dph, respectively, suggest copepod supplementation may positively affect growth of T. goodei. Cassiano et al. (2011) found that a mixed diet of enriched rotifers (B. plicatilis) and copepods (P. pelagicus) resulted in significant increases in body depth and standard length but not in survival when compared to a proportional diet of rotifers alone.

Results of the current study are consistent with other studies and show increased growth and/or survival in fish fed a combination of rotifers and copepods as opposed to feeding with rotifers alone (Hernandez Molejon and Alvarez-Lajonchere

2003; Støttrup and McEvoy 2003; Milione and Zeng 2008; Camus and Zeng 2009;

Conceição et al. 2010). This further justifies the need to develop mass scale copepod culture methods for diversification of marine ornamental aquaculture and to increase productivity of species already in production.

Differences in survival among treatments were not detected on 1-6 dph. A large die off of approximately 70-90% was observed in all culture tanks between 6-7 dph of the experiment. Subsequent termination of the experiment and survival analysis indicated that the die off was far greater in T1 (rotifers alone).

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Larvae were altricial upon hatching and thus relied on endogenous energy reserves until the formation of functional mouth parts and gastrointestinal tract at 3 dph.

These reserves took the form of an oil globule, and a yolk sac. The size of endogenous reserves at parturition has been related to larval survival and growth rates (Berkely et al.

2004). A linear regression model suggested a reduction in oil globule volume of 0.002 mm3/day (Figure 2-3) from 0-3 dph. Egg, oil globule and yolk sizes were similar to those observed in other carangids (Table 2-5).

Similar to other carangids, larval development was fast. T. goodei larvae were very similar but slightly larger in size to T. carolinus with respect to notochord length, body depth, and eye diameter from 1-7 dph (Weirich and Riley 2007). Despite the underdeveloped state of T. goodei at the time of hatching, larval development was rapid. Growth was further accelerated following metamorphosis. The early onset of metamorphosis in this species may make it particularly attractive as a candidate for ornamental aquaculture producers. Development of pigmentation is paramount to the marketability of ornamental fish. The appearance of distinct silvery color, and the extension of dorsal and anal fins as early as 13 dph and 23 dph, respectively, are promising.

This study further refined culture methods for G. speciosus outlined by Broach et al. (2015). To date no replicated comparison of larval culture techniques had been published for G. speciosus. Broach et al. (2015) cultured G. speciosus via co-feeding of copepods and rotifers. Culture of other Carangids has been feasible without the use of copepods (Roo et al. 2014; Experiment 1 herein). This experiment demonstrated the

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feasibility of culturing G. speciosus without the use of copepods, while suggesting provision of P. crassirostris as an additional feed item may increase survival.

In general, the use of copepods in larval feeding regimes is beneficial. The nature of the benefit is less consistent. Contrary to the findings of this study, first feeding

Cynoscion nebulosus larvae fed Acatia tonsa showed increased myotome height, notochord length and condition (myotome height/notochord length). The present studies

(Experiment I-II) show the value of copepods (specifically P. crassirostris) and suggest the importance of their incorporation into commercial aquaculture.

It is important to determine the maximum stocking density for the grow-out phase of any species. This experiment demonstrated that metamorphosed T. goodei can be held at densities of at least 0.7 g/L without adverse effects on growth or feed conversion. Further studies should evaluate higher stocking densities and feeding rates to determine the upper limit of stocking density at various stages. Incorporation of economic aspects such as costs associated with labor, feed, and water usage should be compared to the productivity of different stocking densities. Interactions between stocking density and abiotic environmental conditions (temperature, salinity, dissolved oxygen, TAN, NO2-N) with respect to growth rates should be assessed. Evaluating larval stocking density limitations would be of particular interest due to the implications of conserving live feeds such as copepods.

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CHAPTER 3 OSMOREGULATORY RESPONSE OF T. goodei TO HYPOSALINITY

Foreword

Evaluations of response to salinity change are common when assessing candidate species (Santerre 1976; Tandler 1995 Lemarie et al. 2004). Blaber and Cyrus (1983) examined salinity in 17 carangid species. The oval pompano (Trachinotus ovatus) was reported to withstand acute transfer from full strength sea water (39 g/L) to 7.8 g/L and tolerance of salinities as low as 1.5 g/L when acclimated (Chervinksi 1973). Costa et al.

(2008) found plata pompano (Trachinotus marginatus) to have reduced sensitivity to environmental TAN and nitrite at 10 g/L salinity, and increased sensitivity to TAN at 5 g/L salinity compared to 10 and 30 g/L. Little mention is made of salinity in the limited reviews of culture attempts of T. goodei. However, it was suggested that T. goodei exhibit culturability similar to that of Florida pompano (Trachinotus carolinus) (Jory et al. 1985; Cole et al.

1997).

The ability to culture species at reduced salinities saves money for operations without direct access to saline water supply. Identification of such a species can thus be a means for product diversification at a land locked hatchery otherwise limited to freshwater species.

Furthermore, definition of osmoregulatory limitations for a species is valuable and may have ecological implications (DiMaggio 2009).

Hyposaline conditions have also been reported to increase growth of some species.

Studies with silver sea bream (Sparus sarba), turbot (Scophthalmus maximus), red drum

(Sciaenops ocellatus) and the yellow bellied damsel (Pomacentrus caeruleus) showed increased growth in these species at iso-osmotic salinities (Craig et al. 1995; Woo and Kelly

1995; Boeuf and Payan 2001; Imsland et al. 2003; Prashanth et al. 2012). Conversely,

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juvenile cobia (Rachycentron canadum) cultured at 5 g/L showed lower survival (two trials:

87.5 and 68.3 %) while growth was unaffected (Resley et al. 2006). Many species have exhibited improved growth at intermediate salinities (Boeuf and Payan 2001). This has been attributed to improved digestive efficiency, increased feeding rates and reduced energy allocation to osmoregulation in an isotonic environment (Ferraris et al.1986; Febry and Lutz

1987; Lambert et al. 1994; Gaumet et al. 1995; Boeuf and Payan 2001; Imsland 2001;

Sampaio and Bianchini 2002; Anni and Bianchini 2016). Recently, Anni and Bianchini

(2016) demonstrated improved growth of juvenile T. marginatus held at 3-6 g/L salinities.

Ferraris et al. (1986) observed improved protein digestibility in milkfish (Chanos chanos).

Conversely, Riche et al. (2016) reported reduced digestibility of 6 out of 7 feed ingredients when fed to T. carolinus at 3 g/L versus 28 g/L.

Reduced salinities are implemented as treatments for parasitic infection. The salinity yielding optimal parasite suppression varies by parasite, however, for marine species lower salinities are generally more effective (Dunphy et al. 2003; Fajer-Ávila 2008). Prevention of parasitic outbreak may be an added benefit to low-salinity aquaculture. Knowledge of the lower salinity limits of fish is important for an application of hyposalinity as a parasitic treatment.

Previous pond production trials for T. carolinus indicated potential economic viability

(Tatum and Trimble 1978; Trimble 1980). Trimble (1980) reported monoculture production of 23 kg/ha, and FCR of 5.5, but low survival (38 %) in 0.8 ha ponds stocked with 4925 fish/ha. In polyculture with white shrimp, Trimble (1980) reported total (T. carolinus + P. vannamei) production of 265 kg/ha at a combined FCR of 0.9. Alternatively, Weirich et al.

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(2009) observed higher survival (89.2 – 89.6%) at 5 g/L using intensive recirculating methods (6.5-13.5 kg/m3 in 8 m3 tanks).

Without access to natural sea water, inland marine operations rely on synthetic sea salt mixes. The cost of Instant Ocean sea salt (Spectrum Brands, Middleton WI), converts to

$0.063 per L of water mixed (Pentair-Aquatic Ecosystems, Apopka, FL). At $4.86/18.1 kg

($0.0026/L mixed) rock salt (Morton Salt, Cape Canaveral, FL) would be a cheaper alternative. However, absence of minor constituents of natural sea water, particularly divalent cations (Ca2+ and Mg2+) has yielded deleterious effects. The freshwater, euryhaline

Fundulus seminolis survived acute transfer from freshwater up to 24 g/L natural sea water

(NSW), but only up to 16 g/L using rock salt. When comparing salt sources (NaCl vs. NSW),

F. seminolis exhibited reduced swimming activity and increased mucus production in 16 g/L

NaCl (DiMaggio et al. 2009). Low calcium levels have been linked to elevated mucus production (Potts and Fleming 1970). Lack of divalent cations in hyperosmotic situations has also been associated with osmoregulatory inhibition in the catadromous American eel

(Anguilla anguilla) (Isaia and Masoni 1976). A similar effect was observed in the primarily marine but euryhaline pinfish (Lagodon rhomboids). This was reportedly due to an inability to regulate sodium diffusion (Carrier and Evans 1976). A series of experiments were performed to test the salinity tolerance of T. goodei.

Methods

Feeding

Feed was withheld from the system with experimental fish for 24-48 hours prior to the stocking of any of the following experiments and throughout the course of the experiments.

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Experiment IV.I, Acute Natural Sea Water Salinity Tolerance at a Temperature Gradient

Experimental design and sampling

Twenty-four 86 L glass aquaria were utilized as culture tanks. Each tank was bisected into two equal-volume subsections with a perforated tank divider (Penn-Plax,

Haupauge, New York) allowing continuous water exchange. Four salinity treatments were evaluated: 3, 15, 25, 35 g/L (n=12) (Table 3-9). Tanks were set to treatment salinities (± 0.5 g/L) on 2/16/2016. Salinities were achieved by mixing well water (3 g/L) and sterile Atlantic Ocean water (35 g/L).

Well water was used for the 3 g/L treatment. Sea and well water sources were treated with hypochlorite (bleach) for at least 24 h and dechlorinated with aeration and sodium thiosulfate. Subsequently water was circulated through a UV sterilizer (80 W,

Emperor Aquatics, Pottstown PA) until used.

Forty-eight fish were removed from the stock tank, (T=26.2ºC, Sal=35 g/L) individually weighed (8.5-23.1 g, mean ± SD= 16.5 ± 3.5 g) and photographed next to a ruler (6.6-10.2 cm, mean ± SD= 8.5 ± 0.69 g). No significant differences in mean wet weight were detected among treatments (P= 0.512). A significant difference in total length was detected among treatments (P= 0.042). The range of mean total length among treatments was 8.81-8.26 cm. Differences in length were not biologically significant for this experiment. Immediately after weighing and photographing, fish were transferred into an aquarium subsection (1 fish/subsection). On the day of stocking, mean tank temperature was 22.3 ± 1.3ºC. Mortality was defined as cessation of opercular movement and failure to respond to tactile disturbance. Subsections were observed every hour for the first 12 hours, every 6 hours from 12-36 hours, and every

59

12 hours from 36-96 hours. Fish were removed from their respective subsection and weighed immediately upon discovery of mortality. Water quality parameters

(temperature, salinity, pH, and DO, TAN, and NO2-N) were measured once every 24 hours via previously described instruments. Measurements of hardness and alkalinity of the source water dilutions used for each treatment were conducted after the experiment using a digital titrator (HACH).

Statistical analysis

Survival data was compared via Log-Rank analysis following Kaplan-Meier survival analysis. Wet weight change data were arcsin square root transformed and analyzed with and Kruskall-Wallis test due to violation of the assumption of normality and failure to ameliorate this with transformation. Differences were considered significantly different at P≥0.05. Statistical tests were performed using JMP 13 (Cary, NC).

Experiment IV.II, Acute Natural Sea Water Salinity Tolerance without a Temperature Gradient

Experimental design and sampling

A second experiment was conducted to determine the lower tolerance limit of acute salinity change. The methods followed for this experiment were similar to those described in Experiment IV.I. Aquaria were housed in the same ambient environment as the communal holding tank. At the time of stocking the holding tank temperature was

25.4ºC while experimental tanks were 24.8 ± 0.6ºC. Salinities were attained via mixing of sterile Atlantic Ocean Water and dechlorinated tap water. Six treatments were used:

0.65, 3, 6, 9 and 12 g/L. Dechlorinated tap water was used for the 0.65 g/L treatment water.

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Statistical analysis

Survival data was compared via Log-Rank analysis following Kaplan-Meier survival analysis. Wet weight change data were arcsin square root transformed and analyzed with and ANOVA and subsequent Tukey’s test. Differences were considered significantly different at P≥0.05. Statistical tests were performed using JMP 13 (Cary,

NC).

Experiment V, Acute NaCl Water Salinity Tolerance

Experimental design and sampling

A third experiment was conducted to assess the feasibility of replacing natural sea water with sodium chloride solutions. Methods for this experiment were similar to those described in Experiment IV.II with the following exceptions: Salinity levels (0.57,

3, 6, 9, 12 and 35 g/L) were mixed using NaCl (Morton Salt Chicago, Illinois) and dechlorinated tap water. Observations for mortality took place each hour from 1-13 h, and again at hour 18. Water quality parameters (temperature, salinity, pH and dissolved oxygen, TAN, and NO2-N) were monitored using the previously described equipment and methodology. Temperature and salinity were measured in each replicate of each treatment. TAN and NO2-N were measured in two replicates per treatment and pH and dissolved oxygen were measured in two replicates per treatment.

Statistical analysis

Survival data was compared via Log-Rank analysis following Kaplan-Meier survival analysis. Wet weight change data and water quality data were analyzed via

ANOVA and subsequent Tukey’s test. Differences were considered significantly different at P≥0.05. Statistical tests were performed using JMP 13 (Cary, NC).

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Experiment VI, Stepwise Acclimation to 3 g/L Sea Water

Experimental design and sampling

Four fish were removed from the holding tank (T=25.58 ± 1.36 [23.26-27.16],

Sal=34.638 ± 0.693 33.6-35.56], pH=7.46 ± 0.26 [7.05-7.7], DO=5.368±0.355 [4.97-

5.72]) weighed and placed separately into 4 cylindrical, black walled, white bottomed, fiberglass tanks (Sal=33-35 g/L) for 1 hour (starter tanks). This step served the purpose of spatial acclimation to tank size. Each fish was assigned to one of 4 treatments corresponding to an acclimation duration: 0, 3, 6, and 12 h. After spending 1 hour in the starter tanks, all fish were moved by dip net each hour into tanks of varying salinities

(treatment tanks). Fish assigned to 0, 3, 6, and 12 h treatments experienced hourly salinity reductions of 31.78 ± 0.503, 10.58 ± 0.319, 5.38 ± 0.564, and 2.64 ± 0.195 g/L, respectively, until the target salinity (2.89 ± 0.0889 g/L) was reached. Fish were subsequently held at the target salinity in 105 L tanks (observation tanks) for 48 h. Fish were monitored regularly for mortality. This procedure was repeated 6 times. Treatment salinities were achieved by mixing well water, dechlorinated municipal water and sterile

Atlantic Ocean water. Culture tanks were aerated vigorously to ensure adequate oxygenation. Temperature, salinity, pH, and dissolved oxygen were monitored in all replicates. TAN and NO2-N were measured in at least 4 replicates per treatment. Water quality was measured using the previously described equipment and methodology.

Statistical analysis

Logistic regression was used to evaluate the effect of acclimation time on survival. The P-value associated with the likelihood ratio of the analysis determined if acclimation time significantly affected survival (α=0.05). An odds ratio estimate was calculated for the regression model. Mean wet weight change and water quality

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measurements were analyzed via one-way ANOVA and subsequent Tukey’s means separation tests. If ANOVA assumptions were violated, a Kruskall-Wallis non-parametric test followed by a Dunn’s multiple comparison test was performed. Differences were considered significantly different at P≥0.05. Statistical tests were performed using JMP

13 (Cary, NC).

Results

Experiment IV.I, Acute Natural Sea Water Salinity Tolerance at a Temperature Gradient

Acute transfer from 35 to 3 g/L NSW resulted in 100% mortality. All replicates of this treatment died within 5.50 hours of transfer. Mean survival time was 4.33 ± 0.30 h

(2.5-5.5) (Table 3-1). Survival in the 15, 25 and 35 g/L treatments was 100%. Upon transfer, fish experienced a mean temperature decrease of roughly -3.92ºC (max-min=-

5.86ºC to -2.93ºC). P-values <0.0001 for the Log Rank test indicated survival was significantly different in the 3 g/L treatment than in all other treatments (Table 3-2). Wet weight change data violated the assumption of normality and were compared via a

Kruskal-Wallis test. P-values <0.01 for the Kruskal-Wallis test and Wilcoxon comparisons of each pair indicated a significant difference in wet weight change between the 3 g/L treatment and all other treatments. No significant differences in mean wet weight change between any of the other treatments were detected (Table 3-5).

There were statistically significant differences among treatments with respect to pH and

DO but these were not considered biologically significant for the purposes of this study.

These and all other water quality parameters were maintained within acceptable ranges

(Table 3-8). Lengths of fish at stocking were compared among treatments using a

Kruskal-Wallis test as the normality assumption was violated. Differences in lengths

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among treatments were detected but were not considered biologically significant for the purpose of the study (P=0.042). No significant difference was detected in fish wet weight between treatments (P=0.512).

Experiment IV.II, Acute Natural Sea Water Salinity Tolerance without a Temperature Gradient

No significant difference in wet weight or length at stocking was detected among treatments (P=0.334, P= 0.479). Acute transfer from 35 g/L sea water to dechlorinated tap water resulted in 100% mortality. All replicates within this treatment (0.65 g/L) died within

4.5 hours of acute transfer. Mean survival time was 3.75 ± 0.41 h (1.5-4.5). Survival following acute transfer from 35 to 3 g/L NSW was 75%. Non-surviving replicates of the 3 g/L treatment lived for 10.5-48 h. Survival in the 6, 9, 12, and 35 g/L treatments was 100%.

Upon transfer, fish experienced a mean temperature decrease of -0.74ºC (-1.93-0.01). P- values below 0.001 for the Log Rank test, indicated survival was significantly lower in the

0.65 g/L treatment than in all other treatments (Table 3-2). ANOVA and Tukey’s tests indicated a significant difference in wet weight loss between the 0.65 g/L treatment and all other treatments. These tests revealed no significant differences in wet weight change between any of the other treatments (Table 3-6). Statistically significant differences among treatments with respect to pH and DO were not considered biologically significant for the purposes of this study. These and all other water quality parameters were maintained within acceptable husbandry ranges (Table 3-8).

Experiment V, Acute NaCl Water Salinity Tolerance

No significant difference in length or wet weight of stocked fish was detected among treatments (P=0.555, P=0.479 respectively). Length data was compared using a Kruskal-

Wallis test due to its violation of the normality assumption. Wet weight data was compared

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with an ANOVA test. One hundred percent mortality was observed in all treatments.

Survival times (mean ± SE h) in the 6 NaCl concentrations were 3.13 ± 0.28 (0.57 g/L), 8.38

± 0.83 (3 g/L), 9.38 ± 1.06 (6 g/L), 9.25 ± 1.36 (9 g/L), 9.25 ± 0.72 (12 g/L) and 4.25 ± 0.06 g/L (35 g/L) (Table 3-1). Log rank analysis indicated survival times of the 0 and 35 g/L treatments to be significantly lower than that of all other treatments. No difference was detected between the 0 and 35 g/L treatments (Table 3-4). P-values produced by an

ANOVA test indicated a significant difference in wet weight change between the following treatment pairings: 0.57 and 12, 0.57 and 35, 3 and 6, 3 and 9, 3 and 12, 3 and 35, 6 and 9,

6 and 12, 6 and 35, 9 and 12, 0.57 and 9, 9 and 35, 12 and 35 g/L (Table 3-7). There were no significant differences among treatments with respect to any of the measured water quality parameters. Parameters were maintained within acceptable husbandry ranges

(Table 3-8).

Experiment VI, Stepwise Acclimation to 3 g/L Sea Water

Wet weight and length data were compared using a Kruskal-Wallis test due to violations of the assumptions of normality. No significant difference in length or initial wet weight of fish was detected among treatments (P=0.945, P=0.945 respectively).Twelve and

6 h acclimation times yielded 100% survival. An acclimation time of 3 h yielded 75% survival. An acclimation time of 0 h yielded 33% survival. Results of the logistic regression indicated significant differences in survival among acclimation times (P=0.001). Probability of survival was estimated to increase 1.96 times for each hour added to the acclimation time

(95% CI: 1.22, 4.22). Estimated survival for 12, 6, 63, and 0 hour acclimations was thus

100%, 96%, 75%, and 29% respectively. There were no statistically significant differences among treatments with respect to any of the measured water quality parameters and parameters were maintained within acceptable ranges (Table 3-9). 65

Table 3-1. Kaplan-Meier survival analysis for three acute transfer trials. Survival is expressed as the mean ± SE. Treatment Trial (g/L NSW) survival (h) % survival 3 4.33 ± 0.30 0 Expt. 15 * 100 IV.I 25 * 100 35 * 100 0.65 3.75 ± 0.41 0 3 79.13 ± 11.56 75 Expt. 6 96 100 IV.II 9 96 100 12 96 100 35 96 100 0.57 3.13 ± 0.28 0 3 8.38 ± 0.83 0 Expt. 6 9.38 ± 1.06 0 V 9 9.25 ± 1.36 0 12 9.25 ± 0.72 0 35 4.25 ± 0.06 0

Table 3-2. Log rank comparisons of T. goodei survival following acute transfer to one of four sea water concentrations in Experiment IV.I. 15 g/L 25 g/L 35 g/L 3 g/L P<0.0001 P <0.0001 P <0.0001 15 g/L * * 25 g/L * * 35 g/L * *

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Table 3-3. Log rank comparisons of T. goodei survival following acute transfer to one of six sea water concentrations in Experiment IV.II. 3 g/L 6 g/L 9 g/L 12 g/L 35 g/L 0.65 g/L P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 3 g/L P=0.143 P=0.143 P=0.143 P=0.143 6 g/L P=0.143 * * * 9 g/L P=0.143 * * * 12 g/L P=0.143 * * * 35 g/L P=0.143 * * *

Table 3-4. Log rank comparisons of T. goodei survival following acute transfer to one of six NaCl concentrations Experiment V. 3 g/L 6 g/L 9 g/L 12 g/L 35 g/L 0.57 g/L P<0.001 P<0.001 P<0.001 P<0.001 P=0.082 3 g/L P=0.500 P=0.479 P=0.669 P<0.001 6 g/L P=0.500 P=0.847 P=0.990 P<0.001 9 g/L P=0.479 P=0.479 P=0.720 P=0.006 12 g/L P=0.669 P=0.990 P=0.720 P<0.001 35 g/L P<0.001 P<0.001 P=0.006 P<0.001

Table 3-5. Each pair Wilcoxon t-test comparisons of wet weight changes of T. goodei transferred to one of four natural sea water concentrations in Experiment IV.I. 15 g/L 25 g/L 35 g/L 3 g/L P=0.005 P=0.062 P=0.013 15 g/L * P=1 P=1 25 g/L P=1 * P=1 35 g/L P=1 P=1 *

Table 3-6. Tukey’s Multiple Comparisons of wet weight changes of T. goodei transferred to one of six natural sea water concentrations in Experiment IV.II. 0.65 g/L 3 g/L 6 g/L 9 g/L 12 g/L 3 g/L P<0.001 6 g/L P<0.001 P =0.997 9 g/L P=0.011 P=0.960 P =1 12 g/L P=0.003 P=0.992 P=1 P =1 35 g/L P=0.001 P=0.357 P=0.712 P=0.866 P =0.773

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Table 3-7. ANOVA (P-value) comparisons of wet weight changes of T. goodei transferred to one of six natural NaCl concentrations in Experiment V. 0.57 g/L 3 g/L 6 g/L 9 g/L 12 g/L 3 g/L P=0.994 P=0.042 P<0.0001 P<0.001 6 g/L P=0.141 P =0.042 P=0.00149 P<0.001 9 g/L P<0.0001 P<0.0001 P =0.00149 P<0.001 12 g/L P<0.0001 P<0.0001 P<0.0001 P =0.0229 P<0.001 35 g/L P<0.0001 P<0.0001 P<0.0001 P<0.0001 P<0.001

Table 3-8. Water quality measured during three acute salinity transfer trials, Experiments IV.I – V. Values are expressed as mean ± SD (minimum-maximum). NSW Exp IV.I NSW Exp IV.II NaCl Exp V

22.75 ± 0.58 24.82 ± 0.45 23.14 ± 0.20 T (º C) (20.36-23.43) (23.62-25.56) (22.94-23.55)

8.34 ± 0.14 8.00 ± 0.32 7.07 ± 0.31 pH (8.12-8.68) (7.17-8.58) (6.67-7.45)

DO 7.16 ± 0.55 6.96 ± 0.69 6.81 ± 0.70 (mg/L) (6.19-8.29) (5.33-8.20) (5.69-7.45)

TAN 0.04 ± 0.04 0.03 ± 0.07 0 ± 0 (mg/L) (0-0.115) (0-0.278) (0-0)

NO2-N 0.007 ± 0.013 0.018 ± 0.013 0.026 ± 0.005 (mg/L) (0-0.064) (0-0.05) (0-0.039)

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Table 3-9. Salinity levels and mean changes in salinity per hour during stepwise acclimation from 34.64 ± 0.69 g/L (33.6-35.56) to 2.89 ± 0.089 (2.76-3.16), Experiment VI. Values are reported as mean ± SD (min-max). 0 hours 3 hours 6 hours 12 hours 2.90 ± 0.07 24.75 ± 0.73 29.99 ± 0.66 32.71 ± 0.62 hour 1 (2.80-2.96) (24.31-26.37) (29.58-31.46) (32.26-34.08) 14.11 ± 0.83 24.75 ± 0.73 29.99 ± 0.66 hour 2 (13.62-15.95) (24.31-26.37) (29.58-31.46) 2.93 ± 0.11 19.45 ± 0.76 27.40 ± 0.71 hour 3 (2.7-3.16) (19.07-21.14) (26.96-28.98) 14.11 ± 0.83 24.75 ± 0.73 hour 4 (13.62-15.95) (24.31-26.37) 8.84 ± 0.86 22.00 ± 0.72 hour 5 (8.34-10.76) (21.6-23.62) 2.86 ± 0.06 19.45 ± 0.76 hour 6 (2.78-2.95) (19.07-21.14) 16.87 ± 0.73 hour 7 (16.35-18.49) 14.11 ± 0.83 hour 8 (13.62-15.95) 11.45 ± 0.82 hour 9 (10.97-13.28) 8.84 ± 0.86 hour 10 (8.34-10.76) 6.12 ± 0.89 hour 11 (5.65-8.11) 2.88 ± 0.08 hour 12 (2.76-3.01) change / step (g/L) 31.78 ± 0.50 10.58 ± 0.32 5.38 ± 0.56 2.64 ± 0.19

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Table 3-10. Water quality parameters measured in 48 hour observation tanks following stepwise acclimation to 3 g/L trials, Experiment VI. Values are reported as mean ± SD (min-max). 0h 3h 6h 12h T 27.00 ± 0.93 27.12 ± 0.75 27.22 ± 0.46 27.15 ± 0.82 (º C) (25.90-28.38) (25.66-28.56) (26.22-27.86) (25.60-28.50) Sal 2.90 ± 0.07 2.94 ± 0.12 2.86 ± 0.06 2.88 ± 0.08 (g/L) (2.80-2.96) (2.78-3.16) (2.78-2.95) (2.76-3.01) pH 7.89 ± 0.48 8.03 ± 0.60 8.04 ± 0.54 8.10 ± 0.45 d (7.15-8.52) (6.56 8.58) (6.70-8.61) (7.25-8.63) DO 7.26 ± 1.06 7.12 ± 0.90 7.08 ± 0.78 7.06 ± 0.80 (mg/L) (5.68-8.64) (5.74-8.66) (5.74-8.64) (5.51-8.68) TAN 0.04 ± 0.05 0.04 ± 0.07 0.05 ± 0.09 0.03 ± 0.04 (mg/L) (0-0.104) (0-0.20) (0-0.24) (0-0.09) NO2 0.014 ± 0.013 0.021 ± 0.039 0.014 ± 0.015 0.027 ± 0.05 (mg/L) (0.004-0.032) (0.001-0.131) (0.001-0.048) (0-0.158)

Table 3-11. NSW concentrations (g/L) corresponding to treatments used in Experiment IV.I. Values are reported as mean ± SD (min-max). 3 g/L 15 g/L 25 g/L 35 g/L 2.97 ± 0.38 15.61 ± 0.46 25.32 ± 0.31 35.67 ± 0.31 Expt. IV.I (2.52-3.63) (15.04-16.67) (24.57-25.67) (35.19-36.06)

Table 3-12. NSW and NaCl concentrations (g/L) corresponding to treatments used in Experiments IV.II and V. Values are reported as mean ± SD (min-max). 0-1 g/L 3 g/L 6 g/L 9 g/L 12 g/L 35 g/L Expt. 0.65 ± 0.25 3.27 ± 0.12 5.89 ± 0.18 8.94 ± 0.2 12.19 ± 0.29 34.69 ± 0.16 IV.II (0.33-1) (3.05-3.48) (5.52-6.09) (8.71-9.28) (11.86-12.53) (34.52-35.05) Expt. 0.57 ± 0.23 3.15 ± 0.32 5.88 ± 0.14 9.06 ± 0.37 12.12 ± 0.66 34.69 ± 0.37 V (0.38-0.86) (2.86-3.6) (5.69-6.03) (8.72-9.48) (11.45-12.74) (34.15-35.00)

Table 3-13. Total hardness and total alkalinity of salinities corresponding to treatments in Experiment IV.I. 3 g/L 15 g/L 25 g/L 35 g/L Total Hardness 826.7 ± 23.1 2756.7 ± 93.0 4480.0 ± 78.1 6273.3 ± 86.2 Total Alkalinity 102.3 ± 8.0 108.0 ± 5.6 116.3 ± 7.8 124.7 ± 7.0

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Table 3-14. Total Hardness and total alkalinity of salinities corresponding to treatments in Experiment IV.II. Treatment 0 g/L 3 g/L 6 g/L 9 g/L 12 g/L 35 g/L Total Hardness 120.7 ± 4.6 630.0 ± 61.4 1126.7 ± 5.8 1623.3 ± 49.3 2286.7 ± 58.6 5780 ± 635.0 Total Alkalinity 27.7 ± 0.7 41.1 ± 2.6 47.7 ± 2.7 53.2 ± 1.8 64.3 ±6.4 114.3 ± 1.2

Table 3-15. Total Hardness and total alkalinity of salinities corresponding to treatments in Experiment V. Treatment 0 g/L 3 g/L 6 g/L 9 g/L 12 g/L 35 g/L Total Hardness 123.0 ± 1.0 116.0 ± 1.7 113.1 ± 9.5 109.3 ± 5.9 123.7 ± 10 169.7 ± 9.5 Total Alkalinity 34.1 ± 1.2 34.13 ± 0.6 36.5 ± 3.6 34.4 ± 2.6 35.1 ± 0.6 35.3 ± 2.3

35

30

25 a 20

15

10

5 b b b

0

-5

Change in body (g) in body mass Change -10 3 g/L 15 g/L 25 g/L 35 g/L

-15 Treatment

Figure 3-1. Mean change in T. goodei body wet weight from the time of exposure to four concentrations of natural sea water until mortality or termination of Experiment IV.I.

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35 a 30

25

20

15

10 b b b b b 5

0

Change in body mass (g) mass bodyin Change -5

-10 0 g/L 3 g/L 6 g/L 9 g/L 12 g/L 35 g/L -15

Figure 3-2. Mean change in T. goodei body wet weight from the time of exposure to six concentrations of natural sea water until mortality or termination of Experiment IV.II.

35

30

25 a ab b 20

15 c

10 d d 5

0

-5

Change in body (g) body in mass Change -10 0 g/L 3 g/L 6 g/L 9 g/L 12 g/L 35 g/L Treatment -15 Figure 3-3. Mean change in T. goodei body wet weight from the time of exposure to six concentrations of NaCl until mortality or termination of Experiment V.

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Discussion

Tolerance to hyposaline conditions is species specific in marine fish. Salinity variations may influence metabolic rates, growth, and survival. These effects may be dependent on life stage within a species (Morgan and Iwama 1991; Boeuf and Payan

2001; Hilomen-Garcia et al. 2003; Martinez-Palácios et al. 2004; Barman et al. 2005; de

Hora et al. 2013; Ma et al. 2014; Lisboa et al. 2015; Ishikawa et al. 2016). Reef dwelling fish are generally stenohaline. T. carolinus demonstrated resilience to low salinities in culture settings. McMaster et al. (2007) reported that reductions in salinity from 35 to 19 g/L did not result in reduced growth of T. carolinus. In a study by Riche and Williams

(2010), T. carolinus held at 3 g/L appeared to exhibit improved digestion of two soy based feeds when compared to specimens held at 28 g/L.

The results of these experiments suggest that T. goodei may be a viable candidate for low salinity aquaculture. Preliminary trials indicated survivability following acute transfer from 35 to 3 g/L. Anecdotally, the results of Experiments IV.I and IV.II suggest survivability during salinity drops is affected by temperature. The threshold for complete survival following acute hyposaline transfer was established to be between 3 and 6 g/L. Collectively, results of preliminary trials, Experiment IV.II, and Experiment VI indicated long term survivability of T. goodei in 3 g/L. Experiment VI established 6 h as a minimum acclimation time to ensure 100 % survival of T. goodei in 3 g/L sea water.

Experiment V showed that sodium chloride is not a viable alternative to natural sea water. The results of Experiments IV.I and IV.II suggest a relationship between survivability and salinity (Table 3-1) and between salinity and wet weight change (Figure

3-1; Figure 3-2). Complete mortality across treatments in Experiment V may be linked to dramatically reduced total hardness amongst these treatments when compared to those 73

corresponding to Experiments IV.I and IV.II (Table 3-15; Table 3-13; Table 3-14).

Previous studies have associated reduced hardness with mortality (Grizzle et al. 1985) and more specifically the role of divalent cations in osmoregulatory functionality

(DiMaggio et al. 2009). External calcium is prevents the passive diffusion of sodium and potassium which are required to trigger mechanisms such as cellular contraction.

Sodium-potassium ATPase actively creates diffusional gradients of sodium and potassium as a means of generating electrical potential used to signal these mechanisms. Wurts and Stickney (1989) observed 100% mortality of red drum

(Scianops ocellatusi) held in reduced calcium sea water (<176 mg/L Ca). Forsberg and

Neill (1997) reported increased survival and growth of S. ocellatus in low salinity (0.2 g/L) with supplemented magnesium and calcium, respectively. Likewise, Carrier and

Evans (1976) associated acute mortality of pinfish (Lagodon rhomboides) transferred from calcium supplemented freshwater (400 mg/L Ca) to unaltered freshwater with increased gill permeability and reduced tissue sodium. Thus, low hardness measurements like those in 0-1 and 3 g/L concentrations may be a confounding factor.

Data indicating resilience of T. goodei to salinities as low as 3 g/L is a substantial finding. Contradictory results in Experiment IV.I and IV.II as well as preliminary trials suggest that increased temperature gradients may compound the effects of osmoregulatory shock. Thus, lethal temperature thresholds need to be determined and the influence of temperature variations (acute and acclimated) assessed for their influence on salinity tolerance.

Further research is required to evaluate hyposaline tolerance of palometa at different life stages. Additionally, the effects of low salinity culture on feeding, growth,

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nutrient assimilation, reproduction, and stress response should be evaluated. Finally, the effects of specific divalent cation concentrations will help in understanding the physiological responses observed in these studies.

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CHAPTER 4 SPAWNING, INCUBATION AND LARVAL CULTURE OF Paracanthurus hepatus AND Chaetodon speciosus

Foreword

Captive spawning data on Paracanthurus hepatus is scarce. Robertson (1983) described observations of wild spawning events in P. hepatus. Robertson reported spawning aggregations from January-March along the northern Great Barrier Reef, and noticed spawns between 16:00-18:15 h in 5-8 m of water. Robertson suggested that during spawning events, large aggregations of 25-30 P. hepatus would disperse into 5-8 subgroups containing a sexually mature male and 2-7 smaller individuals suspected to be females. Additionally, Robertson notes only observing paired spawning and no group spawning, but that courtship tended to happen in the same area and at the same time as previous spawns. Captive, volitional spawning has been reported in public aquaria

(Nagano et al. 2001; Cassiano et al. 2015) and research laboratories (Indian River

Research and Education Center, Tropical Aquaculture Laboratory).

All published observations of mating in Chaetodontids (Chaetodontidae) to date indicate paired or harem mating styles (Neudecker and Lobel 1982; Colin and Clavijo

1988; Lobel 1988; Colin 1989; Yabuta and Kawashima 1997; Degidio 2014). Degidio

(2014) presented a thorough evaluation of spawning activity, fecundity, and larval development in the milletseed butterflyfish (Chaetodon miliaris). Degidio reported an influence of sex ratio on egg fecundity. Groups of 3 males and 8 females (3M:8F), as well as 10M:11F both yielded higher fecundity than a 1M:1F pair. The ratio of 3M:8F resulted in significantly higher egg production per fish per spawn when compared to the other two ratios (Degidio 2014). Similar 3:8 (M:F) ratio harem mating was observed with the chevron butterflyfish (Chaetodon trifascialis) and spawning occurred at dusk during

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full or new moon phases (Yabuta and Kawashima 1997). Degidio observed varying results when comparing photoperiod to spawning events. Spawning in populations with sex ratios of 1M:1F and 3M:8F exhibited a positive relationship with increases in day length while the opposite was observed for a 10:11 population. However, Degidio

(2014) noted that this may have been due to the 10M:11F population not spawning until

March 2014, whereas the other two populations began spawning in January 2014.

Spawning may increase with time spent in captivity and time since spawning began.

Thus, evaluations of seasonality in spawning in captivity would require collection of spawning data over several years. Observations of the longnose butterflyfish

(Chaetodon aculeatus), the foureye butterflyfish (Chaetodon capistratus) and the pebbled butterflyfish (Chaetodon multicinctus) suggested possible effects of lunar phase on spawning activity (Tricas 1986; Colin 1989).

Other species of butterflyfish spawn in smaller groups or monogamous pairs

(Colin 1989; Yabuta 1997). Colin reported pair and 1M:2F sex ratios, seasonal spawning (February-May), and per spawn egg production in three Western Atlantic butterflyfishes: longnose butterflyfish (Chaetodon aculeatus; 2090 eggs/female/spawn), the foureye butterflyfish (Chaetodon capistratus; 3710 eggs/female/spawn) and the banded butterflyfish (Chaetodon striatus). Tricas and Hiramoto (1988) indicated seasonal spawning in pebbled butterflyfish (Chaetodon multicinctus) with a peak in spawning in the spring. However, there was evidence that some individuals spawned in the fall.

Establishment of incubation protocols for an aquaculture candidate is a key component to production. Relatively low fecundity and high mortality of altricial pelagic

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larvae further necessitates this development. Abiotic factors such as photoperiod, temperature and salinity can affect hatching success and/or rate (Rana 1990; Marking et al. 1994; Hart and Purser 1995; Barnes et al. 1998; Chen and Zhang 2001;

Moorhead and Zeng 2010). Aeration is commonly applied during incubation to ensure oxygenation and prevent fouling. In pelagic spawners, aeration keeps eggs in suspension to avoid cohesion. Aeration of demersal eggs is also crucial to prevent fouling. Several studies have shown that pre-incubation chemical treatments such as iodine and peroxide can reduce bacterial and fungal colonization of clutches without harming embryos (Douillet and Holt 1994; Harboe et al. 1994; Salvesen and Vadstein

1995; Barnes et al. 1998; Rach et al. 1998; Small and Wolters 2003; Oono et al. 2007;

Rasowo et al. 2007; Stuart et al. 2010; El-Dakour et al. 2013, Jantrakajorn and

Wongtavatchai 2015).

Butterflyfish are attractive, popular aquarium fish whose diversity (129 species,

12 genera) is seen in the aquarium trade. Many butterflyfish species are noted for their hardiness in captivity. In 2015, the first documented successful culture of any

Chaetodontid, the Klien’s butterfly (Chaetodon klieni) occured (Frank Baensch, Reef

Culture Technologies, LLC). Baensch observed flexion between 18-25 dph and metamorphosis at 93 dph. Various diets of rotifers, copepods, and wild plankton were employed with success only coming via predominant feeding of wild plankton. Shortly thereafter, Baensch successfully cultured another Chaetodontid, the schooling banner fish (Heniochus diphreutes). However, no report of the methods or survival have been published. In 2014, P. crassirostris copepod nauplii were fed to C. miliaris grown to 44 dph, producing flexion at 28 dph (Degidio 2014). Despite, successful culture of C. klieni,

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and H. diphreutes, repeated culture success has not been documented. Service of these recent advancements to the industry or imperiled ecosystems depends on the development of commercially practical culture methods. Commercially available butterflyfish species range in retail value from $20-$190. Considering the diversity and value of butterflyfishes in the aquarium trade, protocols that can be modified and applied to different butterflyfish species would be a milestone for the marine ornamental industry.

Methods

Broodstock Acquisition and Maintenance

Paracanthurus hepatus acquisition and husbandry

P. hepatus broodstock were acquired from Sea World Discovery Cove (Orlando,

FL) and Dallas Aquarium. Broodstock were held in 2000 L white, recirculating tanks equipped with wet/dry bio-filters, 80 W UV sterilizers (Emperor Aquatics, Pottstown,

PA), and 1495 W heater chiller units (Aqua Logic, San Diego, CA). Each tank was plumbed with two drains: a central drain at the tank bottom and a surface skimming drain near the tank perimeter. These systems were equipped with egg collection devices (Figure 4-1). Fish were maintained with a mix of commercially prepared frozen foods including LRS Reef Frenzy (Larry’s Reef Services, Advance, NC), various Hikari products (Hikari Sales USA, Hayward, CA), and Otohime dry feed (Marubeni Nisshin

Feed Co., Tokyo, Japan).

Chaetodon sedentarius acquisition and husbandry

A suspected mating pair of Chaetodon sedentarius were collected off the coast of

Marathon, FL on 05/06/2015 (Dynasty Marine, LLC). The fish were immediately transported to the IRREC facility where they were housed in a culture system identical

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to the one used for P. hepatus broodstock. C. sedentarius were fed a diet similar to that provided to P. hepatus.

Water pump

Water level

Tank

100 µm stainless steel screen

300 µm Nitex

PVC pipe

Water flow

5 gallon bucket

Coarse drain sieve

Figure 4-1. Schematic showing the broodstock husbandry tanks and egg collection units used to house and harvest clutches from Paracanthurus hepatus (Populations 1 and 2) and the Chaetodon sedentarius pair.

Spawning and Fecundity

Sampling methodology

Multiple spawns from a population of P. hepatus, and a pair of C. sedantarius were analyzed for fecundity. Fecundity (viable egg quantity) was estimated by quantifying a volumetric subsample of eggs and measuring the quantity of floating viable eggs produced in a spawn. Sinking eggs were assumed to be non-viable.

Fertilization was determined for both floating and sinking eggs by photographing a subsample of eggs and counting fertilized eggs using a dissecting microscope

(Amscope, Irvine, CA).

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Clutches were collected on the morning after fertilization. Eggs were removed from the egg collector, sieved and rinsed into a graduated cylinder to measure the total volume. Three 25 µL subsamples of floating and sinking eggs were mounted and observed for egg quantity and fertilization from clutches spawned by the P. hepatus population and the pair of C. sedentarius (n=45, 35 respectively). Additional spawns were enumerated with different subsample sizes or by comparing total egg volume to previously observed egg densities. Photoperiods were manipulated (14 light:10 dark) with a timer and a single fluorescent bulb. Water quality parameters (T, Sal, pH, DO) were monitored throughout the experimental period using the aforementioned equipment. Regular water changes and additions of well water (approximately 3 g/L salinity) were used to maintain salinity and prevent the accumulation of nitrogenous wastes.

Statistical analysis

A multiple regression procedure was used to evaluate the effects of lunar phase and natural photoperiod on egg production. Models were considered significant at

P≥0.05. Statistical tests were performed using JMP 13 (Cary, NC).

Experiment VII, Hatching Success in P. hepatus

To evaluate hatching success in P. hepatus, 50 eggs were stocked into six 1-L cups with Nitex (30-40 µm) bottoms. Cups were submerged in a common water bath.

Each cup contained approximately 850 mL of Atlantic Ocean water. Eggs were stocked before 14:00 h (18-20 hpf) on the day following fertilization. Three cups were removed from the water bath the following day (before 42 hpf), allowing water to pass through the screen. Unhatched eggs and larvae were then enumerated. Hatch success was defined as the number of larvae observed divided by the number of unhatched eggs observed.

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Hatching success procedures were repeated for 9 clutches spawned between

09/03/2015 and 09/28/2015. Water quality parameters (T, Sal, pH, DO) were monitored via YSI 556A 1-6 times weekly during the experimental period. TAN and NO2-N were measured via a HACH DR/4000U spectrophotometer twice weekly.

Experiment VIII, Effect of Salinity on Hatch Success in P. hepatus

Experimental design and sampling

An experiment was conducted to evaluate the effects of salinity on hatching performance in P. hepatus. Salinities of 25, 30, 35, and 40 g/L (n=8) of sterilized Atlantic

Ocean water were used as treatments. A clutch of eggs was collected at 05:15 (9-11 hpf) on 08/17/2016. Thirty-two 1 L Nitex bottomed cups were inserted into identically shaped closed bottom cups containing 850 mL of treatment water. From 9-15 hpf, 20 eggs were counted and added to each cup. At 21-23 hpf the cups were removed and contents rinsed. The number of larvae and unhatched eggs were enumerated.

Temperature, salinity, pH and dissolved oxygen were measured in each cup upon completion of the experiment (27 hpf). Water samples were also extracted from each treatment for TAN and NO2-N measurements.

Statistical analysis

Hatch success data were arcsine square root transformed prior to analysis. The data violated the normality assumption. A log transformation of the data ameliorated this violation. These data were then compared with and ANOVA test. Water quality data were compared between treatments via ANOVA with the exception of DO due to violation of the normality assumption and failure to ameliorate this violation with transformations. DO data was compared with a Kruskall-Wallis test. Differences were

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considered significantly different at P≥0.05. Statistical tests were performed using JMP

13 (Cary, NC).

Experiment IX, Temporal Variation in Hatch Success of P. hepatus at Various Temperatures

Experimental design and sampling

Two trials were conducted to determine optimal incubation temperature for P. hepatus. Clutches were harvested from the egg collector between 08:00 and 09:00 on the morning after fertilization (12-14 hpf). In the first trial, eight plastic plates consisting of 12 wells each, were filled with 10 mL NSW (32-35 g/l) and stocked with 10 eggs (15-

16 hpf). Plates were subsequently incubated in a water bath held at 27 ± 1º C. One well plate was removed from the water bath every hour from (19-27 hpf). Larvae and unhatched eggs were counted in each well upon removal and mean hatch percentages were calculated.

An experiment with four temperatures as treatments was conducted using a similar procedure. Four water baths with temperatures of 20, 24, 28, and 32 º C were established using aquarium heaters (Marineland, Spectrum Brands, Blacksburg, VA).

Consistency of temperatures in the water baths were confirmed during and in days preceding this experiment with a HOBO data logger (Onset, Bourne, MA). The spawning event was observed between 19:15 and 19:40 on September 16, 2015. Wells were stocked with 10 eggs and incubation commenced by 15.5 hpf on. Hatching was enumerated in six wells from the 32ºC treatment at 17 and 18 hpf. From 19 to 29 hpf hatching was enumerated in 6 wells per treatment every three hours.

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Statistical analysis

Hatch success data were arcsine square root transformed prior to analysis. Data for the 19 hpf sampling time was log transformed to address a violation of the equal variances assumption. ANOVAs were employed to compare hatching percentage sampling intervals. Differences were considered significantly different at P≥0.05.

Statistical tests were performed using JMP 13 (Cary, NC).

Experiment X, Hatching Success in Chaetodon sedentarius

Methods for this experiment followed the procedures described for Experiment

VII using C. sedentarius eggs. These procedures were repeated for 5 clutches spawned between 09/03/2015 and 09/28/2015.

Experiment XI, Effect of Aeration on Hatching Success in Chaetodon sedentarius

Experimental design and sampling

An experiment was conducted to evaluate the effects of aeration on hatch success of C. sedentarius. A clutch of eggs was harvested between 13:00 and 14:00

(15-19 hpf). Eggs were screened, rinsed, and put into a graduated cylinder and allowed to float to the surface. Three 25 µL subsamples of eggs were removed and enumerated

(mean ± SD=32 ± 6.6 eggs/25 µL). Eggs were then stocked into 10 14.75 L round, white bottom fiberglass tanks by adding 100 µL of floating eggs into each tank. Gentle aeration was applied to five of the culture tanks via a centrally placed air stone (1 x 4 cm). Once stocked, a water flow rate of 0.5 mL/s was applied to all ten tanks through a flexible tube (diameter= 0.6 cm) at the perimeter of the tank. On the morning of

09/02/2015 tanks were slowly drained and larvae and eggs were enumerated. Water quality parameters including temperature, salinity, pH, dissolved oxygen, TAN and NO2-

N were measured via a YSI 556A meter and HACH DR 4000U. Temperature, salinity,

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pH and dissolved oxygen were measured after stocking (18-21 hpf) and in every tank upon completion of the experiment.

Statistical analysis

Hatch success data were arcsine square root transformed prior to analysis. A

2-sample T-test was performed to determine possible differences in hatching success percentages between treatments. Differences were considered significantly different at

P≥0.05. Statistical tests were performed using JMP 13 (Cary, NC).

Larval Culture of Chaetodon sedentarius

A clutch was observed in the egg collector on the morning of 04/14/2016. The clutch was collected and contained 3,800 viable eggs. Upon quantification, the eggs were immediately transferred to a 105 L cylindrical, black-walled, white bottomed, fiberglass tank. The tank was equipped with a central drain for a standard 5.08 cm PVC pipe. The tank was arranged with a peripheral water input adjusted to release inflowing water at a level of 0.5-2 cm above the surface of the water. A single rectangular airstone

(3x3x6 cm) was placed on the bottom of the culture tank directly below the water input.

Gentle aeration was provided during incubation to keep eggs in suspension. No water flow was applied during incubation. On the morning of 04/15/2016 (1 dph), a water exchange rate of 2-5 mL/s was applied. Cessation of water exchange during feeding hours (08:00-09:00) resulted in suboptimal dissolved oxygen levels (4.51 mg/L) being observed at 10:15 on April 18, 2016. Thenceforth, water exchange was continuous at rates ranging from 1-9 mL/s. Flow was reduced or stopped on a couple of occasions due to a shortage in water supply. Water quality parameters were measured (T, Sal, pH,

DO) via a YSI 556A meter at least once daily from 04/15/2016 through 05/08/2016 with

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the exception of 05/02/2016 due to equipment malfunction. Total ammonia nitrogen and

NO2-N were measured 2-6 times weekly via HACH DR/4000.

Water was greened using Chaetoceros sp. and/or T-ISO algae. Algal densities were maintained at approximately 50,000-300,000 cells/mL of culture water. Constant flushing of culture water decreased algal density with time. Water was greened by siphoning stock algae of a known cell density into the culture tank. Algae drips were employed in an effort to maintain algal densities during daylight hours. Positioning of the algae input tube into the stream of aeration and water input provided efficient mixing of the high density stock algae into the culture tank. This method of algal addition also functioned to mitigate possible impacts of temperature and salinity gradients on the larvae when algae and culture water were added.

First feeding began on the morning of 3 dph. At this time aeration was increased.

P. crassirostris nauplii were added once or twice daily at mean concentrations of 4.29 nauplii/mL depending on the productivity of nauplii stocks and residual nauplii in the tank. Aeration was adjusted periodically to facilitate feeding, and ensure adequate oxygenation.

Five to six larvae were subsampled daily for photomicrographic documentation between (1-6 dph). Three to four larvae were subsampled on 04/24/2016, 04/26/2016,

04/28/2016, 04/30/2016 (10, 12, 14, 16 dph). One larva was subsampled on 05/07/2016 and 2 moribund larvae were photographed on 05/09/2016. ImageJ (ImageJ, USA) software was used to determine notochord length, eye diameter, body depth and mouth gape of subsampled larvae.

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1900L/h water pump

Water level

Tank

100 µm stainless steel screen

35 µm Nitex

Flexible tube water deposit line

PVC water withdrawal line

5 gallon bucket

Pool noodle

Figure 4-2. Semi-continuous Parvocalanus crassirostris production system. A) Water pump was controlled using an automated timer such that nauplii were collected from ~0600-0900 daily. Turning the pump on started water flow from the 150 L collection tank to the 2000 L culture tank. As the water level in the culture tank rose above the PVC withdrawal line, water began to circulate between the two tanks. B) and C) Screens allowed for the retaining of copepods within the 35-100 µm size range.

Results

Spawning and Fecundity

Paracanthurus hepatus egg production

The first P. hepatus spawn was observed on July 16, 2015. At least 110 spawning events were documented between July 16, 2015 and February 29, 2016.

Spawns occurred between 17:00 and 20:00, around the time the artificial light phase ended. Total clutch size (floating + sinking eggs) ranged from 1342-33167 eggs (mean

± SD=11416 ± 7475) with a mean egg viability of 68.5% (n=44). A mean of 99.7%

(n=54) of floating eggs were fertilized while 42.1% (n=45) of sinking eggs showed

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evidence of fertilization (division). Estimated viable egg production was 5865 ± 109 eggs (mean ± SD) per spawn (1350-25967, n=110)

A regression model was used to assess the effects of lunar phase, water quality parameters and time since the first spawn on estimated spawn size (Model 1). The model was significant and explained 31.36% (R2=0.3136) of variance in spawn size

(Table 4-1, Model 1). Natural photoperiod was the only factor significantly associated with increased spawn size. Consistency of water quality parameters warranted their removal from the model, and their inclusion in the model resulted in lower R2 value. Due to an artificially maintained and constant photoperiod, and the lack of data representing spawns from a full year, this relationship is likely invalid. The association may more accurately be attributed to the amount of time passed since the first spawn occurred.

That is to say, the fish produced larger spawns as time went on, possibly as more females began spawning or females produced more eggs.

Chaetodon sedentarius egg production

The first C. sedentarius spawn was observed on August 03, 2016. At least 60 spawns were documented between August 03, 2016 and February 29, 2016. Neutral buoyancy of some of the early spawns inhibited enumeration. To address this, salinity was gradually raised in the brood stock tanks via the addition of synthetic sea salt mix.

Total clutch size ranged from 8176-23317 eggs (mean ± SD=13819 ± 4153; n=21) with mean egg viability of 52.8%. A mean of 99.8% (n=20) of floating eggs were fertilized while 15.9% (n=21) of sinking eggs showed evidence of fertilization. Total estimated fecundity was 7356 ± 3794 viable eggs/spawn (522-17163; n=54). Water quality

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parameters were maintained within generally acceptable husbandry ranges throughout the experimental period (Table 4-2).

A multiple regression model (Table 4-1, Model 2) was fitted to evaluate the effects of time since first observed spawn, natural photoperiod, moon phase, and water quality parameters on floating egg production. The model was significant (and explained 29.95% (R2=0.2995) of variance in spawn size. Days since the first observed spawn was the only factor significantly associated with increase in spawn size. None of the tested water quality parameters could be associated with egg production. The consistency at which these parameters were maintained warranted their removal from the model.

Experiments VII and X Hatch Success in Paracanthurus hepatus and Chaetodon sedentarius

Mean hatch success for P. hepatus was 98% (± 0.67). Mean hatch success for

C. sedentarius was 91% (± 3.2).

Experiment VIII, Effect of Salinity on Hatch Success in Paracanthurus hepatus

Hatch success in 25, 30, 35, and 40 g/L sea water was 93.0 ± 0.7%, 95.6 ±

0.0%, 93.0 ± 0.7%, 100 ± 0%, 91.9 ± 0.0% respectively. Contrary to preliminary trials utilizing similar methods, no significant difference among treatments with respect to hatching was detected (P=0.533). Water quality parameters were kept within acceptable ranges (Table 4-4). A statistically significant difference in dissolved oxygen was measured in the 35 and 40 g/L treatments (P=0.002) but was not considered biologically significant. With the exception of salinity, no other significant differences in any of the other water quality parameters were detected. Further evaluations should observe for

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temporal effects of salinity on hatching, as well as the effects of salinity on early larval development and feeding.

Experiment IX, Temporal Variation in Hatch Success of P. hepatus at Various Temperatures

Hatching under normal conditions (26-28ºC) began between 14:30 and 15:30 on the day following fertilization. A 95% hatch success was observed at 20:30 and hatching was complete by 21:30 (Figure 4-5).

At 29-31 hours after fertilization (termination), 100% hatching was observed in the three warmest treatments. The 20ºC (± 2.78) treatment exhibited 97.2% hatching at this time. Hatching was first observed in the 32ºC treatment at 14:30 (14.1%). No hatching was observed in the other four treatments at this time. Hatching percentage was significantly higher in the 32ºC treatment (89.1%) than in the 28ºC treatment at 22 hpf (P=0.0017). No hatching was observed in any of the other treatments at this time.

Hatching percentage at 25 hpf was significantly higher in 32ºC treatment (96.1%) than in the 24ºC treatment (53.1%, [P=0.007]). No hatching was observed in the 20ºC treatment and 100% hatching was observed in the 28ºC treatment at this time. At 28 hpf

91.7% hatching was observed in the 20ºC treatment while 100% hatching was observed in all other treatments. At 00:30 hatching in the 20ºC treatment was 97.8% (Table 4-6).

Experiment XI, Effect of Aeration on Hatching Success in Chaetodon sedentarius

Hatching was first observed on 22:38 of 09/01/2016. A mean hatch rate of 57%

(± 21.8) was observed in the aerated treatment. A mean hatch rate of 71.0% (± 34.8) was observed in the static treatment. No significant difference in hatch success was detected between treatments (P= 0.4518). It has been implied that aeration may reduce

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early survival in C. miliaris (Degidio, unpublished data). The results of this experiment suggest that aeration is not necessary during the incubation of C. sedentarius.

Larval Culture of Chaetodon sedentarius

Chaetodon sedentarius eggs were pelagic, non-adhesive and positively buoyant at salinities of at least 35 g/L. Below this salinity, eggs tended to be neutrally buoyant.

Hatching may not be significantly inhibited by slightly lower salinities. However, during preliminary larval culture trials newly hatched larvae were seen congregated at the bottom of the culture tanks. This generally resulted in mortality of these individuals within the first few days post-hatch. Eggs collected the morning after fertilization were

709 ± 4 µm (693-730) in diameter. The chorion was transparent and a single spherical oil globule, homogenous yolk, and perivitelline space were distinguishable in each specimen (Figure 4-12). Oil globule diameter was 182 ± 2 µm (177-197). Perivitelline space measured 15 ± 3 µm (6-34). Yolk volumes were calculated at 0.4204 ± 0.0200 mm3 (0.390-0.446). Oil globule volumes were calculated at 0.0031 ± 0.0012 mm3

(0.00289-0.0040). Ratios of oil globule volume to egg volume were 0.0169 ± 0.0008

(0.0142-0.0213). Ratios of yolk volume to egg volume were 0.8420 ± 0.0165 (0.7340-

0.8840). Ratios of egg surface area to egg volume were 8.4660 ± 0.0490 (8.2215-

8.6561). Ratios of oil globule surface area to oil globule volume were 33.0840 ± 0.4030

(30.5221-33.9504).

Newly hatched (1 dph) larvae were 2124 ± 33 µm (2017-2203) long (notochord length). Body depth measured 289 ± 22 µm (350-2489). Ventral ellipse shaped yolk sacs were approximately 9-16% of the volume of those observed in embryos. Oil globule volume was 60-80% persistent and positioned at the posterior terminus of the

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yolk sac (Figure 4-13). Eyes were unpigmented and a functional gut had not formed, however, an anus was present in 40% of sampled larvae (n=5).

On 2 dph notochord length and body depth measured 2437 ± 25 µm (2379 -

2496) and 337 ± 13 µm (314-388), respectively. Yolk and oil globule volumes were 1-

2% and 35-45% of the embryonic volume respectively. A gut tract opening at the anus and developing gut tissue were observed in all specimens. However, eyes were still unpigmented and mouths unopened. Pigmented spots appeared on dorsal and ventral fin folds and were prevalent along the ventral portion of the notochord. Ventral and anterior tapering of the posterior region formed a distinct paddle shaped caudal fin

(Figure 4-13).

At 3 dph mouths were fully formed and gut tracts completed. Gape heights measured 296 ± 15 µm (262-332). Eyes were pigmented and larvae were deemed capable of feeding. Minor increases in notochord length from 2-3 dph were observed

(45 ± 16 µm). Body depths were 412 ± 12 µm (392-454). Yolk reserves and oil globules were further reduced to 10-20 and <1% of embryonic volume, respectively. Dorsal and ventral fin folds were extended relative to 2 dph, contributing to increased depth.

No significant growth in notochord length or body depth between 3 and 4 dph was seen. Gape height reached 391 ± 22 µm (304-433). No yolk reserves were visible in observed specimens (n=5). Minor oil globules were present in 3 of the observed specimens and one individual maintained what appeared to be an abnormally large oil reserve (7-71% mean embryonic volume).

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Oil and yolk reserves were absent from all observed specimens on 5 dph. Body depth and notochord length from 4 to 5 dph were again negligible. Gape heights were

421 ± 23 µm (345-454).

On 6 dph, increased notochord length and body depth were more noticeable and reached 2802 ± 94 µm (2496-3057) and 513 ± 15 µm (473-554), respectively. Gape heights were 465 ± 27 µm (386-522).

By 10 dph notochord length had increased by a mean of 176.78 ± 32.10 µm

(386-522) since 6 dph. Dorsal and ventral fin folds were reduced giving the larvae a shallower appearance, especially around the gut, resulting in modest increases in body depth relative to 6 dph (Figure 4-13).

At 14 dph the first evidence of swim bladder inflation was observed (33%).

Pigmentation increased dramatically making it difficult to elucidate differentiation of gut sections. Notochord lengths reached 3555 ± 24 µm (3523-3604), body depths were 646

± 15 µm (615-664), and gape heights were 627 ± 15 µm (598-648). Myomeres were extended and made a fuller appearance.

At 16 dph 67% of sampled larvae had inflated swim bladders.

At 18 dph increased body depth was apparent, particularly in the region from the eye through the gut. At this time thiolichthys plates were beginning to form. Previously reduced finfolds migrated to the posterior, forming dorsal and ventral fin-like masses.

Notochord length, body depth and gape height of the sampled larvae measured 4901,

1279 and 857 µm, respectively.

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Flexion was first seen on 23 dph, with a partially formed hypural plate and notochord extending dorsally at a 45º angle. The thiolichthys plate was fully formed.

Cleavage of the fin folds made for more distinct fin structures. Body depth increased.

Table 4-1. Egg production models for P. hepatus and a pair of C. sedentarius. Statistics are derived only from spawns for which a full analysis or floating and sinking eggs was performed (see methods). P Model Equation R2 Parameter Estimate P-value (model) y = 1062.69 + days since first spawn 40.84 <0.001 1 0.326 <0.001 41.46x constant 1158.43 0.169

y = 188.05 + days since first spawn 21.77 <0.001 2 0.441 <0.001 52.64x constant 4759.2 0.896

Table 4-2. Water quality parameters measured from July 2015 – February 2016 in tanks holding a P. hepatus and C. sedentarius broodstock. Population 1 C. sedentarius T 27.66 ± 0.56 (26.42-29.32) 27.58 ± 0.71 (26.11-29.53) Sal 35.31 ± 0.53 (34.40-36.79) 32.28 ± 2.97 (27.96-36.27) DO 5.77 ± 0.38 (4.57-6.7) 5.94 ± 0.60 (4.49-8.73) pH 8.16 ± 0.20 (7.76-8.53) 8.20 ± 0.39 (5.12-8.37)

Table 4-3. Water quality parameters from bath used to incubate P. hepatus eggs in Experiments VII and X between 09/05/2015 and 09/30/2015. NSW Bath T 26.77 ± 0.76ºC (25.37-28.21) Sal 33.55 ± 0.61 g/L (32.80-34.46) DO 5.62 ± 0.44 mg/L (5.03-6.83) pH 7.88 ± 0.17 (7.30-8.03) TAN 0.06 ± 0.07 mg/L (0-0.14) NO2-N 0.010 ± 0 mg/L (0.003-0.012)

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Table 4-4. Water quality parameters measured in cups used to incubate P. hepatus eggs at one of four salinities of Experiment VIII. 40 g/L 35 g/L 30 g/L 25 g/L 26.36 ± 0.07 26.33 ± 0.09 26.34 ± 0.12 26.33 ± 0.07 T (26.22-26.44) (26.22-26.45) (26.22-26.47) (26.24-26.4)

39.83 ± 0.12 34.96 ± 0.1 29.79 ± 0.32 24.77 ± 0.03 Sal (39.60-39.96) (34.87-35.16) (29.05-30.11) (24.73-24.82)

8.33 ± 0.35 8.21 ± 0.03 8.25 ± 0.03 8.21 ± 0.06 pH (8.19-9.19) (8.18-8.27) (8.19-8.28) (8.09-8.27)

5.64 ± 0.16 5.63 ± 0.19 5.94 ± 0.22 5.93 ± 0.16 DO (5.42-5.87) (5.37-5.91) (5.70-6.39) (5.64-6.18)

TAN 0 0 0 0

NO2-N 0.009 0.014 0.010 0.013

Table 4-5. Water quality parameters measured in tanks used to incubate C. sedentarius eggs with and without aeration in Experiment XI and P-values relating the two treatments. P Value T 23.81ºC ± 0.12 (24.05-23.73) 0.350 Sal 33.48 g/L ± 0.03 (33.53-33.45) 0.320 pH 7.9 ± 0.08 (7.95-7.74) 0.531 DO 5.77 mg/L ± 0.17 (5.89-5.47) 0.650 TAN 0 ± 0 mg/L (0-0) NO2-N 0.005 mg/L ± 0.001 (0.0065 -0.0036) 0. 666

Table 4-6. Water quality parameters as measured throughout the larval culture of C. sedentarius trial. Larval culture Trial BOD 04/13/2016 T 26.528 ± 1.0405 (24.89-28.46) Sal 36.158 ± 0.647 (34.72-36.98) pH 8.16 ± 0.07 (7.97-8.27) DO 5.47 ± 0.50 (4.17-6.34) TAN 0.05 ± 0.05 (0-0.1) NO2-N 0.007 ± 0.004 (0-0.018)

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Table 4-7. Embryo characteristics of C. sedentarius (n=8). Parameter Measurement ED 708 µm ± 4 (693-729) OD 181 µm ± 2. (176-196) OV 0.0030 mm3 ± 0.0001 (0.0029-0.0040) YV 0.1570 mm3 ± 0.0037 (0.1421-0.1753) PS 15 µm ± 3 (7-35) EV 0.1870 mm3 ± 0.0033 (0.1742-0.2041) OD:ED 0.0260 ± 0.0039 (0.0242-0.0277) YV:EV 0.8430 ± 0.0164 (0.7343-0.8842)

full moon 30000

25000

20000

15000

Clutch Size Clutch 10000

5000

0

Jul-15

Jan-16

Oct-15

Sep-15 Feb-16

Dec-15 Aug-15 Nov-15 Figure 4-3. Estimates of clutch size (number of floating eggs) as produced by P. hepatus from July 2015 – February 2016. Orange lines represent full moon days.

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30000

25000

20000

15000

Clutch Size Clutch 10000

5000

0

Jul-15

Jan-16

Oct-15

Sep-15 Feb-16

Dec-15 Aug-15 Nov-15 Figure 4-4. Estimates of clutch size (number of floating eggs) as produced by a pair of C. sedentarius from July 2015 – February 2016. Orange lines represent full moon days.

100% 96% 100% 100% 90% 80% 70% 65% 60%

Hatched 50% 40% 35%

30% Percent 20% 16% 10% 5% 0% 1% 0% 19 20 21 22 23 24 25 26 27 Hours after Fertilization

Figure 4-5. Hatching (% hatched) of P. hepatus at (26-28ºC) from 19-27 hour post- fertilization.

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a a 100 20 90 24 a 80 28 b 70 32 60 50 b 40 30

Percent Hatched Percent 20 10 0 18 19 20 21 22 23 24 25 26 27 28 29 Hours Post Fertilization Figure 4-6. Hatching (%) of P. hepatus from 18 – 29 hours post-fertilization at four different temperatures (ºC).

) 0.0035 3

0.003

0.0025

0.002

0.0015

0.001

0.0005 Oil Reserve Volume (mm Volume Reserve Oil 0 0 1 2 3 4 5 6 dph

Figure 4-7. Mean oil reserve volumes (mm3) measured during larval culture of C. sedentarius.

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0.18

0.16 )

3 0.14

0.12

0.1

0.08

0.06

0.04 Yolk Volume (mm Volume Yolk 0.02

0 0 1 2 3 4 5 6 dph

Figure 4-8. Mean yolk volumes (mm3) measured during larval culture of C. sedentarius.

6000 notochord length total length

5000

4000

3000

2000 Length (µm) Length

1000

0 0 5 10 15 20 25 dph Figure 4-9. Mean length measured during larval culture of C. sedentarius.

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1200

1000

800

600

400

200 Mouth Gape Height (µm) Mouth Height Gape

0 0 5 10 15 20 25 dph

Figure 4-10. Mean mouth gape heights (µm) calculated during larval culture of C. sedentarius.

1800

1600

1400

1200

1000

800

600 Body (um) Body Depth 400

200

0 0 5 10 15 20 25 30 dph

Figure 4-11. Mean body depths (µm) measured during larval culture of C. sedentarius.

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Figure 4-12. Hydrated C. sedentarius eggs approximately 10-14 hours post-fertilization.

Figure 4-13. Larval development of Chaetodon sedentarius. A) 1 dph. B) 2 dph. C) 3 dph larvae with pigmented eyes and open mouth. D) 5 dph larvae with absorbed yolk sac. E) 12 dph. F) 14 dph. G) 16 dph. H) 18 dph larvae entering the thiolichthys stage. I) 23 dph larvae with notochord tip at a 45° angle.

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Discussion

This was the first documented evaluation of captive spawning, fecundity, or egg incubation of P. hepatus and C. sedentarius. This was also the first known report of attempted culture of C. sedentarius. All spawns were volitional and handling of brood was avoided following their acquisition. Captive volitional spawning of P. hepatus and C. sedentarius was possible with maintenance of consistent temperature and photoperiod.

P. Hepatus spawning was consistent with reported harem spawning and timing of spawns (Robertson’s 1983). Timing of spawns was also consistent with Robertson’s

(1983) reports. Eggs were generally found in egg collectors from 18:00-20:00.

Between 07/20/2015 (first spawn) and 02/29/2016, spawns were recorded on

110 of 224 days. Not all spawns were collected and it can be assumed that some spawns went unobserved during this time period as well. Mean fecundity measured

5864.87 ± 109.44 eggs/spawn (1350 – 25,966, n =110). Total egg production during this time was approximately 645,134 eggs.

Captive spawning of P. hepatus is feasible. During conditioning, P. hepatus should be closely observed for aggression. Rejection of what was assumed to be subordinate males was obvious and was characterized by persistent and aggressive attacks by one or more individuals on another. Commonly the rejected fish was forced to the water surface along the edge of the tank. Progression of the attacks was rapid; such that, even with frequent monitoring of populations, hemorrhaging and open wounds may be present at first observation of the aggression. Such a fish should be immediately removed from the population and treated as needed for its condition. This is a means of sexing individuals without handling, assuming that primary aggressors and those being attacked are both males.

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Spawning was attained in two identically housed populations. It appeared that sex ratios, population size, and/or brood age could affect viable egg production.

The pair of C.sedentarius began spawning within three months of being harvested and transferred to a captive environment. Spawning occurred in the middle of the night at approximately 20:00-01:00. C. sedentarius accepted a captive diet shortly after acquisition, and were quickly receptive to a wide array of commercial diets including dry feed (Otohime).

Between 08/03/2015 (first spawn) and 02/29/2016 spawns were recorded on 54 out of 210 days. Not all spawns were collected and it can be assumed that some spawns went unobserved during this time period. Estimated fecundities were 7356 ±

516 eggs per spawn (522 – 17,162, n=54). Total egg production during this time was approximately 397,232 eggs for the pair of C. sedentarius.

To date, all indications are that butterflyfish species practice either monogamous pairing or harem spawning. Descriptions of group spawning include small harems of one male and two females, and larger harems (Colin and Clavijo 1988; Lobel 1988;

Colin 1989; Yabuta and Kawashima 1997; Degidio 2014). Degidio (2014) and Lobel

(1988) described a somewhat semi-paired strategy in C. multicinctus and C. miliaris where spawning between an apparent pair is routinely intruded on by what are suspected to be subordinate males. Degidio (2014) determined that exclusive pairs

(1M:1F) of C. miliaris in captivity did not produce as many eggs as harem groupings

(3M:8F; 10M:11F). Mean viable egg production by the C. sedentarius pair in this study

(7201 eggs/spawn) was much higher than that of 1M:1F (57) and 10M:11F (5435) harems and similar to the 3M:8F (7425) harem used by Degidio (2014). Total egg

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production per fish per spawn was much higher in the C. sedentarius pair (7061.5) than in any grouping of C. miliaris employed by Degidio (75-1066).

For such considerable egg output, the C. sedentarius pair required very little maintenance. Small size (10-13 cm), and paired mating allowed low tank biomass and thus minimal feed input and system maintenance.

This was the first reported evaluation of incubation techniques for any Chaetodon or Paracanthurus species. Hatch success under normal conditions of P. hepatus (98%) and C. sedentarius (91%) provided reference for further evaluation of other factors on hatching, as well as a means to judge clutch performance.

Results of Experiment VIII suggest no significant difference in hatch success of

P. hepatus eggs incubated at 25 g/L (64.4%), 30 (63.1%), 35 (66.3%), and 40 g/L

(63.9%) at approximately 22 hpf. A temporal evaluation of hatching under normal temperature conditions (26-28ºC) was conducted to establish a timeline of hatching in

P. hepatus. P. hepatus eggs began hatching between 14:30 and 15:30 on the day after fertilization (~20 hours post fertilization) and were completed by 20:00-22:30 (~27 hours post fertilization) (Figure 4-5). The results of Experiment IX indicated delayed hatching at 20 and 24ºC, and accelerated hatching at 32ºC. However, overall hatch success was unaffected (97.2-100%).

Despite differences in spawning behavior, larval development of C. sedentarius resembled the descriptions of C. miliaris (Degidio unpublished data). C. miliaris eggs were similar in size to other butterflyfish and hatched in an altricial state. The appearance of functional mouth parts and eye pigmentation in C. sedentarius (3 dph) preceded that of C. miliaris (4 dph). Mouth development and eye pigmentation at 3 dph

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was also seen in C. Nippon and C. modestus (Suzuki et al. 1980; Tanaka et al. 2001).

In contrast, initial gape height measurements of C. sedentarius (262-333 µm on 3 dph) were larger than those of C. miliaris (178-184 µm on 4 dph) (Degidio 2014). Absorption of oil and yolk reserves by 5 dph was relatively consistent with observations in other chaetodontids (Tanaka et al. 2001; Degidio 2014). Degidio (2014) first reported absence of these structures on 7 dph but no larval observations were reported on 5 and 6 dph for

C. miliaris.

Notochord growth of C. sedentarius was similar to that of C. miliaris up to first feeding (2435-2528 µm) and through 23-24 dph (4919 µm). Swim bladder inflation was observed later in C. sedentarius (33% at 14 dph) than in C. miliaris (10% 9 dph).

Inflation may have occurred prior to this as larvae were not sampled from 11-13 dph.

Swim bladder inflation increased (66%) on 16 dph, which was lower than the reported

13 dph inflation percentage for C. miliaris. Increases in body depth in C. sedentarius aligned temporally with similar observations in C. miliaris (Degidio 2014). Signs of flexion and thiolichthys plate formation were discovered earlier in C. sedentarius (23,

18-23 dph) than in C. miliaris (28, 24-26 dph). Extensive mortalities occurred between

16 and 23 dph. This is consistent with such die-offs observed by Degidio (2014) and may be associated with major developmental events.

Egg and larval morphology, and larval development of C. sedentarius seems typical of chaetodontids. While further replicated and more frequent sampling is needed for validation, C. sedentarius may exhibit slightly accelerated development with respect to some growth parameters. This paired with high fecundity suggest C. sedentarius may be a good candidate for marine ornamental aquaculture.

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Recently, P. hepatus and C. sedentarius were successfully cultured. Further research is required to facilitate their commercialization. Further development of live feed productivity, as well as economic analysis of production costs and establishment of profitability thresholds will be necessary. However, high fecundity and resilience of embryos to abiotic fluctuations are characteristics critical to future commercial production of these species.

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CHAPTER 5 CONCLUSION

Experiments conducted evaluated critical questions for production efficiency of candidate species. The necessity and benefits of P. crassirostris copepods as a first feed to T. goodei and G. speciosus was determined. Development of T. goodei to the juvenile stage was documented for the first time. Fecundity, and spawning conditions were documented for C. sedentarius and P. hepatus. Growth and development of C. sedentarius to 25 dph was documented for the first time.

Experiment III was the first evaluation of stocking density done on palometa. This experiment was performed on fish shortly after metamorphosis. While high levels of

TAN and NO2-N were detected, no significant differences in the measurements of these or in growth or survival were detected among treatments. Unaffected growth and total survival among treatments suggests palometa possess characteristics conducive to commercial aquaculture. Growth and survival of T. goodei at a range of stocking densities further supports their aquaculture candidacy.

Experiments IV.I-VI were the first reported salinity tolerance trials with palometa.

Palometa had 100% survival following acute transfer from 35 g/L to 6 g/L sea water.

Complete mortality in Experiment V indicated NaCl was not an adequate substitute for sea water with this species. A minimum acclimation time of 6 h ensured 100% survival of T. goodei in 3 g/L sea water (48 h post-acclimation). Acclimation times of 0 and 3 hours yielded 33 and 67% survival, respectively. Such tolerance may also facilitate this species’ potential for pond based culture.

This is the first known report of fecundity measurements for P. hepatus. A population of P. hepatus was conditioned and began spawning within 18 months of their

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transfer from public aquaria. After their initial spawning event, populations spawned frequently and regularly, often for several days in a row. Regular spawning may be interrupted by periods of one to several weeks without spawns. Conditioning occurred under a simple feeding regime and required minimal manipulation of the brood animals.

With maintenance of temperature and photoperiod, year round spawning was observed

(July 2015-August 2016). Future replicated broodstock studies could investigate effects of population size, sex ratio, and abiotic factors on fecundity and spawn frequency.

One pair of Chaetodon sedentarius was acquired and spawning began within three months of collection from the wild. Labor involved in conditioning and general maintenance of brood was minimal. Brood fish responded to a wide array of diets.

Spawns occurred regularly with spawning events often taking place 2-3 times weekly.

Photoperiod and temperature consistency permitted year round spawning of C. sedentarius. Regular spawning may be interrupted by periods of one to several weeks without spawning. Consistent spawning of C. sedentarius at temperatures above 26ºC contrasts reports by Colin (1988). There was no apparent effect of lunar phase on spawn occurrence or size.

Fecundity and fertilization percentage of floating eggs was high. Total fecundity was very similar to the most productive sex ratio (3M:8F) utilized by Degidio (2014) with millitseed butterflyfish.

Continuous volitional spawning, ease of conditioning, and low maintenance husbandry of C. sedentarius and P. hepatus are characteristics of these species that show their potential for future commercial production.

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BIOGRAPHICAL SKETCH

Carter Cyr was born in 1992 in Portland, Maine. In his youth, he participated in several different team sports and enjoyed hunting and fishing in northern Maine.

Upon graduating high school, Carter attended Roger Williams University where he majored in marine biology. He worked as a research assistant for 1.5 years in the

Roger Williams University Wet Lab and also volunteered at the New England Aquarium

Rescue and Rehabilitation Center in Quincy, Massachusetts while working as a recreational fisheries observer for the Massachusetts Division of Marine Fisheries.

After graduating with a Bachelor of Science degree from Roger Williams

University, Carter accepted a fully funded graduate assistantship under Dr. Cortney Ohs at the University of Florida. In August of 2016, Carter accepted a full-time position as a

Biological Scientist II at the University of Florida’s Shellfish Aquaculture Extension office where he has worked while completing his Master of Science degree.

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