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EVALUATION OF LARVAL CULTURE METHODS FOR THREE MARINE FINFISH, MONODACTYLUS SEBAE, LAGODON RHOMBOIDES, AND SELAR CRUMENOPHTHALMUS

DANIEL JAMES ELEFANTE

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

2019

To my wife, Amanda, who has been a constant source of support and encouragement during the challenges of graduate school and life. This work is also dedicated to my parents, who have always loved and encouraged me to reach my goals.

ACKNOWLEDGMENTS

I would like to thank my advisor Dr. Cortney Ohs for his support and guidance during this process. Through the duration of the writing process, he kept me motivated and made sure that I was ready to defend the work that I had completed. Besides my advisor, I would like to thank the rest of my committee: Dr. Ruth Francis-Floyd and Dr.

Charles Cichra, for their encouragement, helpful comments, and constructive questions.

I would also like to thank everyone at the University of Florida IRREC: Dr. Jason

Broach, Andrew Palau, Scott Grabe, Audrey Beaney, John Marcellus, and Bryan

Danson. Without their help, none of this would have been possible.

Last but not the least; I would like to thank my parents and my wife for their constant encouragement and support throughout this process.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

LIST OF ABBREVIATIONS ...... 9

ABSTRACT ...... 10

CHAPTER

1 INTRODUCTION ...... 12

2 THE CULTURE OF MONODACTYLUS SEBAE ...... 14

Methods ...... 15 Broodstock Conditioning and Egg Collection ...... 15 Egg Stocking Density ...... 16 Salinity Effects on Egg Hatching Success ...... 18 Larval Rearing Experiments ...... 18 Stocking Eggs vs Stocking Larvae ...... 20 Effects of Different Live Feeds on Larval Growth and Survival...... 22 Statistical Analysis ...... 24 Results ...... 24 Egg Stocking Density ...... 24 Salinity Effects on Egg Hatching Success ...... 25 Stocking Eggs vs Stocking Larvae ...... 26 Effects of Different Live Feeds on Larval Growth and Survival...... 27 Discussion ...... 31

3 CULTURE OF LAGODON RHOMBOIDES ...... 36

Methods ...... 37 Broodstock Conditioning and Egg Collection ...... 37 Water Quality ...... 39 Larval Culture Experiments ...... 39 Live Food and Algae Culture Preparation...... 40 Effects of Different Live Feeds on Larval Growth and Survival...... 41 Effects of Different Stocking Densities on Larval Growth and Survival ...... 42 Statistical Analysis ...... 43 Results ...... 44 Effects of Different Live Feeds on Larval Growth and Survival...... 44 Effects of Different Stocking Densities on Larval Growth and Survival ...... 47

Discussion ...... 51

4 CULTURE OF SELAR CRUMENOPHTHALMUS ...... 54

Methods ...... 55 Broodstock Conditioning and Egg Collection ...... 55 Larval Culture Experiments ...... 55 Green Water Effects on Growth and Survival ...... 56 Aeration Intensity Effects on Growth and Survival ...... 56 Effects of Different Live Feeds on Larval Growth and Survival...... 57 Results ...... 58 Green Water Effects on Growth and Survival ...... 58 Aeration Effects on Growth and Survival ...... 59 Effects of Different Live Feeds on Larval Growth and Survival...... 59 Discussion ...... 61

5 CONCLUSION ...... 64

LIST OF REFERENCES ...... 68

BIOGRAPHICAL SKETCH ...... 76

LIST OF TABLES

Table page

2-1 Mean standard length ±SD (mm) of five M. sebae larvae randomly selected from each replicate tank and each treatment...... 29

3-1 Standard length measurements (mm) of five Lagodon rhomboides larvae randomly selected from each tank in each treatment...... 46

3-2 Mean standard length and body depth measurements (mm)...... 49

4-1 Mean standard length (mm) of 8 Selar crumenophthalmus larvae selected at random from each tank in each treatment (n=16)...... 59

4-2 Mean standard length (mm) of 8 Selar crumenophthalmus larvae selected at random from each tank in each treatment (n=16)...... 60

LIST OF FIGURES

Figure page

2-1 Egg salinity and stocking density experimental setup...... 17

2-2 Experimental system utilized in all larval experiments described in this master’s thesis...... 19

2-3 The larvae of M. sebae on a Sedgewick Rafter counting cell...... 21

2-4 Feeding schedule for the live feed experiment with M. sebae over 9 days post hatch...... 23

2-5 Mean hatching percentage ±SD of M. sebae eggs in stocking densities of 10, 20, 30, and 40/L at 2 dph (p = 0.0212)...... 25

2-6 Mean hatching percentage of M. sebae eggs incubated in water salinity of 0, 5, 10, 15, 20, 30, 40, 45, and 50 g/L...... 26

2-7 Mean percent survival of all M. sebae stocked either as eggs or larvae after 5 days...... 27

2-8 Survival (mean ± SE) of M. sebae larvae after 9 days...... 28

2-9 Mean standard length (mm) measurements of 5 M. sebae larvae selected at random from each of the 6 tanks in each treatment...... 30

3-1 Feeding schedule for live feed experiment conducted with pinfish over 11 days post hatch...... 42

3-2 Survival (Mean ± SD) of Lagodon rhomboides larvae at 11 dph...... 45

3-3 Percent survival at 11dph of Lagodon rhomboides larvae for each of the four densities tested based on 5 replicate tanks...... 48

3-4 Mean standard length (mm) and body depth (mm) of five Lagodon rhomboides larvae selected at random from each tank in each stocking density treatment (40, 60, 80, 100/L) at 0, 6, and 11 dph...... 50

4-1 Feeding schedule for live feed experiment conducted with goggle eye over 5 days post hatch...... 58

LIST OF ABBREVIATIONS dph Days post hatch. The day on which an egg hatches is considered 0 days post hatch and each twenty-four-hour period following hatching will add one day to this value.

IRREC Indian River Research and Education Center

UMEH University of Miami Experimental Hatchery

µm Micrometer

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

EVALUATION OF LARVAL CULTURE METHODS FOR THREE MARINE FINFISH, MONODACTYLUS SEBAE, LAGODON RHOMBOIDES, AND SELAR CRUMENOPHTHALMUS By

Daniel James Elefante

May 2019

Chair: Cortney Ohs Major: Fisheries and Aquatic Sciences

Most aquaculturists obtain their stock as fingerlings, because of this, they know little about the lives of their fish prior to this developmental stage. The environmental conditions that larvae are cultured in can have significant long-term effects on growth and survival. Experiments were conducted to determine factors that impact the larval development of three fish species: Mono Sebae (Monodactylus sebae), Pinfish

(Langodon rhomboides), and Goggle Eye (Selar crumenophthalmus).

The effects stocking densities and salinities have on egg hatching success of M. sebae were evaluated. Four stocking densities were examined (10, 20, 30, and 40/L).

Results indicated that the two lowest stocking densities had significantly higher hatching success. Nine salinities of embryo incubation water (0, 5, 10, 15, 20, 30, 40, 45, and 50 g/L) were evaluated and hatching success was determined. Results indicated that salinities from 5 to 15 g/L had significantly higher hatching success than the other salinities. A third experiment was conducted with M. sebae to evaluate the developmental effects of feeding one of three live feed organisms (Parvocalanus crassostris nauplii, Pseudodioptamus pelagicus nauplii, and the rotifer Brachionus

plicatilus). Results indicated there were no significant differences in larval survival among treatments.

Larvae of the marine baitfish Pinfish (Langodon rhomboides) were cultured at four stocking densities (40, 60, 80, and 100 larvae/L) and the effects on growth and survival were determined. No significant differences in survival were observed, but in one of two trials, the lowest stocking density resulted in greater larval growth. No significant differences in survival were observed in larvae fed two different live feeds for

11 days, but larvae in both trials had increased growth rates when fed a diet of live P. crassirostris copepod nauplii.

Several experiments were performed with Selar crumenophthalmus larvae to determine the effects of aeration, the presence of phytoplankton, and two different live feed blends. However, only the experiment involving live feeds was completed due to early complete mortality in all other trials. A mixed diet of P. crassirostris copepods and

Brachionus plicatilis. rotifers resulted in a statistically significant increase in growth

(larval length) and survival.

CHAPTER 1 INTRODUCTION

Fish species are generally classified as foodfish, baitfish, and ornamental fish species to reflect how they are most commonly utilized by humans. The aquaculture industry is steadily growing in the United States and worldwide, and has experienced large increases in the last two decades (USDA 2014). In the same period of time, the world’s demand for ornamental aquarium fishes has also grown into a U.S. $15-30 billion a year industry (Penning et al. 2009). As a direct result of research conducted at the University of Florida IRREC, culture methods have been established for the brackish water ornamental fish M. sebae, and production in Florida is now well over 200,000 individuals per year. This amounts to a yearly farm gate value of about $100,000 as of

2014 (Pers. Comm. Dr. Cortney Ohs 2018).

The production of marine baitfish for recreational and commercial fishing is an area of aquaculture that is expanding in the United States. Sales of marine bait and tackle are a part of a $2.3 billion dollar a year industry. As of 2011, the estimated revenue generated by the sale of saltwater fishing bait in the United States was approximately $546 million dollars (U.S. Department of Interior, U.S. Fish and Wildlife

Service, & U.S. Department of Commerce, U.S. Census Bureau 2011, Ohs et al. 2018).

Wild caught baitfish are seasonal which leaves fishermen with limited bait or with the wrong size of fish available at certain times of the year (Staugler 2016). Having a constant market supply of a seasonal baitfish species, at an assortment of desirable sizes, could allow a farmer to remain in the market year round and take advantage of seasonal supply shortages. While pinfish can net between $1 and $1.50 per fish, goggle eye can command prices well over $100 USD per dozen during a shortage in the wild or

when supplying billfish tournaments. The ornamental and baitfish species evaluated in the following studies are commercially important to the southern United States may allow for significant potential profits for farmers.

CHAPTER 2 THE CULTURE OF MONODACTYLUS SEBAE

Not all fish species are suitable for aquaculture due to inherent traits of the fish themselves. Species with a small mouth, overly long larval phase, difficult or expensive food requirements, or extreme sensitivity to water quality changes are some of these traits. These traits also have the potential to increase costs of production (Webber and

Riordan 1976, Liao and Huang 2000). For marine ornamental aquaculture, the larval phase is a period of high mortality due to live food requirements and unknown culture requirements (Fiksen et al. 2002, Herath and Atapaththu 2013). Decisions, made during the larval phase, can affect growth, development, and survival well into the future

(Hamre et al. 2013).

Further research is needed to determine the causes and methods to overcome high mortality during the larval phase of any potential aquaculture candidate. Something as simple as a 10% change in stocking density could increase the survival to metamorphosis of a fish species and consequently may increase the profit margin (El-

Sayed 2002). Identifying easily obtainable and less-expensive food items that have equal ability to provide larval fish with adequate nutrition could result in a greatly improved cost to profit ratio (Le Ruyet et al. 1993, Støttrup 2000). The experiments conducted in this study will focus on the larval phase of fish production and will evaluate the effects of food quality and quantity, egg and larval stocking densities, and water quality on egg hatching success, growth and survival.

The Mono Sebae, Monodactylus sebae, is one of six species that belong to the

Monodactylidae family. Common names for the species include Sebae Mono, African

Moony, and the Striped Fingerfish. It is commonly marketed as a brackish water fish to

the aquarium industry which limits its market size since brackish aquariums only represent 7% of the market. It has a laterally compressed body shape and is primarily silver in color with four long, black, vertical stripes that stretch from the dorsal to the ventral side of the fish; one crosses over the eye, another over the operculum, the next occurs from the rear of the dorsal fin to the rear of the anal fins, and the last crosses the caudal peduncle (Panfili et al. 2006; Gning et al. 2008). Prior to research at the

University of Florida, almost all M. sebae sold in the aquarium industry were collected from their natural habitat in coastal western Africa.

M. sebae is euryhaline and can survive in salinities from 0 g/L to 40 g/L. They are catadromous, meaning they migrate from rivers to estuaries to spawn; all recorded spawning events have been at salinities greater than 19 g/L (Akatso et al. 1977, Panfili et al. 2006, Gning et al. 2008). This salinity tolerance of M. sebae could have effects on eggs and larvae. Nothing is reported about egg and larval salinity tolerance for this species.

When investigating a new fish species for aquaculture, it is crucial to optimize each step of the culture process beginning with spawning and then hatching methods for eggs. In this study, we investigated the effects of several factors on egg hatching success and larval survival to first feeding in an effort to determine which parameters were best.

Methods

Broodstock Conditioning and Egg Collection

M. sebae broodstock were imported by a wholesaler and stocked into ponds at the University of Florida Tropical Aquaculture Laboratory (UF-TAL) in 2009 and held for about nine months until they reached maturity (approximately 10 cm total length). The

broodstock were then moved to the University of Florida Indian River Research and

Education Center (UF-IRREC) and were housed in several rectangular 2000-L tanks equipped with a 3-horsepower pump, a 200-L trickle biofilter, a 145 watt UV sterilizer, and three airstones. Brood systems were filled with a mixture of sterilized natural seawater from the Atlantic Ocean and dechlorinated tap water to attain a salinity between 19 and 28 g/L, temperature of 26°C, and a 12:12 L:D photoperiod were used to simulate the fish’s natural, spawning habitat. A commercially available pelleted diet consisted of a 2.0 mm slow sinking pellet (Zeigler Bros. Inc., Gardners, PA, USA, 50% protein, 15% fat, 2% fiber, 12% moisture, and 8% ash) was fed once daily to satiation.

During spawning experiments, this maintenance diet was supplemented with frozen chopped krill (approximately 5 cm in length when whole), Euphausia superba, fed once daily to satiation.

Each day, a floating egg collector (Ohs et al. 2018) in each tank was checked for newly spawned eggs (Emata et al. 1994). Spawned eggs were rinsed out of the collectors with water taken directly from the broodstock tanks and placed into 3-L containers, then volumetrically quantified in a 500-mL graduated cylinder. After adding eggs, the graduated cylinder was topped off to 500-mL with 35-g/L filtered seawater to ensure that the eggs remained buoyant. All floating eggs were assumed to be viable and were stocked into a cylindrical, aerated, 110-L fiberglass tank.

Egg Stocking Density

The optimal stocking density of eggs during incubation can affect hatching success. In this experiment, different egg stocking densities (10, 20, 30, and 40/L) were counted by hand and stocked directly into identical 100-µm screen bottomed 1-L (Top

Diameter: 11.4 cm, Bottom Diameter: 8.4 cm, Height: 14.6 cm) clear plastic containers

placed inside of an identical solid bottomed cup filled with 800 mL of water per treatment for a total of 5 reps of each treatment (Figure 2-1). Each cup was then submerged into a water bath filled with sterile 35-g/L seawater and kept at a constant temperature of 26.2 °C ± 0.02 by an ambient climate control system (insulated room with air conditioning unit). A B

Figure 2-1. Egg salinity and stocking density experimental setup. All egg experiments were performed in the cups displayed in photo A. In photo A, the screen bottom cup (left) was placed inside of the solid bottom cup (right). The complete experimental setup is displayed in photo B. Photos courtesy of Daniel Elefante.

A floating foam grid ensured that each cup remained floating in the bath at the same level. Each cup was aerated gently with its own identical (1.27 cm x 4.5 cm) airstone placed at the bottom/center with an airflow rate of 0.3 to 1.7 L³/min. These incubation methods were maintained until all eggs hatched and the larvae had reached first feeding at 2 dph. After hatching, the total number of surviving larvae in each cup were hand counted and photos of five larvae from each cup were taken to determine

final mean larval standard length. Water quality (temperature, salinity, dissolved oxygen, and pH) measurements were taken daily from each individual cup to ensure that there were no major changes in water quality through the duration of the experiment.

Salinity Effects on Egg Hatching Success

To determine the effects that different salinities have on hatching success, eggs were stocked in the same manner as the previously documented egg stocking density of 20 eggs/L. Nine replicates of the following salinities, 0, 5, 10, 15, 20, 30, 40 g/L and 5 replicates of the following salinities, 45 and 50 g/L were added at a volume of 800 mL volumes identical 100-µm screen bottom 1-L (Top Diameter: 11.4 cm, Bottom Diameter:

8.4 cm, Height: 14.6 cm) clear plastic containers resting inside of an identical solid- bottom cup per treatment. Each cup was then submerged into a water bath filled with sterile 35-g/L seawater and kept at a constant temperature of 23.3 °C ± 0.005 by an ambient climate control system (insulated room with air conditioning unit). A floating foam grid ensured that each cup remained floating in the bath at the same level. Each cup was aerated gently with its own identical (1.27 cm x 4.5 cm) airstone placed at the bottom/center with an airflow rate of 0.3 to 1.7 L³/min. These incubation methods were maintained until all eggs hatched and the larvae had reached first feeding at 2 dph. The total numbers of surviving larvae in each cup were hand counted and photos of 5 larvae from each cup were taken as a subsample of the hatched eggs. Water quality

(temperature, salinity, dissolved oxygen, and pH) measurements were recorded once daily from each cup (a total of 9 measurements per day for 4 days).

Larval Rearing Experiments

All experiments involving larvae were performed in the same flow-through experimental system, which consisted of 26 identical cylindrical fiberglass 19-L tanks

containing 15-L of water. Each was equipped with internal screened standpipes made of a 35.56-cm length of 2.54-cm ID PVC pipe with 15-20, 1.27-cm holes drilled into each.

To retain or flush out different sized food particles while always retaining larvae, standpipes were constructed with two different sizes of nylon screen, 35 µm and 100

µm.

Figure 2-2. Experimental system utilized in all larval experiments described in this master’s thesis. Photo courtesy of Daniel Elefante.

Each of the tanks was supplied with air from a ceramic airstone (1.27 cm x 4.5 cm) placed in the center next to the standpipe. Airflow was adjusted through the use of a plastic L-valve above each tank and dissolved oxygen content was maintained at 5.45

± 0.5 mg/L. Each tank contained one adjustable water input tube (0.48-cm I.D. vinyl tubing) equipped with a plastic L-valve and a roller clamp for fine adjustments of the

flowrate that supplied sterile seawater. All the seawater came from the same reservoir to ensure that there was no variation in water quality among treatment tanks. The flow rate of each tank was adjusted twice daily, once in the morning and again twelve hours later. To flush uneaten food particles at night, a higher flow rate of approximately 90 mL/min (three exchanges in 24 h) was used. To retain food particles during the day, a lower flow rate of approximately 20 mL/min (one exchange in 24 h) was used. The temperature of each tank was maintained at 26°C ± 0.39 by an ambient climate control system (insulated room with air conditioning unit) (Figure 2-2).

Stocking Eggs vs Stocking Larvae

Newly collected eggs were rinsed out of their collectors and placed inside of a

500-mL graduated cylinder which contained 35 g/L sterile seawater. Ten 1-mL samples of eggs were used to volumetrically estimate the number of eggs spawned. All collected eggs were then transferred to a 3-L plastic container with similar water quality to that of the broodstock tanks before being stocked into a 110-L cylindrical tank where they were again volumetrically sub-sampled (the mean of 10, 30-mL samples) and stocked into six cylindrical 19-L experimental tanks filled with 15-L of sterilized seawater at the previously determined optimal density of 20 eggs/L. After the eggs, remaining in the

110-L tank hatched, the newly hatched larvae were quantified again (the mean of 10,

30-mL samples) to determine the hatching percentage. The newly hatched larvae were then stocked into six cylindrical 15-L fiberglass tanks at the previously determined optimal density of 20/L.

Figure 2-3. The larvae of M. sebae on a Sedgewick Rafter counting cell. The red line shows the standard length measured of larvae. The blue line shows body depth of the larvae. All larvae measured throughout this master’s thesis were measured using the same guidelines. Photo courtesy of Daniel Elefante.

To determine potential effects of stocking method on long term survival and growth of larvae, larvae were fed and evaluated for 9 days post hatch. At the onset of first feeding, both treatments were fed s-strain rotifers (Brachionus sp.) enriched with

Ori-Green (Skretting Canada, Inc., Bayside, New Brunswick, Canada) at a density of

15/mL for five days. The rotifers were cultured in 200-L cylindrical tanks containing 25 g/L salinity water and were fed Nannochloropsis sp. paste (Nanno 3600, Reed

Mariculture, Inc. Campbell, CA) daily. Prior to feeding, rotifers were enriched with Ori-

Green in a 26-L bucket for three hours at 25-27 °C according to the manufacturer's instructions. For 5 days, water quality in the system was maintained constant and daily feedings of enriched rotifers were administered to each tank. At the end of this period, the number of surviving larvae in each tank was counted by hand and photos were taken of 25 larvae from each tank in each treatment on a sedgewick rafter counting cell

to provide a grid for measurement reference (Figure 2-3). The standard length and body depth of each larvae was then measured through the use of ImageJ analysis software

(Rossband 2014).

Effects of Different Live Feeds on Larval Growth and Survival

The standard reference diet of enriched rotifers is the most widely used for larval production of marine finfish (Aragão et al. 2004). This reference diet was compared to two other diets, copepod nauplii of Parvocalanus crassirostris or Pseudodiaptomus pelagicus, using six replicate tanks per treatment. To prevent cross contamination, each live food organism was cultured in a separate section of the hatchery at UF-IRREC.

P. crassirostris was cultured in 200-L, cylindrical, fiberglass tanks filled with 25-

26 g/L seawater and housed in an isolated, temperature-controlled room maintained at

28 °C. Each tank received 3-6 L of live Tisochrysis lutea (Tiso) algae at a density of approximately 250,000 cells/mL daily and was moderately aerated. Every fifteen days, each tank had a complete water exchange and was inoculated with newly collected nauplii from one of the other tanks. P. pelagicus was cultured in a similar manner, but without full water exchanges. These copepods were instead given a 20% water exchange every 10 days. Nauplii stages of copepods were harvested with air lift collectors (Ohs et al. 2019).

All rotifers utilized in this experiment were housed, cared for, and enriched by the same method described in Chapter 2. All live feeds (Copepods and Rotifers) were counted using the following method. Live food orgnisms were first homogenized in a 3-L plastic container, three 1-mL samples were taken, and each sample was placed in its own 30-mL cup. These samples were then diluted (5:1) with 35-g/L salinity filtered

seawater, sterilized with iodine to stain the food organisms, and counted inside of a sedgwick rafter counting chamber.

Figure 2-4. Feeding schedule for the live feed experiment with M. sebae over 9 days post hatch. Different treatments are labeled A, B, and C above.

Prior to stocking, larvae were first quantified by taking 10, 30-mL samples to determine the mean larval density/mL. Larvae were stocked at 20/L using previously described methods. At the onset of first feeding (1 dph), approximately 250,000 cells/mL of live Tiso algae were added once daily to every tank to green the water. Four subsamples were taken from a 19-L algae bottle each day to determine the mean algal density with the aid of a hemaocytometer (Perez 2006). Three treatment diets were examined, labeled A, B, and C in Figure 2-4. Each live food organism was fed at the same density of approximately 10/mL once daily until 5 dph. At 5 dph, both copepod treatments (A and B) were weaned onto rotifers and received 5/mL copepod nauplii and

5/mL enriched rotifers. From 6-9 dph, all treatments received 10/mL enriched rotifers once daily. At the end of the 10-day experiment, growth in standard length was

measured by taking photos of 30 larvae from each tank, and all surviving larvae from each tank were hand counted.

Statistical Analysis

All collected data for hatching success percentage, larval growth, and larval survival were statistically analyzed with JMP Statistical Analysis Software (JMP®,

Version 10. SAS Institute, Inc., Cary, NC) to determine any difference in treatment means. Experimental data were first assessed for normality through the use of a histogram plot and a Shapiro-Wilkes test. After determining that data were normal, experiments involving more than two treatments were tested for homogeneity of variance with a Levene’s Test. Next, statistical significance was tested through the use of an ANOVA. If the ANOVA indicated a statistically significant difference among treatments, a Tukey’s HSD analysis was used to determine which treatment means were significantly different. Experiments involving only two treatments were statistically analyzed with a T-test. All percentage data were arcsine square root transformed prior to statistical analysis. A P-value ≤0.05 was considered statistically significant for all analyses. All numerical data are presented as the treatment mean ± standard deviation.

Results

Egg Stocking Density

Mean egg hatching success was significantly different (p = 0.0212) among the four stocking densities ranging from 44.4% to 65%. The lowest egg stocking density of

10 eggs/L had significantly higher hatching percentage (65 ± 18.54) than the 30 and 40 eggs/L treatments, but did not vary significantly from the 20 eggs/L treatment. The highest two egg stocking densities of 30 and 40 eggs/L resulted in the lowest two

hatching percentages (44.17% and 44.38%, respectively) although they did not vary from the 20 eggs/L treatment (Figure 2-5).

100

90 A

80 70 AB 60 B B 50 40 30

20 Mean Hatching Percentage HatchingMean 10 0 10/L 20/L 30/L 40/L Treatment

Figure 2-5. Mean hatching percentage ±SD of M. sebae eggs in stocking densities of 10, 20, 30, and 40/L at 2 dph (p = 0.0212). Means with the same letters are not significantly different.

Salinity Effects on Egg Hatching Success

Egg hatching success was significantly different (p = 0.0001) among treatment salinities. Hatching percentage ranged from 0% in the 50 g/L salinity to 97.9% in the 5 g/L salinity treatment (Table 2-6). Standard deviation of the total number of eggs hatched was relatively low within all treatments. In general, lower salinities (0 g/L–30 g/L) resulted in higher rates of hatching success than higher salinities.

Egg hatching success in the 40 and 50 g/L treatments was significantly less than all other treatments (Figure 2-6). Hatching success in the 5 g/L treatment was significantly higher than all other treatments except for the 15 g/L treatment, which was

not statistically different from the 0 g/L and 10 through 30 g/L treatments. The 0, 10, 20, and 30 g/L treatments were not statistically different than the 45 g/L treatment.

Figure 2-6. Mean hatching percentage of M. sebae eggs incubated in water salinity of 0, 5, 10, 15, 20, 30, 40, 45, and 50 g/L.

Stocking Eggs vs Stocking Larvae

Survival after a 5-day period was not statistically different (p = 0.5920) between the stocked egg and stocked larvae treatments. Larvae had a slightly higher numerical survival percentage of 77 ± 9.2% v.s. 75.8 ± 8.7% for eggs (Figure 2-7).

100 90 A A

80 70 60 50 40 30 Mean Larval Survival Larval Mean 20 10 0 Larvae Egg Treatment

Figure 2-7. Mean percent survival of all M. sebae stocked either as eggs or larvae after 5 days. Means with the same letters are not significantly different.

Water quality parameters in the larval stocking treatment tanks were maintained at the following values throughout the experiment: temperature, 23.21 ± 0.05 °C; salinity, 28.40 ± 0.44 g/L; dissolved oxygen, 7.1 ± 0.05 g/L; pH, 7.94 ± 0.07. Water quality parameters for the egg stocking treatment were maintained at the following values throughout the experiment: temperature, 23.18 ± 0.02 °C; salinity, 28.23 ± 0.04 g/L; dissolved oxygen, 7.0 ± 0.05 g/L; pH, 7.95 ± 0.07. No significant differences were found in temperature (p = 0.3482), salinity (p = 0.4347), dissolved oxygen (p = 0.1166), or pH (p = 0.8346) between treatments for the duration of the experiment.

Effects of Different Live Feeds on Larval Growth and Survival

Survival after 9 days was not statistically significantly different (p = 0.8785) among treatments. Larvae, fed Brachionus sp. exclusively, had the highest numerical percent survival at 64.17 ± 11.92%. The treatment fed P. pelagicus exclusively for 5

days had the lowest numerical percent survival (50.90 ± 15.86%) of all treatment diets

(Figure 2-8).

100 A 90 A 80 A 70 60 50 40 30 Mean Larval Survival Larval Mean 20 10 0 P. crass. P. pelag. B. plica. Treatment

Figure 2-8. Survival (mean ± SE) of M. sebae larvae after 9 days. Treatments were as follows: Parvo (larvae fed P. crassirostris exclusively for 5 days), Pseudo (larvae fed P. pelagicus exclusively for 5 days), and Roti (larvae fed Brachionus sp. exclusively for the experiment's duration). Means with the same letters are not significantly different.

Growth (Standard Length) over 9 days was not significantly different among treatments. Larvae, fed P. pelagicus exclusively for 5 days, had the greatest numerical increase in standard length. The treatment with the lowest numerical increase in standard length was the treatment fed Brachionus sp. exclusively. Initially, treatments fed Brachionus sp. exclusively exhibited higher rates of growth after two days post hatch before slowing down on the final day. The treatment fed P. crassirostris had the lowest rate of growth through the first two measurement days before becoming the treatment with the highest rate of growth on the final day of the study (Table 2-1).

Table 2-1. Mean standard length ±SD (mm) of five M. sebae larvae randomly selected from each replicate tank and each treatment. Mean growth per day is based on the difference between sequential larva length measurements. All data points were means of 30 total length measurements per treatment. Treatments were as follows: P. crass. (larvae fed P. crassirostris exclusively for 5 days), P. pelag. (larvae fed P. pelagicus exclusively for 5 days), B. plica. (larvae fed Brachionus sp. exclusively for the duration of the experiment). Significnce is indicated for treatment means of the same day. Day Treatment Significance Mean Standard Dev. Growth Per Day (mm) 0 All - 1.6621 0.1591 - 3 P. crass. B 2.4218 0.2511 0.2532 3 P. pelag. B 2.4277 0.2920 0.2552 3 B. plic. A 2.4416 0.2467 0.2598 6 P. crass. A 3.0129 0.4277 0.1970 6 P. pelag. A 3.2217 0.4258 0.2647 6 B. plic. A 3.5546 0.3568 0.3710 9 P. crass. AB 4.0904 0.3535 0.3592 9 P. pelag. A 4.2794 0.4230 0.3526 9 B. plica. B 3.9810 0.3214 0.1421

There was a significant difference (p = 0.0001) in final standard length measurement among treatments at 3 dph. Larvae fed Brachionus sp. treatment had a significantly higher mean standard length than both of the treatments fed either species of copepod. No significant differences (p = 0.9568) were found in mean standard length among treatments at 6 dph. At 9dph, the P. pelagicus treatment had a significantly higher final mean standard length (p = 0.0085) than the treatment fed Brachionus sp. exclusively.

4.5000

4.0000

3.5000

3.0000 P. crass. P. pelag. 2.5000 B. plica.

2.0000 Mean standard length (mm) length standardMean

1.5000 0 2 4 6 8 10 Days post hatch

Figure 2-9. Mean standard length (mm) measurements of 5 M. sebae larvae selected at random from each of the 6 tanks in each treatment. Data were means of 30 standard length (mm) measurements per treatment. Treatments were as follows: P. crass. (larvae fed P. crassirostris exclusively for 5 days), P. pelag. (larvae fed P. pelagicus exclusively for 5 days), B. plica. (larvae fed Brachionus sp. exclusively for the duration of the experiment).

Water quality parameters for the P. crassirostris treatment were maintained at the following values throughout the experiment: temperature, 28.4 ± 0.5 °C; salinity,

28.5 ± 0.02 g/L; dissolved oxygen, 5.4 ± 0.5 g/L; pH, 7.9 ± 0.01. Water quality parameters for the P. pelagicus treatment were maintained at the following values throughout the experiment: temperature, 28.6 ± 0.32 °C; salinity, 28.5 ± 0.03 g/L; dissolved oxygen, 5.4 ± 0.53 g/L; pH, 8 ± 0.01. Water quality parameters for the

Brachionus sp. treatment were maintained at the following values throughout the experiment: temperature, 28.5 ± 0.36 °C; salinity, 28.5 ± 0.02 g/L; dissolved oxygen, 5.5

± 0.4 g/L; pH, 8 ± 0.01. No significant differences among treatments were found with temperature (p = 0.7610), salinity (p = 0.9636), dissolved oxygen (p = 0.8831), or pH (p

= 0.5932).

Discussion

When stocking eggs of the M. sebae to attain high hatching success, results indicate that lower stocking densities may result in higher survival. The lower stocking density of 10 eggs/L resulted in a significantly higher hatching success than higher densities of 30 and 40 eggs/L (Figure 2-5). Lower stocking densities also resulted in a higher hatching success in other studies with bay anchovy and lined sole (Houde 1977).

Although not experienced in the current study, higher stocking densities can result in higher levels of nitrogenous waste from hatching eggs and less available dissolved oxygen for the newly hatched larvae (Montero et al. 1999, Szczepkowski et al. 2010,

Miyashima et al. 2012). Lower stocking densities will require more tank space and a larger footprint in a hatchery, and may lower the number of marketable fish because of space limitations. Commercial breeders of this species will have to weigh the costs and benefits of choosing higher hatching success versus the ability to stock more eggs in less space.

Recently, further research has investigated the hatching success of eggs transported distances at higher densities. The methods described in these studies could be used to increase hatch success independent of transportation. High water salinity has been found to decrease bacterial infection mortality in artificially incubated eggs of the sand goby, Pomatoschistus minutus, a brackish water species, but only at higher densities (Lehtonen and Kvarnemo, 2015). A combination of these two egg-based studies similar to those performed in the current study with increasing salinity and egg density may yield similar results as those with M. sebae, and should be investigated in the future. The use of pH stabilizers has also been reported to allow higher egg stocking densities to be transported successfully (Stuart et al. 2018). Since the current

experiments were performed in stagnant systems, it is unlikely that the small changes in salinity and pH recorded could have negatively affected the hatch success of the more densely stocked treatments. Performing these experiments again in a flow-through system may improve water quality and may allow for higher stocking densities.

M. sebae have a wide salinity tolerance. It is documented that this fish moves from lower salinity estuaries, where it usually resides, into full-strength salinity ocean water during spawning season (Akatso et al. 1977). Results of the salinity experiments outlined above, however suggest that eggs hatch with greater success in lower salinities. Many species of marine fish, including gobies and anchovies that spawn in marine environments with variable salinities, also exhibit this trait (Beck et al. 2001;

Mookkan et al. 2014). The eggs are released into a marine environment during spawning and are carried by currents to the lower salinity water of nearby estuaries where they settle and hatch (Lasker et al. 1972; Norcross and Shaw 1984). Eggs in all tested salinities from 0 to 30 g/L did result in hatching success and survival percentage to first feeding between 70.80 ± 18.5 and 97.9 ± 4.40% (Figure 2-6). This may be because lower salinity water has a higher capacity for dissolved oxygen and may inhibit the growth of detrimental bacteria and fungi (Hoar and Randall 1969; Stuart et al. 2018).

A large range of salinity tolerance is a beneficial trait for a potential candidate species for aquaculture. A higher hatching success and juvenile survival post hatch in freshwater was observed in Gulf killifish, Fundulus grandis, possibly due to unfavorable conditions that the freshwater created for bacteria and other detrimental microbes to proliferate (Ramee and Allen 2016). Eggs of another brackish water pufferfish species,

Takifugu flavidus, were found to have a similar hatching percentage in salinities from 5

g/L to 45 g/L (Zhang et al. 2010). The positive effects that lower salinity water has on egg hatching success could also lower costs for hatcheries that utilize artificial salt to make seawater.

Producers and researchers must make a decision regarding when to stock larvae to maximize survival. Stocking newly hatched larvae into a culture system is useful because the hatching success is already known. The disadvantage of stocking newly hatched larvae is that they are much more delicate than eggs, and can be easily damaged by handling methods. Using newly collected eggs to stock culture tanks does not allow for an accurate assessment of hatching success, but will prevent larvae from being damaged during the stocking procedure.

It was hypothesized that, due to handling stress on the fragile, newly hatched larvae, it might be advantageous to stock eggs directly. Within a few hours of fertilization, eggs of pelagic fish seem to become relatively resistant to mechanical stressors like handling (Bunn et al. 2000). Larvae of M. sebae have been shown to be fairly hardy, but larval fish in general seem to be more negatively impacted by handling stress than eggs (Pers. Comm. Scott Grabe 2014). However, results of the current experiment did not support improved hatch and survival when stocking using either method (Figure 2-7).

Understanding how the stocking method affects larval survival is important.

Stocking eggs into an experimental system makes calculating the hatching percentage of the fish difficult, but it is thought to decrease stress-related complications in the hatched larvae (Bunn et al. 2000). Stocking larvae into a system post hatch allows a culturist to more accurately estimate the number of larvae in each tank, but may cause

unnecessary stress to the larvae (Pers. Comm. Scott Grabe 2014). The positives and negatives of each method are mitigated when dealing with a fish species that is more resistant to handling stress like M. sebae. An aquaculture candidate that exhibits a higher than average tolerance for handling related stressors is a benefit to the culturist.

When several different live foods were compared, it seemed that the most commonly used larval food (Brachionus sp.) had the highest larval survival (64.17 ±

11.92%) (Figure 2-8). Copepods, since the improvement of their culture techniques

(VanderLugt and Lenz 2009), have largely been found to improve larval growth and survival in ornamental and foodfish species alike (Schipp et al. 2006, Ajiboye et al.

2011). However, some species of fish respond better to rotifers at first feeding than any other type of feed, even if copepods are also offered (Wilcox et al. 2006, Beack and

Turingan 2007, Maehre et al. 2013). This may be because of the ease at which rotifers are captured by less agile larvae fish or because rotifers are a better fit for the mouth gape of the larval fish (Kraul 2006).

Overall larval growth was greater in the treatments fed copepods than those fed rotifers. Treatments fed P. pelagicus, the species of copepod with the larger naupliar stage, showed significantly greater overall growth than the treatment fed rotifers exclusively (Table 2-1). This could be due to the superior nutritional and fatty acid profile of copepods or ease of digestion (Shields et al. 1999, Van der Meeren et al. 2009).

There are many benefits from a production standpoint for a fish that exhibits an acceptable survival and growth percentage when offered rotifers at first feeding. Overall, rotifers require less time to culture, are more resistant to changes in environmental parameters, and can be cultured at much higher densities in less space than copepods

(Dhont et al. 2013). Rotifers also readily accept prepared foods and algal concentrates, which can eliminate the need for dedicated live phytoplankton culture in a hatchery. The cost savings gained from feeding rotifers may outweigh the increased growth rate that copepods can provide to a culturist (Suantika et al. 2003).

CHAPTER 3 CULTURE OF LAGODON RHOMBOIDES

The pinfish, Lagodon rhomboides, is a member of the Sparidae family that is a widely used baitfish in the southeastern United States and is the most popular bait in FL for near shore, offshore, and bottom fishing (Ohs et al. 2017). Pinfish are silver colored with 6 dark vertical bars and alternating yellow and blue stripes that run down the length of the oval shaped body. Pinfish are sexually mature at 1 to 2 years of age and are known to spawn several times during fall and winter months. This spawning season can be artificially lengthened through the use of chillers to maintain lower water temperature in broodstock tanks. Pinfish can grow to lengths of 30 cm, but fish between 7.5 and 15 cm are preferred by anglers (Ohs et al. 2011).

The aquaculture potential of pinfish has received much attention in the past decade. Pinfish can tolerate a wide range of temperatures (10-30 °C) and a wide range of salinities (0-75 g/L) (Ohs et al. 2011). They can also be cultured at relatively high densities and are known for their hardiness as baitfish by anglers. They will accept a wide range of food items and rapidly grow to their relatively small market size that takes approximately 90 days to reach. This decreases time to market and return on investment (Broach et al. 2015).

When investigating a new fish species for aquaculture, it is crucial to optimize each step of the culture process. A fish’s larval phase is often the time period associated with the highest mortalities due to increased nutrient demand, overcrowding, and the completion of several major morphological changes. Therefore, the effects of stocking density and several larval feeding regimens on the growth and survival of pinfish larvae were evaluated.

Methods

Broodstock Conditioning and Egg Collection

In trial 1, approximately 100 pinfish broodstock were collected in January 2009 by commercial fishermen (J & J Bait, LLC, Indialantic, Florida) from the Indian River

Lagoon near Sebastian, Florida, USA and transported to the University of Florida Indian

River Research and Education Center (UF-IRREC) in Fort Pierce, Florida. These fish were quarantined and housed as described by DiMaggio (2012) were help for up to 4 years. In June 2013, eight fish were moved from a greenhouse, to inside the hatchery building and stocked into each of four cylindrical 2000-L tanks. To determine the sex of the broodstock and ensure an even sex ratio in each spawning tank, pressure was applied to the sides of the coelom, anterior to the urogenital opening, to force milt from mature male fish and in some cases, eggs from female fish. Any specimen that could not be sexed in this manner was cannulated with a Teflon catheter tube (0.97-mm inside diameter, 1.27-mm outside diameter). This tube was inserted into the urogenital opening of anaesthetized fish (tricaine methanesulfonate, MS-222, at 100 mg/L), and a sample was removed via suction. All collected samples were viewed under a dissecting microscope to determine sex and the size and stage of egg maturation.

Each broodstock tank was equipped with a 3.2-amp pump, a 200-L trickle biofilter, a 145-watt UV sterilizer, and three airstones to ensure adequate dissolved oxygen content. These tanks were covered with a cone lid to block out ambient light and illuminated with a single 60-watt compact fluorescent bulb used to control photoperiod.

An Aqua Logic Delta Star Chiller (1.5HP 230V DS-10) was used to manipulate water temperature and mimic spawning conditions. Each tank was also equipped with a surface skimming upwelling egg collector (Broach et al. 2016) that used 500-µm screen

to retain spawned eggs and keep them in suspension until they could be collected and quantified.

All brood systems were maintained with a mixture of sterilized natural seawater from the Atlantic Ocean and sterilized well water to attain a salinity of approximately 34 g/L. The temperature in the tanks was maintained at 23 °C. A 12:12 hour light:dark photocycle was used. A commercially available pelleted diet consisted of a 2.0-mm slow sinking pellet (Zeigler Bros., Inc., Gardners, PA, USA, 50% protein, 15% fat, 2% fiber,

12% moisture, and 8% ash) and was fed once daily to satiation. During spawning experiments, the maintenance diet was supplemented with frozen chopped krill (approx.

5 cm in length when whole), Euphausia superba, and frozen chopped squid, Loligo duvauceli, each fed once daily to apparent satiation.

Each day, the egg collectors in all tanks were checked for newly spawned eggs.

When spawns occurred, eggs were removed from the egg collectors and placed into 3-L containers, then volumetrically quantified in a 500-mL graduated cylinder. After adding eggs, the graduated cylinder was topped off to the 500-mL mark with 35-g/L filtered seawater to ensure that the eggs remained buoyant. All floating eggs were assumed to be viable and stocked into a cylindrical, 110-L fiberglass tank to be used for experimentation.

The broodstock used in trial 2, were collected in January 2012 by commercial fishermen (J & J Bait, LLC, Indialantic, Florida) from the Indian River Lagoon near

Sebastian, Florida, USA and moved to the Florida Oceanographic Society in Stuart,

Florida. Approximately 100 fish were acclimated and stocked into a 2,270-L, opaque, cylindrical tank equipped with flow-through filtered seawater from the Atlantic Ocean.

These fish were provided with the same diet as the broodfish in the previous experiment, fed once daily to apparent satiation. Eggs were collected through the use of a 100-µm filter sock attached to the surface skimming filter outflow of the tank.

Water Quality

A YSI 556 multiparameter meter (YSI, Inc., Yellow Springs, Ohio, USA) was used to measure salinity (g/L), temperature (°C), pH, and dissolved oxygen (mg/L) in one tank from each treatment daily for a total of four measurements per day. Water was removed from each tank through the bottom drain to ensure that no larvae were removed. After testing water quality, excess water was returned to the tank that it was removed from to ensure that minimal changes were made to tank volumes. At 0, 6, and

11 dph, a 50-mL sample was taken from every tank and refrigerated in a sterile, 50-mL polypropylene centrifuge tube for measuring total ammonia nitrogen (TAN) and nitrite- nitrogen. These parameters were measured with a HACH DR 4000 spectrophotometer

(Hach Company, Loveland, Colorado, USA) within 24 h of collection.

Larval Culture Experiments

All larval experiments were conducted in the same flow-through experimental system (Figure 2-2). This system consisted of 26 identical 15-L cylindrical fiberglass tanks with internal screen standpipes made of a 35.6-cm length of 2.5-cm ID PVC pipe with 15-20, 1.3-cm holes drilled in each. To retain or flush out different sized food particles while always retaining larvae, standpipes were covered with nylon screens, either 35-µm and 100-µm-diameter mesh. Each of the tanks was supplied with air through a single ceramic airstone (1.3-cm diameter by 4.5-cm long) positioned next to the standpipe. Airflow was adjusted with a plastic L-valve for each tank and dissolved oxygen was maintained. Each tank contained one adjustable water input tube (0.48-cm

I.D. vinyl tubing) equipped with a plastic L-valve and a roller clamp for fine adjustments to the flowrate that supplied sterile seawater to each tank. All of the water came from the same reservoir to ensure that there was no variation in water quality among replicant tanks. The flow rate of each tank was adjusted twice daily, once in the morning and once in the late afternoon. To flush uneaten food out during the night, a higher flow rate of approximately 90 mL/min. (three exchanges in 24 h) was used. To retain food particles during the day, a lower flow rate of approximately 20 mL/min (one exchange in

24 h) was used. The temperature of each tank was maintained in a climate controlled room.

Live Food and Algae Culture Preparation

Each copepod species was cultured in a separate area of the hatchery at UF-

IRREC Aquaculture Research Facility. Nauplii of the copepod species P. crassirostris and P. pelagicus utilized in the following experiments were cultured using methods identical to those discussed in the methods section of Chapter 2. No enrichment was used with either species of copepod nauplii before they were fed to the larvae.

All rotifers utilized in this experiment were housed, cared for, and enriched in the manner discussed in the methods section of Chapter 2. All live feeds were counted with an identical method. All collected food organisms were first homogenized in a 3-L plastic container, three 1-mL samples were taken, and each sample was placed in its own 30-mL cup. These samples were then diluted (5:1) with 35-g/L filtered seawater, sterilized with iodine to stain the organisms, and counted inside of a Sedgwick Rafter counting chamber. Four samples were taken from a 19-L algae bottle each day to determine the mean algal cell density with the aid of a compound microscope and hemaocytometer.

Effects of Different Live Feeds on Larval Growth and Survival

Larvae were first quantified by taking 10, 30-mL samples homogenized with air to determine the mean larval density per mL before being stocked into the experimental system at a density of 40/L. Ten tanks were stocked, with five replicate tanks per treatment. At the onset of first feeding (1 dph), five randomly selected larval tanks were supplied with once daily feedings of 10/mL enriched rotifers and the five remaining tanks were supplied with once daily feedings of 10/mL P. crassirostris nauplii.

P. crassirostris was cultured in 00-L, cylindrical, fiberglass tanks filled with 5-

g L salinity water and housed in a temperature-controlled room maintained at .

Each tank had 3-6 L of live Tisochrysis lutea (Tiso) algae added at a density of approximately 250,000 cells/mL daily and were moderately aerated. Every 15 days, each tank was given a complete water exchange and inoculated with newly collected nauplii from one of the other tanks. Nauplii were collected from the tanks every morning with the use of an airlift collector (Cassiano et al. 2015) that separated nauplii from other developmental stages within the tank. All material retained by the collector was then passed through a 55-µm sieve to ensure that only nauplii were retained. Nauplii were then held in a gently aerated 3-L plastic container filled with 26-g/L-salinity filtered seawater until they were fed. All collected food organisms were first homogenized in a

3-L plastic container, three 1-mL samples were taken, and each sample was placed in its own 30-mL cup. These samples were then diluted (5:1) with 35-g/L filtered seawater, sterilized with iodine to stain the live food organisms, and counted inside of a Sedgwick

Rafter counting chamber (1 mL total volume) to determine density/mL. This was used to accurately determine the numbers of live food organisms fed.

Figure 3-1. Feeding schedule for live feed experiment conducted with pinfish over 11 days post hatch. Different treatments are labeled A and B above.

Approximately 250,000 cells/mL of live Tisochrysis lutea (Tiso) algae were added once daily to every larval culture tank to green the water for the duration of the experiment. Two treatment diets were examined, labeled A and B in Figure 3-1. At 5 dph in treatment A, tanks receiving only P. crassirostris nauplii were fed once daily at a density of 5/mL of each nauplii and enriched rotifers. There was no change in the feed supplied to treatment B, the rotifer only treatment. From 6 dph until the final day of the experiment at 11 dph, all tanks in both treatments received once daily feedings of 10/mL enriched rotifers. At the end of the 11-day experiment, all surviving larvae were hand counted and photographed on a Sedgwick Rafter cell for measurement of total length.

Effects of Different Stocking Densities on Larval Growth and Survival

Newly collected eggs were rinsed out of their collectors and placed inside of a

500-mL graduated cylinder with 35 g/L filtered seawater. Ten, 1-mL samples were taken from the cylinder to determine the number of eggs spawned. All collected eggs were then transferred to a 3-L plastic container with similar water quality as in the broodstock tanks. They were then stocked into a 110-L cylindrical tank with gentle aeration until

hatch. After all eggs were hatched, the larvae were volumetrically sub-sampled (the mean of 10, 30 mL samples) and stocked into 20 cylindrical 19-L fiberglass tanks filled with 15-L of filtered seawater at four different treatment densities (40, 60, 80, and 100 larvae/L) with 5 replicates for each density.

At the onset of first feeding, all treatments were fed s-strain rotifers (Brachionus sp.) enriched with Ori-Green at a density of 10/mL daily for the duration of the experiment. All photos of larvae were taken on a Sedgewick Rafter counting cell (Figure

2-3). At 0 dph, photos were taken of 25 larvae from the 110-L tank before stocking the larvae into the experimental system. At 6 dph, photos were taken of 5 randomly selected larvae from each tank in each treatment. At 11 dph, the final day of the experiment, photos were taken of a subsample of 25 randomly selected larvae from each tank in each treatment. All surviving larvae in each tank were then hand counted.

The standard length and body depth of each larvae was then photographed and measured with ImageJ analysis software (Rossband 2014).

Statistical Analysis

All data for hatching success percentage, larval growth, and larval survival were statistically analyzed with JMP Statistical Analysis Software (JMP®, Version 10. SAS

Institute, Inc., Cary, NC) to determine differences among treatment means.

Experimental data were first assessed for normality through the use of a histogram plot and Shapiro-Wilkes test. After determining the data were normal, experiments involving more than two treatments were tested for homogeneity of variance with a Levene’s

Test. Next, statistical significance was tested through the use of an ANOVA. If the

ANOVA indicated a statistically significant difference among treatments, a Tukey’s HSD analysis was used to determine which treatments were significantly different.

Experiments involving only two treatments were statistically analyzed with a T-test. All percentage data were arcsine square root transformed prior to statistical analysis. A P- value ≤0.05 was considered statistically significant for all analyses. All numerical data are presented as the treatment mean ± standard deviation.

Results

Effects of Different Live Feeds on Larval Growth and Survival

Trial 1 was conducted from March 15, 2014 to March 25, 2014. Trial 2 was conducted from March 27, 2014 to April 7, 2014. Survival to 11 dph was not statistically significantly different (p = 0.7097) between treatments, but one treatment had numerically higher survival in each of the two trials. Overall, percent survival was higher in both treatments during trial 2 than trial 1. The percent survival of the copepod treatment in both trials was similar with a difference of only 2.1%, while the difference between trials for the rotifer treatment was 22.2%. In addition to the higher difference between trials, a high SD of 20.7 was present in the rotifer treatment for trial 1 (Figure 3-

2).

100

Trial Trial

80 1 2

60 A A 40 A A

0 Mean Larval Survival Percentage Survival Larval Mean Rotifers Copepods Rotifers Copepods -20 Treatment

Figure 3-2. Survival (Mean ± SD) of Lagodon rhomboides larvae at 11 dph. Survival percentage is based on an initial mean stocking density of 40 larvae/L. Means with the same letters are not significantly different.

Growth (standard length) over an 11-day period was similar between treatments with larvae fed P. crassirostris having the greatest standard length at 11dph in trial 1, as well as the greatest increase in body depth between days 6 and 11 in both trials. Larvae fed rotifers had the lowest standard deviation from the mean in all measurements except for the body depth measurements in trial 1. Larvae in the copepod treatment were larger than those in the rotifer treatment in terms of standard length at 11 dph in both trials (Table 3-1).

Table 3-1. Standard length measurements (mm) of five Lagodon rhomboides larvae randomly selected from each tank in each treatment. Growth per day was mean growth per day based on the difference between sequential measurments. All data points are means of 25 length measurements (mm) per treatment. Treatments were: Copepod (larvae fed P. crassirostris exclusively for 4 days then rotifers) and Rotifer (larvae fed Brachionus sp. exclusively for the experiment). Standard Length Body Depth Growth/Day Growth/Day Trial dph Treatment (mm) (mm) (Length) (mm) (Depth) (mm) 1 0 All 1.75 ± 0.07 0.49 ± 0.06 - - 6 Rotifer 2.98 ± 0.26 0.49 ± 0.08 1.23 0 6 Copepod 2.91 ± 0.31 0.46 ± 0.07 1.16 0.03 11 Rotifer 3.63 ± 0.38 0.63 ± 0.11 0.65 0.14 11 Copepod 3.64 ± 0.41 0.63 ± 0.10 0.73 0.17 2 0 All 2.49 ± 0.1 0.44 ± 0.0 - - 6 Rotifer 3.03 ± 0.2 0.47 ± 0.0 0.54 0.03 6 Copepod 3.23 ± 0.2 0.45 ± 0.0 0.74 0.01 11 Rotifer 3.70 ± 0.6 0.69 ± 0.1 0.67 0.22 11 Copepod 3.89 ± 0.6 0.73 ± 0.2 0.66 0.28

In trial 1, there was a significant difference in standard lengths at 11 dph between treatments (p= 0.0189). There was no significant difference in body depth at 11 dph between treatments (p= 0.1558).

In trial 2, there was a significant difference in standard length between treatments

(p= 0.0103). Body depth was significantly different between treatments (p= 0.0400).

In trial 1, mean ± SD of water quality parameters for the P. crassirostris treatment were: temperature 23.6 ± 0.46 °C, salinity 35.1 ± 0.5 g/L, dissolved oxygen 6.6 ± 0.18 g/L, and pH 7.9 ± 0.8. Mean water quality parameters ±SD for the rotifer treatment were: temperature 23.7 ± 0.4 °C, salinity 35 ± 0.6 g/L, dissolved oxygen 6.6 ± 0.2 g/L, and pH 7.8 ± 0.1. No significant differences were found in temperature (p = 0.7916), salinity (p = 0.8111), dissolved oxygen (p = 0.6755), or pH (p = 0.0795) between treatments for the duration of the experiment.

In trial 2, mean ± SD of water quality parameters for the P. crassirostris treatment were: temperature 23.5 ± 0.38 °C, salinity 34.4 ± 0.6 g/L, dissolved oxygen 6.8 ± 0.15 g/L, and pH 8 ± 0.1. Mean water quality parameters ±SD for the rotifer treatment were: temperature 23.5 ± 0.4 °C, salinity 34.5 ± 0.6 g/L, dissolved oxygen 6.8 ± 0.2 g/L, and pH 8 ± 0.1. No significant differences were found in temperature (p = 0.8370), salinity (p

= 0.9102), dissolved oxygen (p = 0.1923), or pH (p = 0.3221) between treatments for the duration of the experiment.

Effects of Different Stocking Densities on Larval Growth and Survival

In trial 1, survival after 11 days was similar among the four treatments with a difference of only 2.89% between the highest and lowest mean percent survival. The highest stocking density of 100 larvae/L had the highest percent survival and lowest deviation from the mean, while the lowest stocking density had the lowest percent survival and highest deviation from the mean (Figure 3-3).

100 Trial 100

Trial 90 1 2 80 80 70 60 60 A 50 A 40 AB 40 AB A A A 30 B 20 20

Mean Larval Survival Percentage Survival Larval Mean 10 0 Mean Larval Survival Percentage Survival Larval Mean 40/L 60/L 80/L 100/L 0 40/L 60/L 80/L 100/L -20 Larvae per Liter Larvae per Liter

Figure 3-3. Percent survival at 11dph of Lagodon rhomboides larvae for each of the four densities tested based on 5 replicate tanks. Survival percentage was based on an initial mean egg stocking density that differed among density treatments (40, 60, 80, 100/L). Means with the same letters were not significantly different.

There were no significant differences (p=0.7017) in survival percentage among treatments in trial 1. There was a significant difference in trial 2 (p = 0.0364) among treatments.

Standard length at 11 dph was not significantly different (p = 0.4018) among stocking densities in trial 1 (Table 3-2). Body depth at 11 dph was not significantly different (p = 0.8364) among stocking densities in trial 1. In trial 1, larvae stocked at a density of 60/L displayed the largest increase in standard length and body depth, and larvae stocked at a density of 80/L resulted in the smallest increase in standard length.

Standard length at 11 dph was significantly different (p = 0.0006) among treatments in trial 2 (Table 3-2). Growth in body depth at 11 dph was significantly different (p = 0.0317) among treatments in trial 2. Larvae stocked at a density of 40/L

had the greatest standard length and body depth, and larvae stocked at a density of

80/L displayed the smallest standard length and body depth.

Table 3-2. Mean standard length and body depth measurements (mm). Growth per day was mean growth per day based on the difference between the previous and current mean larval length measurements. All data points were means of 25 measurements (mm). Means with the same letters are not significantly different among treatments within the day and trial.

Treatment Standard Body Depth Trial dph Significance Significance (#/L) Length (mm) (mm)

1 0 All 1.75 ± 0.07 - 0.49 ± 0.06 - 6 40 2.98 ± 0.26 A 0.49 ± 0.08 A 6 60 2.97 ± 0.29 A 0.48 ± 0.06 A 6 80 2.95 ± 0.24 A 0.48 ± 0.07 A 6 100 2.89 ± 0.28 A 0.46 ± 0.07 A 11 40 3.55 ± 0.46 A 0.61 ± 0.12 A 11 60 3.68 ± 0.42 A 0.64 ± 0.11 A 11 80 3.61 ± 0.45 A 0.62 ± 0.11 A 11 100 3.62 ± 0.45 A 0.61 ± 0.12 A 2 0 All 2.49 ± 0.10 - 0.44 ± 0.03 - 6 40 3.03 ± 0.16 A 0.47 ± 0.05 A 6 60 2.92 ± 0.15 A 0.47 ± 0.05 A 6 80 2.96 ± 0.13 A 0.47 ± 0.04 A 6 100 3.01 ± 0.19 A 0.46 ± 0.05 A 11 40 3.70 ± 0.55 A 0.69 ± 0.14 A 11 60 3.63 ± 0.52 AB 0.68 ± 0.13 AB 11 80 3.46 ± 0.47 BC 0.65 ± 0.12 AB 11 100 3.52 ± 0.46 C 0.66 ± 0.11 B

In trial 1, there was no significant difference in mean standard length among treatments at 6 dph or 11 dph (p = 0.0809; p = 0.1456). There was no significant difference in mean body depth among treatments at 6 dph or 11dph (p = 0.5463; p =

0.1798).

In trial 2, there was a significant difference in mean standard length among treatments at 11 dph (p = 0.0006), but not at 6 dph (p = 0.0518). There was a significant

difference in mean body depth among treatments at 11 dph (p = 0.0317), but not at 6 dph (p = 0.1375).

Figure 3-4. Mean standard length (mm) and body depth (mm) of five Lagodon rhomboides larvae selected at random from each tank in each stocking density treatment (40, 60, 80, 100/L) at 0, 6, and 11 dph. A and B are trial 1 data. C and D are trial 2 data. All data are means of 25 length measurements (mm) per treatment.

In trial 1, mean ± SD of water quality parameters between treatments were: temperature 23.6 ± 0.4 °C, salinity 35.2 ± 0.4 g/L, dissolved oxygen 6.7 ± 0.2 g/L, and pH 7.9 ± 0.1. No significant differences were found in mean temperature (p = 0.9355),

salinity (p = 0.9575), dissolved oxygen (p = 0.8980), or pH (p = 0.5392) between treatments for the duration of the experiment.

In trial 2, mean ± SD of water quality parameters between treatments were: temperature 23.8 ± 0.4 °C, salinity 34.2 ± 0.2 g/L, dissolved oxygen 6.3 ± 0.2 g/L, and pH 7.9 ± 0.1. No significant differences were found in mean temperature (p = 0.9930), salinity (p = 0.9745), dissolved oxygen (p = 0.9798), or pH (p = 0.6030) between treatments for the duration of the experiment.

Discussion

The type and quantity of food fed to larvae within the first few days of first feeding has a major impact on survival of many larval fish. Often, fish larvae fed copepods at first feeding show increased growth and a higher survival percentage during the larval phase. In the current study, pinfish fed the copepod, P. crassirostris at first feeding, did not exhibit significantly higher survival than those fed exclusively the S-strain rotifer

Brachionus sp.

Although the use of copepods seems to significantly improve the growth and survival of many species including pink snapper (Pagrus auratus), Atlantic cod (Gadus morhua), and Clark's anemonefish (Amphiprion clarkii), enriched rotifers are still the preferred choice for most commercial hatcheries (Payne et al. 2001; Imsland et al.

2006; Olivotto et al. 2008). Copepods are often only used if feeding rotifers to larval fish at first feeding is not successful or does not result in suitable survival. If no significant growth or survival differences are observed in fish fed copepods at first feeding, rotifers will be the preferred choice for most culturists because they are more easily cultured than copepods in large numbers. The more labor intensive culture methods involved in

growing most copepod species could offset minor increases in growth or survival that that may occur with fish larvae (Kraul 2006).

In the two experiments, eggs were obtained from different broodstock. In general, survival was higher in the second experiment. A possible explanation for this discrepancy between trials may have been related to variable quality in the diets fed to broodstock in the experiment. Trial 1 took place after the beginning of spawning season for the broodstock, so several weeks had passed before these fish were weaned on to the high EFA (essential fatty acid) diet typically given to broodstock for several weeks prior to this time period. The broodstock in trial 2 had been provided a high EFA diet for several weeks prior to the beginning of their spawning season because they were part of a pilot-size fish hatchery that depended on large, high quality spawns. The importance of a consistent, high quality EFA component in the diet of brood fish is well documented, as the addition of EFAs to fish feed can result in an improvement in egg quality and hatch percentage (Navas et al. 1997; Izquierdo et al. 2000).

Multi-batch spawners are also known to have variable spawn quality over time, even with the use of gonadotropin-releasing hormone. The fish in trial 1 were several years older than the fish in trial 2, and had been in captivity much longer. The age of the experimental broodstock may have also contributed to the lower larval survival observed, however, experienced broodstock can also result in higher survival in larval fish (McAvoy and McAvoy 1992; Mylonas et al. 2003).

When culturing any fish in a commercial setting, it is important to consider the culture parameters and the effects that they may have on growth and survival so profit can be maximized. Stocking density is a major concern for culturists as higher densities

are often associated with lower survival percentages and growth rates over time

(Chambel et al. 2015). When stocked at densities as high as 100 larvae/L, pinfish do not seem to exhibit any significant decreased growth or survival. A difference of slightly more than 15% was observed in trial 2, between the highest and lowest stocking densities. These results were similar to those observed by Broach et al. (2016) with their pinfish egg stocking density study wherein a mean egg stocking density of 250 eggs/L was recommended to achieve the highest survival to first feeding. The experiment performed by Broach et al. (2016) ended at 4 hours after the initiation of hatching; however, we were able to show similar effects up to 11 dph. Longer-term studies of stocking density and its effects on larvae would be beneficial; however, a lot of factors contribute to larval survival.

Many commonly cultured fish species perform equally well at high and low densities. Atlantic cod showed no differences in growth or survival when cultured at a density of 300 larvae/L versus a density of 50 larvae/L (Baskerville-Bridges and Kling

2000). Larval fish that survive well at higher stocking densities are good candidates for aquaculture because they allow culturists to produce more fish within a smaller amount of tank space (Webber and Riordan 1976). The results of the current experiments clearly indicate that pinfish adapt to multiple densities. This quality makes the pinfish a desirable candidate species for aquaculture.

CHAPTER 4 CULTURE OF SELAR CRUMENOPHTHALMUS

The goggle eye, Selar crumenophthalmus, is a schooling bait and foodfish species with high demand. Goggle eye are mostly silver and elongate and have a mean fork length of 25 cm in the wild and were given the common name of goggle eye because they have large eyes relative to their body size. These mainly nocturnal fish travel in large schools and prefer shallow inland waters. Goggle eye are most commonly used as a foodfish species in the areas around the Indian Ocean, but are also used by saltwater anglers worldwide for game fish in tropical and sub-tropical waters. Global harvest for this species exceeded 200,000 kg in 2014 with most of that catch entering the foodfish market (Food and Agriculture Organization of the United Nations 2017).

Spawning generally occurs from April to September with females spawning every three days during the night. Sexual maturity is documented in fish of 20.2cm total length

(Fauzi et al. 2018). Spawn sizes of 92,000 eggs have been reported and this species grows rapidly, as much as 13 cm in a single year (Clarke 1995; Iwai et al. 1996; Roos et al. 2007; Espino-Barr et al. 2016). Researchers at the University of Miami Experimental

Hatchery (UMEH) in Miami, FL have had success spawning this species in captivity, but have had limited success culturing larvae (Welch et al. 2013). A collaboration was established to investigate larval culture methods.

In this study, all experiments were small scale so a large number of variables could be evaluated with high replication at one time. Thus, we investigated the effects of multiple parameters on growth and survival of larvae including green water and water movement to determine the optimal culture methods. Two live feeds for newly hatched larval fish were also investigated to test for their effects on growth and survival. At

UMEH, rotifers were the only available live larval food. Therefore, researchers at UF-

IRREC were provided with goggle eye eggs to perform food studies with copepods.

Methods

Broodstock Conditioning and Egg Collection

Goggle eye broodstock were housed and conditioned at UMEH. Egg collectors within broodstock tanks were checked daily for spawns. Upon collecting and quantifying a spawn, researchers in Miami notified UF-IRREC staff, who then drove to Miami to pick up the eggs and return them to UF-IRREC. Eggs were transported in an aerated, 25-L bucket for approximately 3 hours in a climate controlled truck. Upon arriving at UF-

IRREC, the water containing the eggs was tested for temperature, salinity, and pH to make sure that water in the 110-L cylindrical egg incubation tank was similar. Once water parameters were identical, eggs were enumerated and stocked into this tank. This tank was then gently aerated with a ceramic airstone (1.27 cm diameter by 4.5 cm long) positioned near the bottom, center.

Spawn 1 consisted of approximately 3,342 eggs and was received in September

2013. Spawn 2 consisted of approximately 3,765 eggs and was received in December

2013.

Larval Culture Experiments

All experiments involving larvae in this chapter were performed in the same experimental setup described in the methods section of Chapter 2. This research is the result of two spawns at UMEH. Experiments were designed to test effects of greenwater and aeration intensity using eggs from spawn 1. Experiments were designed to test the effects of different live foods on larval growth and survival using eggs from spawn 2.

Green Water Effects on Growth and Survival

After thoroughly mixing the tank water, eggs in the 110-L tanks were volumetrically sub-sampled (the mean of 10, 30 mL samples) and stocked into four cylindrical 15-L experimental tanks at a density of 10/L. At the onset of first feeding, all treatments were fed S-strain rotifers (Brachionus sp.) enriched with Ori-Green (Skretting

Canada, Inc., Bayside, New Brunswick, Canada) at a density of 15/mL for five days. All rotifers utilized in this experiment were cultured and enriched as described in Chapter 1.

Two of the tanks were provided with live Tisochrysis lutea (Tiso) algae at a density of approximately 250,000 cells/mL for the duration of the experiment, while the remaining two tanks were clear water with no algae added.

For 5 days, water quality parameters in the system were kept constant and measured daily. Daily feedings of enriched rotifers were administered to all four tanks.

Daily photos were taken of 8 randomly selected larvae from each tank in each treatment. At the end of this period, the number of surviving larvae in each tank was hand counted and photos were taken of all surviving larvae from each tank in each treatment on a Sedgewick Rafter counting cell (Figure 2-3). The standard length and body depth of each larva was then measured with ImageJ analysis software (Rossband

2014).

Aeration Intensity Effects on Growth and Survival

After thoroughly mixing the tank water, eggs in the 110-L tanks were volumetrically sub-sampled (the mean of 10, 30-mL samples) and stocked into four cylindrical 15-L experimental tanks at a density of 10/L. Tank water was mixed by gently swirling the water with an air stone back and forth across the bottom of the tank in a grid pattern for approximately 30 seconds. All treatments were fed S-strain rotifers

(Brachionus sp.) according to the parameters identified in Chapter 1. All treatments were provided with once daily additions of live Tiso algae at a density of approximately

250,000 cells/mL for five days.

Two of the tanks in this experiment were provided with slow rates of aeration of 4 bubbles per second coming to the surface of each tank. The other two tanks were provided with faster rates of aeration, 10 - 12 bubbles per second coming to the surface.

Sweetwater AS1 Silica air diffusers, with a width of 1.3 cm and length of 3.8 cm, were used.

For 5 days, water quality in the system was kept constant and measured daily.

Daily feedings of 10/mL enriched rotifers were administered to all four tanks. At the end of the experimental period, data were collected as described in Chapter 1.

Effects of Different Live Feeds on Larval Growth and Survival

In this experiment, rotifers only were compared to a diet consisting of an equal ration of enriched rotifers and copepod nauplii of P. crassirostris. The copepods were cultured in a separate section of the hatchery at UF-IRREC. Parvocalanus crassirostris, utilized in the following experiments, were cultured using methods identical to those discussed in the methods section of Chapter 2. No enrichment was given to the copepods before feeding to the larvae.

Two treatment diets were examined, labeled A and B in Figure 4-1. At the onset of first feeding, all three replicates in treatment A were fed 10/mL of both enriched

Brachionus sp. and P. crassirostris nauplii daily for five days. All three replicates of treatment B were provided with enriched, S-strain rotifers (Brachionus sp.) at a density of 20/mL for five days. All rotifers utilized in this experiment were housed, cared for, and enriched as described in Chapter 2 of this document. All rotifers utilized in this

experiment were housed, cared for, and as described in Chapter 2. All live feeds were counted by following the same method outlined in Chapter 2.

Figure 4-1. Feeding schedule for live feed experiment conducted with goggle eye over 5 days post hatch. Different treatments are labeled A and B above.

At the onset of first feeding (1 dph), approximately 250,000 cells/mL of Tiso algae were added once daily to every tank to green the water. Daily, four 10-mL subsamples were taken from a 19-L algae bottle to determine the mean algal density with the aid of a hemaocytometer. Daily, photos were taken of 8 randomly selected larvae from each tank in each treatment. At 5 dph, all remaining larvae in each tank were hand counted to determine survival percentage and growth was measured by taking photos on a sedgewick rafter cell of all remaining larvae from each tank (Figure 2-3).

Results

Green Water Effects on Growth and Survival

Although the experiment was planned to continue to 5 dph, all larvae were deceased at the beginning of 4 dph.

Table 4-1. Mean standard length (mm) of 8 Selar crumenophthalmus larvae selected at random from each tank in each treatment (n=16). Growth per day was based on the difference between the previous and current larvae mean lengths. Treatments were as follows: Algae (larvae reared in tanks containing approximately 250,000 cells/L of Tiso algae for the duration of the experiment) and Clear (larvae reared in tanks without any algae added for the duration of the experiment). dph Treatment Standard Length (mm) Body Depth (mm) 0 All 2.17 ± 0.04 0.30 ± 0 1 Algae 2.42 ± 0.01 0.36 ± 0 1 Clear 2.36 ± 0.00 0.35 ± 0 2 Algae 2.25 ± 0.02 0.34 ± 0 2 Clear 2.26 ± 0.00 0.31 ± 0 3 Algae 2.31 ± 0.03 0.34 ± 0 3 Clear 2.30 ± 0.00 0.30 ± 0

Throughout this experiment, there were no sigificant differences in the standard length (p=0.9230) measurements from each treatment. There was a significant difference in mean body depth (p=0.0244) between treatments.

Aeration Effects on Growth and Survival

Although the experiment was planned to continue to day five post hatch, all larvae were deceased at 2 dph. All larvae, in the low aeration treatment, were deceased on the morning of 1 dph and all larvae, in the high aeration treatment, were deceased at the beginning of 2 dph. No data were collected from this trial beyond 0 dph.

Mean ± SD of dissolved oxygen in the high aeration treatment was 6.41 ± 0.2 g/L. Mean ± SD of dissolved oxygen in the high aeration treatment was 6.38 ± 0.2 g/L.

No significant differences were found in mean dissolved oxygen (p = 0.8768 between treatments for the duration of the experiment.

Effects of Different Live Feeds on Larval Growth and Survival

The effect of live feeds on larval growth and survival was the only treatment in which larvae were observed with full guts. Larvae in treatment B were provided with

only enriched rotifers and did not seem to be eating at any point in time based on visual analysis of the guts under a dissecting microscope. In contrast, food was observed in the gut of larvae fed diet A.

Table 4-2. Mean standard length (mm) of 8 Selar crumenophthalmus larvae selected at random from each tank in each treatment (n=16). Mean growth per day was based on the difference between the previous and current larvae lengths. Treatments were as follows: Mix (larvae fed an even ratio of P. crassirostris and Brachionus sp. for 4 days) and Roti (larvae fed Brachionus sp. exclusively for 4 days. dph Treatment Standard Length (mm) Body Depth (mm) 0 All 2.18 ± 0.03 0.30 ± 0 1 A 2.13 ± 0.03 0.32 ± 0 1 B 2.40 ± 0.20 0.31 ± 0 2 A 2.51 ± 0.02 0.38 ± 0 2 B 2.42 ± 0.03 0.37 ± 0 3 A 2.50 ± 0.01 0.41 ± 0 3 B 2.26 ± 0.03 0.31 ± 0

As the study progressed to day 3, larvae fed a mixed diet had a significantly higher mean rate of growth than larvae fed only rotifers. Larvae fed a mixed diet also had significantly greater length (p=0.0144) and body depth (p=0.0080) than those fed only rotifers at the end of this experiment (Table 4-2).

In the treatment A, mean ± SD of water quality parameters were: temperature

28.8 ± 0.3 °C, salinity 35 ± 1 g/L, dissolved oxygen 6 ± 0.2 g/L, and pH 8.1 ± 0.1. In the treatment B, mean ± SD of water quality parameters were: temperature 28.8 ± 0.2 °C, salinity 34.8 ± 1 g/L, dissolved oxygen 6 ± 0.2 g/L, and pH 8.1 ± 0. No significant differences were found in mean temperature (p = 0.7344), salinity (p = 0.6018), dissolved oxygen (p = 0.4527), or pH (p = 0.3682) between treatments for the duration of the experiment.

Discussion

Every experiment outlined in this section was performed with fish eggs that endured several hours of transportation in a covered and aerated bucket. Human handling of broodstock fish and eggs or larvae is generally kept to a minimum because it may have a negative effect on the viability or hatchability of the egg (Conte 2004).

Temperature variation especially is known to have adverse effects on larval fish development (Pepin 1991). The need for and method of transportation of the eggs used in these experiments could have affected egg quality before the experiments began.

There was also no record of egg quality within this breeding population over time, so there was no way to be certain that they had been producing high quality eggs to begin with. Establishing a group of broodstock goggle eye at UF-IRREC and maintaining their diet while monitoring spawn size/quality may alleviate these concerns in future experiments.

Larval fish cultured in green water did not seem to exhibit any advantages in growth or survival over those cultured in clear water. There were no significant differences in the standard length, depth, or overall survival of developing larvae between treatments for the duration of the study. When their culture methods were first under investigation, most marine finfish species were placed in green water as it often improved feeding success and survival (Naas et al. 1992, Reitan et al. 2003). Cobia,

Rachycentron canadum, showed a significantly higher survival percentage when cultured in water greened with either T. lutea or N. oculata (Faulk and Holt 2005).

Greenwater has also been attributed to decreased deformities in striped trumpeter ,

Latris lineata, larvae (Cobcroft et al. 2012) and increased percentages of swim bladder inflation in California yellowtail, Seriola lalandi, larvae (Stuart and Drawbridge 2011).

The use of greenwater in the culture of goggle eye does not seem to affect survival or development based on our limited experiment, but more research should be conducted in this area.

Larvae cultured in more turbulent water did not survive after hatching, which could be due to many different factors. Determining an optimum level of water movement for larval fish development can be beneficial to feeding success of the larvae

(Kato et al. 2008). The turbulence avoidance theory suggests that both fish and potential prey congregate in areas of low flow to avoid turbulence, which results in higher concentrations of prey and a higher encounter rate for larvae (Franks 2001). The current experiment was proposed and carried out because goggle eye reside close to the surface in areas where currents routinely move at speeds between 1 and 1.5 km/hour which suggests that an optimum level of turbulence for larval development may be higher than average (NOAA 1998).

Though no useable larvae were collected, the larval culture tanks, with a higher level of turbulence, had a higher dissolved oxygen content. Seven-band grouper cultured in higher rates of aeration resulted in greater survival due to increased dissolved oxygen and the additional water movement it provided (Sakakura et al. 2006).

It was hypothesized by Miyashima et al. (2012) that newly hatched, quickly developing larvae metabolize food at a faster rate than more developed or mature larvae. All four of the species examined in their experiment required a higher concentration of dissolved oxygen to support their accelerated metabolism and were adversely affected by dissolved oxygen concentrations slightly below normal.

Providing larvae with a varied diet was one reported method to achieve a more uniform rate of growth and survival (Sales 2011). By supplying many sizes of food particles with different nutritional profiles and swimming patterns, it was proposed that larvae eat and develop more uniformly (Le Ruyet et al. 1993, Rosenlund et al. 1997). In the current study, the lack of food in the gut of larvae provided with only one source of food seems consistent with this hypothesis. Larval body depth was measured as a way to assess ingestion of food. A significantly higher body depth at the conclusion of the experiment could indicate that larvae fed diet A were ingesting more food than those fed diet B.

Although no goggle eye were raised through metamorphosis as a result of these experiments, a large amount of information was gathered that will help to improve their culture methods. Though these were not large scale experiments, three important aspects of goggle eye larval culture were addressed that should guide future research; larval food preference, greenwater effects on growth and survival, and aeration intensity preference. A study of stocking density and its effects on growth and survival like those performed with M sebae and pinfish in this thesis should be pursued. Investigations into water quality parameters during egg incubation and larval hatching could also improve the quality of larvae for experiments and growout.

CHAPTER 5 CONCLUSION

When investigating the effects of egg stocking density on hatch success of M. sebae, densities over 20 eggs/L led to significantly lower hatching success. These results were consistent with other studies of this type with a stocking density on the lower to middle end of the norm for a given species producing significantly higher hatching successes (Broach et al. 2016). There has been a great deal of research conducted related to transportation of fish eggs and yolk sac larvae that could possibly be applied here to increase hatch success at higher stocking densities (Lehtonen and

Kvarnemo 2015; Stuart et al. 2018).

Lower stocking densities in pinfish larvae resulted in significantly better growth in standard length but no significant difference in larval survival to 10 dph. Much higher stocking densities of pinfish eggs (250, 500, 1000, and 2000 eggs/ L) were investigated by Broach et al. (2016). This experiment showed that the lowest stocking density of 250 eggs/L had a significantly higher hatching success than any of the other treatments. It would be valuable to use similar stocking densities in a larval growth and development study. This study could also be extended to metamorphosis to see the long-term effects that denser stocking may have on larval development.

Environmental factors like salinity seem to be more important than originally anticipated when dealing with the brackish water species M. sebae. The species has been identified as completely euryhaline and their eggs can also tolerate a wide range of salinities, although in the current study the greatest hatching success occurred at a salinity of 5 g/L. Other euryhaline species exhibit a similar relationship between egg hatching success and salinity. The Gulf killifish, Fundulus grandis, was found to have

higher embryo survival to hatching in saltwater, but a higher rate of hatching and juvenile survival post-hatch in freshwater (Ramee and Allen 2016). Embryo survival was not recorded in the current study, but in the future, it could be added as an additional data set. Egg development in a euryhaline species was examined by Dugue et al.

(2014) who found that both low (0 gL) and natural (35 g/L) salinity levels had little to no effect on reproductive investment. While an artificial manipulation of salinity was used for spawning in our M. sebae broodstock, looking at the effects that different salinity levels have on reproductive investment would be valuable. It may be feasible to raise these fish entirely in low salinity or freshwater. A longer term study that examines the continued effects of different salinities on M. sebae larvae as they develop is justified to determine if their entire larval phase could be completed in the same lower salinity environment.

Larval stocking density followed the same trend as egg stocking density with lower densities resulting in significantly higher rates of growth, but there were no significant differences in larval survival to 10 dph. Although higher larval stocking densities sometimes result in greater larval survival (Hasenbein et al. 2016), this is not commonly the case (Sahoo et al. 2010; Aidos et al. 2018). The advantage of using lower stocking density based on these slightly higher growth numbers would have to be considered by a farmer.

A great deal of research is being performed concerning the effects of different live feeds on larval growth and survival. Using a less costly feed is always preferable, but not if it leads to a significantly slower rate of growth or survival. Pinfish and goggle eye were the only species examined here that had significantly higher instances of

growth in either body depth or standard length when offered copepods in place of, or in addition to, rotifers. When compared to rotifers as a first food for larval fish, many researchers have found that copepods are generally superior and result in higher rates of growth and overall survival (Payne et al. 2001; Imsland et al. 2006; Olivotto et al.

2008). However, these differences must be large enough to outweigh the added costs of copepod culture. For instance, Zeng et al. (2018) found that P. crassirostris nauplii and copepodites added to a diet of enriched rotifers fed to Synchiropus splendidus larvae led to a 50% increase in larval survival to 11 dph. As copepod culture methods improve, the small differences in growth and survival revealed by these experiments may become cost effective enough for commercial hatcheries to pursue.

Even though M. sebae did not display a significantly higher growth rate or overall survival when offered copepods at first feeding, other potential differences between larvae in each treatment could be examined. In recent years, nauplii of the copepod P. crassirostris has been widely used at first feeding in instances when enriched rotifers did not elicit a feeding response from larvae (Degidio et al. 2017; Callan et al. 2018).

Offering copepod nauplii at first feeding may not be necessary when rearing M. sebae as both food types were readily consumed, although they did seem to have a preference for rotifers overall (Lee et al. 2018). The addition of copepods to the diet of larval fish has been linked to a decrease in deformities (Hansen 2011) as well as decreased transfer related mortality, an important factor when dealing with baitfish and ornamentals. A longer-term study that examines deformities in juvenile fish up to metamorphosis would allow us to explore the effects of food types on a wide range of characteristics like swim bladder inflation, physical deformities, and transfer stress.

There is a great deal more research needed for the larval culture of goggle eye.

The experiments performed here with small sample sizes and low replication helped determine future areas to explore when larger scale studies are possible. The turbidity experiment for instance, did not show any significant differences in growth between treatments, but an overwhelming amount of scientific publications support the idea that greenwater, or just increased turbidity in larval rearing tanks of many fish species, improve growth and survival (Naas et al. 1992; Reitan et al. 2003; Faulk and Holt 2005).

The possibility of using clay to darken larval tank water has been evaluated in other species with mixed results (Broach et al. 2015; Lee et al. 2017). If this method were to be evaluated with goggle eye, it could further reduce the cost of culturing the species for potential farmers.

In the aeration intensity experiment, larvae survived longer in the high aeration treatment than in the low aeration treatment. Differences in aeration intensity are seen as both a positive and a negative environmental factor in the literature. Aeration is used as a cue for morphological changes (Kurata et al. 2017), a method of raising dissolved oxygen (Miyashima et al. 2012), as a way to reduce predation among larvae (Benetti et al. 2008), and for many other diverse reasons (Sakakura et al. 2006). Both of these environmental factors have been explored enough to identify them as valuable candidates for future larger scale studies.

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BIOGRAPHICAL SKETCH

Daniel Elefante was born in 1990 in Lincoln Park, New Jersey where he enjoyed spending time outdoors and taking care of several diverse aquariums. After graduating high school, Daniel attened Roger Williams University where he majored in Maine

Biology. While attending Roger Willaims, Daniel worked in the Marine Science Lab for four years and with the OGRE and CEED Grant programs for three summers after regular classes had ended.

Daniel accepted a graduate assistanceship under Dr. Cortney Ohs at the

University of Florida in August 2012. In July of 2015 Daniel completed his master’s research at the University of Florida, and moved to the northeastern United States to seek employment in his chosen field. In June of 2016, Daniel accepted a position as the laboratory manager at the Southampton High School Marine Ornamental Lab where he has been working since.