UNIVERSITY OF HAWAI'I LIBRARY

EFFECTS OF BROODSTOCK DIET AND ENVIRONMENTAL IODIDE CONCENTRATIONS ON LARVAL GROWTH, SURVIVAL, EGG AND WHOLE BODY CONCENTRATIONS OF THYROID HORMONES AND CORTISOL IN PACIFIC THREADFIN, POLYDACTYLUS SEXFIUS

A THESIS SUBMI'I"I'ED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

ANIMAL SCIENCES

AUGUST 2008

By Elisha M. Witt

Thesis Committee:

E. Gordon Gran, Chairperson Douglas Vincent Tetsuya Hirano We certify that we have read this thesis and that, in onr opinion, it is satisfaetory in scope and qnality as a thesis for the degree of Master of Science in Animal Science.

THESIS CO,Ml\.nT,g..

ii I dedicate this thesis to Dr. Paul Brown, who sparked my interest in aquaculture.

iii ACKNOWLEDGEMENTS

1 would like to thank my thesis committee, and especially Prof. E. Gordon Grau for allowing me the opportunity to pursue this degree in his lab. 1 would also like to thank Dr. Tetsuya Hirano for his steadfast mentorship. Further, 1 thank those persons who assisted with my research, including Dr. Andy Pierce, Dr. DIlrren Lerner, Dr. Lori

Davis, Dr. Kai Fox, Jason Breves, Anna Kosztowny, and Masa Yoshioka from the

Hawai'i Institute of Marine Biology; Dr. Charles Laidley, Chris Demarke, Ken Liu, Don

Delapena, Joe Aipa, and Chad Callan from the Oceanic Institute; Randy Cates, Ryan

Murashige, Aaron Moriwake, Augustine Molnar, Jon Ginoza, and Gary Germano from

Hukilau Foods. This study has been supported in part by Binational Agricultural

Research and Development Fund (BARD, 18-35695-04).

iv ABSTRACT

Pacific threadfin (Polydactylus sexftlis), known locally by its Polynesian name "moi", is rapidly becoming a premier aquaculture species in Hawai'i and throughout the Indo­

Pacific. Nevertheless, threadfin culture is suffering from extraordinary loss of seed stock during the pre-metamorphic and metamorphic periods, and from dramatic size variation among animals after metamorphosis that leads to cannibalism. The objectives of my studies were to: 1) determine the relationship, if any, between diet of threadfin broodstock and thyroid hormone content of fertilized eggs; 2) examine the effects of potassium iodide (KI) supplementation to broodstock rearing tanks on thyroid hormone concentrations in fertilized eggs; and 3) examine effects ofiodide concentration of rearing tanks on larval growth, survival, metamorphosis, and whole body concentrations of thyroid hormones and cortisol. Broodstock fed a diet rich in iodine produced fertilized eggs with significantly higher thyroxine (T4) and triiodothyronine (T3) levels than eggs produced by broodstock fed either a raw diet of squid, lake smelt and shrimp, or a commercial marine broodstock feed. Fertilized eggs from broodstock fed the iodine-rich feed had a T4 concentration of 3.29 nglg, significantly greater than 0.09 nglg and 0.59 nglg found in eggs from broodstock fed either the raw diet or the commercial feed respectively. Fertilized eggs produced from broodstock receiving the iodine-rich feed had T3 concentration of 6.43 nglg compared with 0.05 nglg and 0.21 nglg for eggs from broodstock fed raw and commercial diets respectively.

Broodstock held in tanks supplemented with KI (0.08 mgIL) also produced fertilized eggs with elevated concentrations ofT4 and T3. Eggs from KI-treated broodstock had T4 concentration of 1.91 nglg, significantly greater than 0.09 nglg found in eggs from

v untreated broodstock. Eggs from K1-treated broodstock bad T3 concentration of 7.81 ng/g compared with 0.05 ng/g found in eggs from untreated broodstock.

Threadfin larvae reared in ocean water grew significantly larger, and showed increased survival compared with larvae reared in water from a seawater-injected well that bad lower iodide concentrations. Larvae reared in ocean water developed more mpidly than in well seawater. At 14 days post-hatch, 50% of the larvae reared in ocean water reached flexion-stage, compared with 15% in well seawater. In ocean water-reared larvae, whole body T4 concentrations increased sharply from 0.4-0.5 nglg at hatching through day 13 post-hatch to 1.9 nglg on day 15, and declined gradually to 0.5 nglg by day 23. Larvae reared in well seawater did no show a peak in T4, as levels increased gradually from 0.2 nglg on day 1 to 2.4 nglg by day 25. Profiles OfT3 were similar between the two groups, decreasing from 0.17 nglg at 8 h before hatching to 0.02-0.04 nglg by day 7 post-hatch, and then gradually increasing to 0.45 nglg by day 23. Whole body concentration of cortisol was 0.5 nglg prior to hatching for both groups, increasing to 27-30 nglg by day 5. Cortisol levels fluctuated during days 7 to 25 post-hatch between

12 and 26 nglg for ocean water-reared larvae, and between 14 and 32 ng/g for larvae reared in well seawater. Absence of a peak in the T4 profile in well seawater-reared larvae may indicate incomplete synchronization of development or metamorphosis.

Threadfin larvae reared in K1-supplemented well seawater grew significantly larger, and developed more rapidly than larvae reared in well seawater with lower iodide concentration. At 13 days post-hatch 42% of larvae reared in K1-treated seawater reached flexion-stage compared with 26% ofuntreated larvae. By 15 days post 79"10 of larvae reared in iodide rich seawater had reached flexion-stage compared with 66% of

vi larvae reared in seawater without iodide supplementation. Survival to day 25 however, did not differ statistically between control and treated larval groups. Significant difi.erences in whole body T4 and T3 profiles were seen in larval cohorts from broodstock reared in untreated and KI-treated seawater and between larvae reared in untreated and

KI -treated rearing water.

These results suggest the importance of environmental iodide in maternal deposition of thyroid hormones in eggs, larval metamorphosis, and subsequent survival and growth. possibly through the synthesis of thyroid hormones.

vii TABLE OF CONTENTS

I>I1I>IC}llrlO~ ...... •...... ili

}lCKNOWLIIDGIIMIlNTS ...... " ...... iv

.ABSTRA.CT ...... " ...... '" ...... v

LIST OF T.ABLIIS ...... ix

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

CHAPTIlR 1: ~OI>UClrIO~ ...... 1

CHAPTIlR 2: MIlrnOI>s ...... •...... 5

CHAPTIlR 3: RIlSULTS ...... 14

CHAPTIIR 4: I>ISCUSSIO~ ...... •.. 67

RIlFIIRII~ CIIS ...... 79

viU LIST OF TABLES

Table Page

1. Iodine, total ammonia, nitrite, and nitrate levels (mg/L)

in larval rearing water ••••••••••••••••••••••••••••••••••••••.••••••••• 22

ix LIST OF FIGURES

Figure ~ 1. Elution profiles ofT4, T3, and cortisol through SepPak Light C 18 cartridge ...... •...... 24

2. Displacement curves for T4 standard and serial dilution of whole body extract ofthreadfin larvae ...... 26

3. Displacement curves for T3 standard and serial dilution of whole body extract ofthreadfin larvae ...... •...... 28

4. Displacement curves for cortisol standard and serial dilution of whole body extract of threadfin larvae ...... 30

5. Effect ofbroodstock diet on T4 concentration in fertilized threadfin eggs ...... •...... 32

6. Effect of broodstock diet on T3 concentration in fertilized threadfineggs .....•.•...... 34

7. Effect ofbroodstock diet on body length and survival of resultant threadfin larvae at 25 days post-hatch ...... •...... 36

8. Effect ofKI supplementation ofbroodstock rearing water on T4 concentration in fertilized threadfin eggs .•...... •...... 38

9. Effect ofKI supplementation ofbroodstock rearing water on T3 concentration in fertilized threadfin eggs ...... •...... 40

x Figure Page 10. Effect ofKI supplementation ofbroodstock rearing water on growth and survival of resultant tbreadfin larvae at 25 days post-hatch ...... 42

11. Development of tbread:fin larvae in ocean water. The time of development was expressed as days post-hatch. Day 0; unhatched embryo, 0.76 mm, Day 1; hatched larvae, 2.9 mm, Day 7; pre-flexion stage larvae, 3.2 mm, Day 14; pre-flexion stage larvae, 4.8 mm, Day 17; larvae at flexion stage, 5.6 mm, Day 23; post-flexion stage larvae, 12.3 mm, Day 25; juvenile tbreadfin, 14.6 mm ...... 44

12. Effect of rearing water on percent of larvae at flexion stage days 14 and 17 post-hatch ...... 46

13. Growth oftbreadfin larvae in ocean water and well seawater ...•. .48

14. Body length. body weight, and survival oftbreadfin larvae at day 25 post-hatch in ocean water and well seawater ...... 50

15. Effects of larval rearing water on developmental changes in whole body concentrations ofT4. T4. and cortisol ...... 52

16. Effect ofKI supplementation to rearing water on percent of larvae at flexion stage on days 13 and 15 post-hatch ...... 54

17. Standard length of tbreadfin larvae reared in well seawater and KI-supplemented well seawater ...... •...... 56

xi Figure Page 18. Wet weight ofthreadfin larvae reared in well seawater and KI-supplemented well seawater ...... 58

19. Standard length and wet weight ofthreadfin larvae (cohorts 1 & 2) reared in well seawater and KI-supplemented well seawater ...... 60

20. Effect ofKI supplementation oflarval rearing water on survival of threadfin larvae at 25 days post-batch ...... 62

21. Effects of KI supplementation oflarva1 rearing water on developmental changes in whole body concentrations ofT4 and T3 (Cohort I) ...... 64

22. Effects ofKI supplementation oflarval rearing water on

developmental changes in whole body concentrations ofT4 and T3 (Cohort 2) ...... 66

xii CHAPTER I

INTRODUCTION

Information pertaining to the life history of Pacific threadfin (Polydactylus sexfilis), known locally as "moi" is gathered from Lowell (1971), Kanayama (1973), May (1976) and Rao (1977). Juveniles "moi-lii" and adults inhabit the surf zone, reef faces, and mud­ sand areas across the Hawaiian Islands. They have a conical snout, inferior mouth, and filamentous lower pectoral rays, making the animal well adapted to benthic feeding. Moi are characterized as opportunistic feeders, having a diet composed mainly of sma1l fishes

(holocentrids and labrids) and crustaceans (crabs and shrimp). Moi are protandric hermaphrodites, redifferentiating from reproductively functional males into functional females in their second year (22-40 cm in length). During the transformation, which lasts approximately eight months, the fish are hermaphroditic. Self-fertilization during this period is impossible, however, since the testes is the only functional component of the gonad.

Pacific threadfin is the only polynernid found in Hawai'i (Gosline and Brock, 1965).

The species is not limited to Hawai'i, however, and has been reported in Madagascar

(Bleeker, 1875), Pakistan (Qureshi, 1960), India and into the Malay archipelago (Day,

1958), and Johnston Island (Jordan and Evermann, 1905). Surprisingly, neither the eggs, nor hatched larvae have ever been captured from the wild.

Captive broodstock spawn for 3-6 consecutive days between 6 pm and 12 am during the third lunar quarter (Ostrowski and Molnar, 1997). An estimated 150,000 to 200,000 eggs are thought to be released at each spawning. The embryonic stage is relatively short

1 as fertilized eggs, measuring approximately 0.78 mm in diameter, hatch within 24 hours.

Larvae absorb their yolk in 2-3 days after which time they begin to feed on zooplankton.

Moi is rapidly becoming a premier aquaculture species in Hawai'i and throughout the

Indo-Pacific. It is highly prized for its excellent flesh quality, fast growth, and adaptability to conditions of captive culture. The culture of threadfin has progressed rapidly over the past four decades. Investigation of moi as a potential for culture began at the Hawai'i Institute for Marine Biology (HIMB) in the early 1970's. By 1973, Mayet al. demonstrated the successful spawning of captive rooi in net pens, and by 1976 research progressed to focusing on larval culture. In these initial trials, poor larval survival was observed, with less than 3% of animals reaching the juvenile stage (May,

1976). In addition to very low survival, larvae had high rates of deformed operculi (May,

1976; May and Hashimoto, unpublished data). Research on improving hatchery techniques continued at Oceanic Institute (Waimanalo, Hawai'i) through the 1980's and

90's and by 1999 moi were successfully being farmed in the off-shore net pen of the

University of Hawai'i Sea Grant College Program (Ostrowski et al., 1996; Kam et al.,

2003). Nevertheless, threadfin culture suffers from extraordinary loss during the pre­ metamorphic and metamorphic periods and from dramatic size variation among animals after metamorphosis that leads to cannibalism. Availability of fingerlings is among the most critical factors for commercial success in marine fish farms, and the lack of knowledge of optimal environmental conditions during the early life stages of marine fishes is identified as a primary bottleneck to improved larval rearing (planas and Cunha,

1999).

2 Growth and development in fish, as in all vertebrates, are governed through orderly release of hormones from the neuroendocrine system, which integrates environmental, physiological, and genetic information. Many of the developmental processes that occur during early larval stages of teleost fish, including growth and metamorphosis, are regulated by the thyroid hormones. thyroxine or tetraiodothyronine (T4) and triiodothyronine (T3) (Eales, 1979; Power et aI., 2001; Blanton and Specker, 2007).

Thyroid hormone synthesis occurs in the thyroid follicle. which consists of a single layer of epithelial cells enclosing a colloid-filled space. In fish, the predominant, perhaps only, secretagogue of the thyroid gland, T4, is formed by the molecular addition of iodide (f) to the amino acid tyrosine (Grau, 1987). Fish accumulate iodide from the surrounding water by active transport at the gills, and through absorption in the gut (Eales, 1979).

Thyroid function is largely carried out by TJ, the biologically active form. at target tissues. The conversion ofT4 to T3 occurs in peripheral tissues by enzymatic deiodination ofT4. Egg yolk is also known to contain thyroid hormones, indicating that maternal thyroid hormones are utilized by developing embryos (Tanaka et aI., 1995;

Power et aI., 2001; Blanton and Specker, 2007).

The Oceanic Institute is a major fingerling supplier of Pacific threadfin in Hawai'i. It has been reported that seawater (well seawater) used at Oceanic Institute's hatchery contains less iodide than ocean water. White-tip reef sharks. Triaenodon obesus, kept at

Sea Life Park located adjacent to the Oceanic Institute using the same well seawater, develop goitre, a syndrome typical of severe hypothyroidism that results from iodide deficiency (Crow et aI., 1998). When two sharks. expressing both goitre and suppressed plasma T4 and T3, were transferred from rearing tanks at Sea Life Park to a coastal lagoon

3 with natural seawater at the Hawai'i Institute of Marine Biology, plasma T4 and T3 increased dramatically within 2 weeks, and goitre diameter was reduced by two thirds within 3 months. Thus, a major contributor to reduced survival during the larval stage in

Pacific threadfin at the Oceanic Institute may be the reduced availability of iodide in the water which is likely to result in low thyroid hormone levels and attenuated development, that in turn results in increased mortality and reduced hatchery production. Likewise, an inadequate supply of iodide may compromise thyroid function in adult animals, possibly altering deposition of thyroid hormones in produced eggs. The occurrence of goiters identified in broodstock moi at Oceanic Institute has been correlated with increased mortalities (C. Laidley, unpublished data). The following studies were undertaken to examine the relationship between 1) broodstock diet and 2) environmental iodide with thyroid hormone levels of fertilized eggs and whether the concentration of iodide in rearing water is related to larval performance, and whether profiles for whole body T4 and T3 reflect enviromnental iodide levels.

4 CHAPTER II

MATERIALS AND METHODS

2.1 Effects ofbroodstock diet on thyroid hormone concentrations in fertilized threadfln eggs, growth and survival ofresultant larvae at day 25 post-hatch (Experiment #1.)

Broodstock of Pacific tbreadfin (Polydactylus sexfllis) were kept at Oceanic Institute's

Waimanalo facility, in 25,000 L flow-through tanks supplied with water from a seawater­ injected well. They were fed by hand daily to apparent satiation with a standard diet of raw squid and lake smelt with shrimp supplemented once per week, or one of two experimental diets, either a commercial marine broodstock feed (Skretting, Vita1is) or a pelleted feed formulated at Oceanic Institute rich in iodine (Oceanic). Initially, broodstock fed the experimental diets consumed less feed than the fish fed the raw diet, however, after approximately five months feed consumption was similar among all groups. Egg production from the broodstock fish fed the experimental diets also djmjnjghed during the transition between feeds. For this reason, the collection of spawned eggs for thyroid hormone analysis was accomplished following the animals return to normal spawuing pattern. Fertilized eggs were collected during the night from tank eflluent using a 0.5 mm mesh net suspended in water. Larval rearing trials were completed to examine the possible effects ofbroodstock diet on hatching rate, growth and survival of thread:fin larvae to day 25 post-hatch.

5 2.2 Effects ofKI supplementation to broodstock rearing water on thyroid hormone concentrations offertilized threadfin eggs, growth and survival ofresultant larvae at day

25 post-hatch (Experiment #2).

A potassium iodide (Kl) drip was installed on two threadfin broodstock tanks supplying the rearing water with a continuous 80= iodide (0.096 ± 0.001 mgIL). Prior to the treatment, and after forty five days ofK! supplementation, egg samples were collected as described above and analyzed for thyroid hormone content. Larval rearing trials were completed to examine the possible effects ofbroodstock diet on hatching rate. growth and survival ofthreadfin larvae to day 25 post-hatch.

2.3 Effects ofenvironmental iodide in rearing water on larval survival, growth, and profiles ofthyroid hormones and cortisol (Experiment #3)

At 8 am, the eggs from broodstock fed a raw diet were transferred into an aerated 5 gallon bucket and sodium chloride was added to raise the salinity from 32%0 to 36%0, thus increasing the buoyancy of viable eggs. Twelve 1,000 L tanks (6 tanks with recirculating ocean water and 6 with flow-through well seawater) were each stocked with 50,000 eggs

(50 eggsIL), referred to as day O. A seawater drip was in place throughout the study to account fur evaporative loss from recirculating ocean water tanks. Fresh ocean water was added to the recirculating system on day 12, replacing approximately one-fifth of the total volume.

Larval threadfin were fed 4 times daily with rotifers measuring 170 nm in lorica length and 127 nm in width (Florida Aquafarms, FL) beginning on day 3 post-hatch.

Prior to feeding, rotifers were enriched for 3 h with Algamac 2000 (Bio-Marine,

6 Hawthorne, CAl and DC DHA Selco (INVE Aquaculture Nutrition, Thailand).

Beginning on day 10 post-hatch, they were fed Artemia naupli enriched overnight with

Algamac 2000 and DC DHA Selco. Algal paste of Nannochlorpsis sp. (Reed

Mariculture, Campbell, CAl was added to the larval tanks daily as background feed for the live zooplankton. Feeding was supplemented with a dry crumble (Skretting,

Vancouver, BC) beginning on day 12 post-hatch.

2.4 Effect ofKI supplementation ofrearing water on larval growth, survival, thyroid hormone profiles (Experiment #4)

Two trials were conducted to investigate effects of the supplementation of iodide to the rearing water on larval performance and thyroid hormone profiles. In the first experiment, larvae were sampled on days 1,3, and 5 post-hatch to evaluate possible treatment effects during early development In the second trial, larvae were sampled beginning on day 9 post-hatch and every other day thereafter. For both trials, four 4,000 liter tanks were each stocked with 160,000 fertilized threadfin eggs (40 eggsIL) from two broodstock groups. Two tanks were stocked with eggs from either broodstock reared in untreated well seawater (cohort 1) or from broodstock reared in KI-supplemented well seawater (cohort 2). Tanks were supplied with flow-through well seawater that was degassed, and passed through a sand filter, cartridge filter, and UV sterilizer. A KI drip was insta1led on two of the four tanks (one tank from each larval cohort) supplying iodide at (0.045 ± 0.001 mgIL) to the rearing water. Feeding protocols were similar to those described in experiment #3.

7 Sampling

For each experiment, aliquots of eggs (500 mg) were collected at the time of stocking and stored in 2 m1 Eppendorftubes at -80°C for future analyses. For experiments #1 and

#2, larvae were sampled on day 25 post-hatch, at the termination of the larval stage. For experiment #3, larvae were sampled beginning on day 1 post-hatch and every other day until day 11 post-hatch, subsequent sampling was conducted on days 14, 17,20,23 and

25 post-hatch. Within each treatment, 3 replicate samples of 100 mg wet weight were taken by pooling larvae from two tanks. For experiment #4, larvae were sampled on days

1, 3, and 5 post-hatch (trial 1) and every other day from day 9 to 25 post-hatch (trial 2).

For each tank, 5 replicate samples of 100 mg wet weight were taken. Larvae were sampled by dipping a 5 L beaker into the tank and filtering the contents over a 200 f.lID mesh screen. An over-dose ofMS 222 (Sigma, St. Louis, MO) was used to anesthetize the animals within seconds of sampling. Larvae were minced using a scalpel and stored in 2 ml Eppendorftubes at -80°C; larvae smaller than 5 rom did not require mincing, and were frozen whole.

Water chemistry

Water samples from the holding facilities were examined for temperature, pH, salinity, dissolved oxygen, and nutrients (total ammonia nitrogen, NOi, N03·, total I, r, and 103"). Nutrient water samples were collected with a syringe and then filtered through in-line CF/C glass fiber filters. Water samples were held in Nalgene bottles, placed on ice, and frozen at _20°C within 4 h of collection. Nutrients were measured on a

8 Tecbnicon II Autoanalyzer as described by Crow et al. (1998). Analyses of total iodine, iodide (f) and iodate (103) were performed by ENC Labs (Albuquerque, NM).

Hormone Extraction

To extract T4, T3, and cortisol from tissue, 100 mg whole body samples were homogenized for 30 sec in 0.5 ml ice-cold ethanol with a Polytron homogenizer

(Kinematica, Littau, Switzerland). An additional 0.5 ml of ethanol was used to wash residual tissue from the blade. Combined samples were mixed using a vortex mixer for

30 sec and stored overnight at 4°C. On the following day, samples were centrifuged for

15 min at 12,000 rpm.

The ethanol extracts were purified using a Sep-Pak Light C18 cartridge solid-phase extraction device (Waters, Milford, MA). The cartridge was preconditioned with 2 ml ethanol, followed by 2 ml of 0.1 % trifluoroacetic acid (TFA, Sigma). In order to establish elution profiles through Sep-Pak cartridge, either 5,000-10,000 cpm of 1251_ labeled T4 (L-3' ,5' J~ thyroxine, 1,500 ~CiI~g, Amersham, Piscataway, NJ), 1251_ labeled T3 (L-3' ,3, 5J2s1 triiodothyronine, 3,076 ~CiI~, Amersham) or 3H-Iabeled cortisol (1,2,6,7-3H(N) cortisol, 70-100 Cilmmol, Perkin Elmer, Boston, MA) were dissolved in 1 ml of20% ethanol in 0.1 % TF A, and applied to the cartridge at a flow rate of 0.3-1 mlImin. The cartridge was first washed with 2 ml of20% ethanol in 0.1% TFA, followed by 1 ml of20-80% ethanol in 0.1 % TFA. As shown in Fig. 1, all the hormones were eluted at between 30-50% of ethanol.

The sample (1 ml ethanol extract) was diluted to 20% ethanol in 0.1 % TF A, by adding

4 ml of 0.1 % TF A, and applied to the cartridge at a flow rate ofless than 0.2 m1!min. 9 The cartridge was first washed with 2 ml of20"/O ethanol in 0.1% TFA, followed by 1 ml of30% ethanol in 0.1% TFA. The hormones. T", T3 and cortisol, were eluted with 1 ml of 50% ethanol in 0.1 % TF A. The samples were lyophilized in 3 h using Automatic

SpeedVac (Savant Instruments, Holbrook, NY). Dried samples were reconstituted at 1 mg/fJl with 100 fJl of 0.1 I M sodium barbital (Sigma), 0.1% gelatin, 0.05% sodium azide

(Sigma), pH 8.6.

To assess recovery of the hormones, each of the tissue homogenates were spiked with

10,000 cpm of 12sI_labeled T4, 12SI_labeled T3, or 3H-labeled cortisol. The tissue content of the hormones were determined by radioinImonoassays and corrected for the extraction efficiency of each sample. Typical extraction efficiency was 60 ± 2% for T4 (n=6), 66 ±

1% forT3 (n= 6) and 41 ± 1.7% for cortisol (n=6). Low recovery of cortisol seenJS to be due to a significant fraction of the hormone being lost in the 30% ethanol wash (Fig. 1).

Radioimmunoassays

Whole body concentrations ofT4 and T3 were measured using radioimmonoassays following Tagawa and Hirano (1987) with slight modifications. Reconstituted samples were added in duplicate to polystyrene tubes with 100 fJl barbital buffer; 0.11 M sodium barbital, 0.15% bovine gamma globulin (Cohn Fraction n, Sigma), 0.1 % gelatin, 0.03%

8-anilino-l-naphthalene sulfonic acid ammonium salt (Sigma), 0.05% sodium azide, pH adjusted to 8.6, and 50 fJl of T4 or T3 antibody (Fitzgerald Industries International,

Concord, MA) diluted with barbital buffer to 1:50,000 and 1:30,000, respectively. Tubes were mixed with a vortex mixer for 10 sec and incubated at room temperature for 30 min to allow the antibody and hormone to reach equilibrium. Then, 50 fJl (15,000 cpm) of 10 1251_1abeled T4 (L-3' ,5,)251 thyroxine, 1,500 IlCilJ,tg, Amersham) or 1251-labeled T3 (L-

3',3, 5_1~ triiodothyronine, 3,076 IlCilJ.Lg, Amersham) were added, and the tubes were incubated at 37°C for 1 h. After incubation, the tubes were cooled for 5 min at room temperature (24 0e), followed by 5 min at 4°C before adding 250 III of 20% polyethelene glycol in barbital buffer to form a precipitate. Tubes were mixed with a vortex mixer for 10 sec followed by 30 min incubation at 4°C. Samples were centrifuged for 30 min at 3,000 rpm at 4 °C, and after aspiration of the supernatant, tubes were counted in a gamma counter (Cobm II, Packard, Merideo, CT). The intra-assay coefficients of variation were 9% (n=9) for T4 and 10% (n=10) for T3.

Whole body concentrations of cortisol were also measured by a mdioimmunoassay.

Reconstituted samples were run in duplicate with 150 J1l 3H-labeled cortisol (7,000 cpm), and 100 J1l cortisol antibody (Fitzgerald Industries International) at 1:20,000 dilution in

0.0 I M phosphate buffered saline; 1% bovine serum albumin, 0.9"10 NaCI, 0.1 % sodimn azide, pH 7.3. Borosilicate glass tubes containing sample, label, and antibody were incubated at room temperature for 2 h. Following incubation, tubes were placed on ice for 5 min before adding 400 J1l of dextran-ooated charcoal (Sigma) to remove unbound cortisol. Tubes were centrifuged for 30 min at 3,000 rpm at 4°C, and 0.5 m1 of supernatant was then added to 4 m1 scintillation fluid (Scinti Safe Econo 1, Fisher

Scientific, Pittsburgh, PA) and counted in a beta counter (LS 3801, Beckman Coulter,

Fullerton, CA) for 2 min. The intra-assay coefficient of variation was 14% (n=IO).

The validity of ethanol extract and Sep-Pak purification was assessed by the parallel displacement curves with T40 T3, or cortisol standard and serial dilution of purified tissue extracts. An aliquot of the tissue extract after Sep-Pak purification was stripped by 11 incubating at room temperature for 15 min with 2% activated charcoal. As shown in Fig.

2, the displacement curves for Sep-Pak extracts were parallel with T4 standard, and there was no cross reaction in the stripped extracts. Similar results were obtained for T 3 (Fig.

3) and cortisol assays (Fig. 4). The ethanol extract of whole embryos without Sep-Pak purification also produced parallel displacement, but significant cross-reaction was observed after stripping (data not shown).

Morphometric measurements

For experiment #3, standard length oflarvae in ocean water and well seawater (30 larvae each) were measured using a digital camera equipped with Q Capture Pro software

(QImaging Designs, Surrey, Be, Canada) until day 11 post-hatch, and larvae at days 14,

17,20,23, and 25 post-hatch were measured using digital calipers. Gut content and gill deformities were analyzed using a dissection microscope, and survival at day 25 post­ hatch was determined by live counts during tranSfer of the larvae from the hatchery to nursery tanks. For experiment #4, standard length (50 larvae) was measured with Q

Capture Pro software for all sampling days and survival at day 25 was determined by estimated densities in transfer containers compared with a standard with a known count

Statistical analysis

Statistical analysis was conducted using two-way analysis of variance (ANDV A) to determine treatment differences and changes in T4, T3 and cortisol concentrations over time, followed by the least significant difference (LSD) test Dunnett's test was performed to determine significant differences in hormone concentrations compared with

12 values on day 1 or day 9 post-hatch (JMP IN 5.1 software; SAS Institute, Cary, NC).

Percent survival, wet weight, and standard length were analyzed by one-way ANDVA, followed by Student's (-test. Data are expressed as means ± SEM in the text and figures.

Statistical significance was accepted when P :s 0.05.

13 CHAPTERID

RESULTS

3.1 Effects ofbroodstock diet on thyroid hormone concentrations offertilized threadfin eggs, hatching rate, growth and survival ofresultant larvae at day 25 post-hatch

(Experiment #1.)

Thyroid hormone content in fertilized tbreadfin eggs varied significantly among broodstock groups fed three experimental diets. Broodstock fed a raw diet of squid and smelt daily and shrimp once weekly produced eggs containing 0.09 ± 0.018 nglg T4. significantly lower than 0.59 ± 0.099 nglg T4 for eggs from broodstock fed a commercial marine broodstock diet (Skretting) and 3.29 ± 0.11 nglg T4 found in eggs from broodstock fed a diet rich in iodine (Oceanic) (Fig. 5). Triiodothyronine levels in the same eggs showed even greater treatment effects. Broodstock receiving the raw diet produced eggs containing 0.40 ± 0.052 nglg T3. Significantly lower than 1.01 ± 021 nglg, and 6.43 ± 0.35 nglg T3 found in eggs from broodstock fed the commercial diet and iodine rich feed respectively (Fig. 6). Hatching rates of fertilized eggs were 78.5 ± 4.3% for the broodstock group fed the raw diet, 93.9 ± 4.2% for commercial feed group, and

75.4 ± 8.8% for broodstock fed iodine rich feed. Larvae hatched from eggs produced by broodstock fed the commercial diet grew to 14.97 ± 1.13 mm by day 25 post-hatch, and were significantly larger than larvae hatched from eggs produced by broodstock fed the raw diet, 11.30 ± 0.54 rom, and iodine rich feed 10.33 ± 0.43mm. Survival oflarvae to day 25 post-hatch, however, was not affected by broodstock diet (Fig. 7).

14 3.2 Effects ofKl supplementation to broodstock rearing water on thyroid hormone concentrations offertilized threadfin eggs, hatching rate, growth and survival of resultant larvae at day 25 post-hatch (Experiment #2).

Thyroid hormone concentration in fertilized threadfin eggs was significantly altered by

KI treatment of tank water. Broodstock fish held in tanks receiving a KI supplementation of 0.096 ± 0.001 mgIL iodide for forty-five days produced fertilized eggs containing 1.91

± 0.01 nglg T4. greater (P ~ 0.001) than 0.59 ± 0.01 nglg T4 which was found in eggs from the same broodstock reared prior to the study in seawater with an iodide concentration of 0.01 ± 0.001 mgIL (Fig. 8). Triiodothyronine levels were also elevated in eggs from the treated animals with a concentration of7.81 ± 0.80 nglg T3, higher (P ~

0.001) than 0.40 ± 0.02 nglg T3 for eggs spawned previous to KI supplementation (Fig.

9). Hatching rates offerti1ized eggs were 93.7 ± 4.2% for broodstock reared in KI enriched seawater and 78.5 ± 4.3% for the broodstock group reared in untreated well seawater. Standard length and wet weight of larvae at day 25 post were not different (P ~

0.05) among treatment groups. Larvae hatched from eggs produced by broodstock reared in KI-supplemented well seawater grew to 13.7 ± 0.63 mm and 59 ± 5.9 mg by day 25 post-hatch compared with 10.96 ± 0.8 mm and 40.6 ± 8.9 mg for larvae hatched from eggs produced by broodstock reared in untreated well seawater. However, the survival rate at day 25 post-hatch of 13 ± 2.8% was lower (P ~ 0.01) for larvae from KI-treated broodstock than the 32 ± 4.7% survival rate for larvae from untreated broodstock (Fig.

10).

15 3.3 Effects ofenvironmental iodide in rearing water on larval survival, growth, and profiles ofthyroid hormones and cortisol (Experiment #3)

Both pH (PH 8.2) and salinity (35.0%0) were higher for ocean water compared with well seawater (PH 7.8 and 31.8%0). Average water temperatures were 26.8 and 26.7 °C for ocean water and well seawater, respectively. As shown in Table I, total iodine concentration was not significantly different in ocean water and well seawater. Iodide (I) concentration however, was higher (p ~ 0.001) in ocean water than in well seawater, whereas iodate (103) concentration was higher (p ~ 0.001) in well seawater. Total ammonia nitrogen and nitrite concentrations were significantly higher in ocean water than in well seawater. There was no difference in nitrate concentrations between ocean water and well seawater. For both ocean water and well seawater, total ammonia nitrogen, nitrite, and nitrate concentrations remained within tolerable ranges for larval systems (Russo and Thurston, 1991).

There was no difference in hatching rate between the two groups, 74.9 ± 8.6% (n=6) in ocean water and 67.1 ± 4.4% (n=6) in well seawater. At time of stocking, eggs measured 0.78 ± 0.02 mm (n=20) in diameter, and standard length at hatching was 2.75 ±

0.17 mm (n=20). Yolk sac absorption was complete by days 3 and 4 post-hatch, at which time the larvae first began to feed. There was no difference in number of rotifers or

Artemia in the gut between larvae reared in well seawater and ocean water throughout the experiment (data not shown).

To evaluate developmental progress, larvae were subdivided into fom stages according to development of the axial skeleton (Kendall et al., 1984; Kim, 1999). These

16 stages included pre-flexion (2.9-5.3 mm in standard length, 3-15 days post-hatch), flexion

(5.3-7.01IlllL, 15 to 18 days post-hatch), post-flexion (7.0-141I1llL, 18 to 25 days post­ hatch) and juvenile (> 141I1llL, after day 25). Pre-flexion larvae had a straight notochord, fin envelop, and pigmentation limited to the anterior head region and yolk sac. Threadfin larvae at pre-flexion stage were generally transparent and poorly developed, but capable of swimming with pectoral fins and undulation. The flexion stage was marked by the posterior end of the notochord being deflected dorsally allowing development of the caudal fin, while dorsal and ventral fins became discrete rather than continuous.

Substantial pigmentation was seen along the entire body length. The post-flexion larvae gained improved locomotion with complete sets offins and lateral line. The end of the larval period was marked by transition to juvenile characteristics including ctenoid scale formation, vertically barred body pigmentation, and increased antagonistic behavior within the same cohort (Fig. 11).

The larvae reared in ocean water developed more quickly than those in well seawater.

On day 14 post-hatch, 50% of ocean water larvae reached flexion stage compared with

15% for those in well seawater, and by 17 day post-hatch 93% of ocean water and 80% of well seawater larvae reached flexion phase (Fig. 12). For both treatments, body length increased gradually during the first 2 weeks, followed by more rapid growth beginning on day 17. From day 14 to day 25, ocean water-reared larvae were significantly larger than those in well seawater (Fig. 13).

On day 25, larval growth, as measured by standard length and wet weight, was significantly greater in ocean water than in well seawater. Survival of the larvae in ocean

17 water at 25 days post-hatch was significantly greater than in well seawater, showing a nearly two-fold difference (Fig. 14).

Threadfin embryos 8 h before hatching (day 0) had 0.41 ± 027 ngfg T4. For larvae reared in ocean water, whole body T4 concentrations were maintained at 0.1-0.5 ng/g during the first 13 days post-hatch. On day 15, they showed a marked and significant increase to above 1 ngfg. The high levels were maintained until day 21. Thyroxine levels then declined by days 23 and 25. Larvae reared in well seawater showed a gradual rise in whole body T4 concentrations beginning on day 3. During days 7 and 13 post­ hatch, whole body T4 concentrations were significantly greater in the larvae reared in ocean water than those in well seawater. Larvae from both treatments had similar whole body T4 levels on day 15 post-hatch (Fig. 15A).

Whole body T3 concentrations were similar for both treatments throughout the study.

Threadfin embryos 8 h before hatch had 0.17 ± 0.02 ng/g T3. Triiodothyronine levels declined during the first week, reaching their lowest concentration of 0.02-0.04 ngfg on day 7 post-hatch. Whole body T3 concentrations then increased gradually to 0.3-0.4 ngfg by day 25 post-hatch (Fig. 15B).

Whole body concentrations of cortisol were low at 0.8 ngfg 8 h before hatch (day 0) and on day 1 post-hatch for both the larvae in ocean water and those in well seawater.

On day 3 post-hatch, the concentrations increased to 5-8 ngfg, and increased markedly and significantly to 28-30 ng/g on day 5, and high levels were maintained until day 25

(Fig. 15C).

18 3.4 Effect ofK1 supplementation ofrearing water on larval growth. survi1la~ and thyroid hormone profiles (Experiment #4)

Average water temperature was 26.5 °C for aIllarvaI rearing tanks with no one tank deviating significantly from the average. Total ammonia nitrogen and nitrate and nitrate levels in rearing water did not vary significantly among rearing tanks, and were within tolerable ranges as described in experiment #3. For both cohorts, those larvae reared in

KI-supplemented well seawater grew significantly larger, and developed more rapidly than larvae reared in well seawater with lower iodide concentration. At 13 days post­ hatch 42% of larvae reared in KI-treated seawater reached flexion stage compared with

26% of untreated larvae, and by 15 days post 79"10 oflarvae reared in iodide supplemented seawater had reached flexion stage compared with 66% oflarvae reared in well seawater without iodide supplementation (Fig. 16). In the first cohort (eggs from broodstock reared in untreated well seawater), larvae reared in well seawater with elevated iodide concentration (0.045 ± 0.001 mgIL) reached 11.76 ± 0.25 mm in length and 49.6 mg in weight by day 25 post-hatch, compared with 10.16 ± 0.11 mm and 31.7 mg for larvae reared in untreated well seawater with a lower iodide concentration (0.01 ±

0.001 mgIL). In the second cohort (eggs from broodstock reared in KI-treated seawater), larvae reared in KI-supplemented seawater reached 13.79 ± 0.27 mm in length and 64.8 mg in weight at day 25 post-hatch, compared with 12.54 ± 0.27 mm and 53.1 mg for larvae reared in untreated well seawater (Fig. 17 & 18). By day 25 post-hatch average length and weight of larvae reared in KI-supplemented seawater were 12.78 ± 1.02 mm and 57.2 ± 10.8 mg, while larvae reared in untreated seawater reached 11.35 ± 1.19 mm

19 and 42.4 ± 10.7 mg (Fig. 19). Survival to day 25 post-hatch however, was not different

(P:-:; 0.05) between treated and untreated larvae as 27.1 ± 7.3 % of larvae reared in KI­ treated seawater survived to day 25 compared with 29.3 ± 4.3% of untreated larvae (Fig.

20).

Thread:fin embryos from the first cohort at 8 homs before hatching (day 0) had 1.35 ±

0.54 nglg T4. lower (P:-:; 0.001) than 7.68 ± 1.69 nglg T4forthe second cohort. A significant decline from 7.68 ± 1.69 nglg T40n day 0 to 2.75 ± 0.27 nglg (control) and

3.55 ± 0.23 nglg (KI) by day 1 was observed in animals from the second cohort, while no decline in T4 was seen in the first cohort between day 0 and day 1. For larvae in the first cohort, whole body Tdevels rose significantly from 1.18 ± 0.54 nglgon day 1 to 3.38 ±

0.18 nglg T4 (control) and 5.27 ± 0.18 nglg (KI) by day 5 post-hatch. while no changes in whole body T4 were observed in larvae from the second cohort during the same period.

On day 5 post-hatch. larvae from the first cohort reared in KI-supplemented seawater had

5.27 ± 0.18 nglg T40 significantly higher than 3.38 ± 0.18 nglg for larvae in untreated well seawater. On the same day, larvae from the second cohort reared in KI-treated seawater also had a greater whole body T4 concentration of3.13 ± 0.15 nglg compared with 2.07 ±

0.14 nglg for larvae reared in well seawater not supplemented with KI. No consistent effect ofKI treatment to rearing water on whole body T4concentrations was observed past day 5 post-hatch. However, marked increases in T4 were seen on days 13 and 19 post-hatch in untreated and KI-treated larvae from the first cohort, and on day 13 post­ hatch for untreated larvae and days 13 and 21 post-hatch in KI larvae from the second cohort (Fig. 21).

20 Whole body T 3 profiles were similar to those for T4 from hatching to day 5 post-batch.

Threadfin embryos (day 0) from the first cohort bad 2.36 ± 0.10 ng/g T3, lower (P ~ 0.01) than 3.71 ± 0.31 ng/g T3 found in embryos from the second cohort. By day I post-hatch, a significant decline in whole body T3 to 1.22 ± 0.01 ng/g (control) and 1.3 ± 0.08 ng/g

(KI) in the first cohort and 1.57 ± 0.13 ng/g (control) and 1.82 ± 0.15 ng/g (KI) for the second cohort occurred. Triiodothyronine levels then increased to 2.01 ± 0.11 ng/g by day 3 and 1.79 ± 0.12 ng/g by day 5 in larvae from the first cohort reared in KI­ supplemented well seawater. No significant changes in T3 were observed between days I and 5 post-batch in larvae reared in untreated well seawater from the first cohort, or in larvae from the second cohort reared in either untreated or treated well seawater.

Triiodothyronine concentrations increased significantly after day 11 post-hatch in treated and untreated larvae from both cohorts, reaching their maximum levels on day 21 post­ batch (Fig 22).

21 Table 1. Iodine, total ammonia nitrogen, nitrite, and nitrate levels (mgIL) in larval rearing water.

Ocean water Well seawater

Total iodine 0.051 ± 0.010 (n=3) 0.050 ± 0.007 (n=3)

Iodide (f) 0.020 ± 0.001 *** (n=3) 0.010 ± 0.001 (n=3)

Iodate (103) 0.039 ± 0.001 ** (n=3) 0.046 ± 0.001 (n=3)

Total ammonia nitrogen 0.098 ± 0.019*** (n=6) 0.036 ± 0.009 (n=6)

Nitrite (NOi) 0.048 ± 0.014*· (n=6) 0.024 ± 0.004 (n=6)

Nitrate (N03) 2.615 ± 1.043 (n=6) 3.362 ± 0.734 (n=6)

Mean ± SEM (n=4) **, *** Significantly different from well seawater at P ::5 om, and

0.001, respectively

22 Fig. 1. Elution profiles of T4, T3. and cortisol through

SepPak Light CIS cartridge

23 T4 cortisol T3 ,... .000 ;-- "" """ ~ ''''' .... ""'" I-- E E E Q. Q. "'" e- - ~ ''''''' u ."""'" ''''' ''''''' lO'

10 10 JO 40 SO 600 70 10 20 JO 40 5(1 60 10 20 JO .w 5(1 60 70 10 ethanol (-;0) ethanol (%)

24 Fig. 2. Displacement curves for T4 standard and serial dilution of whole body extract oftbreadfin larvae.

25 Tissue extract

0.625 2.5 10 40mg 100

Sep-Pak extract,• stripped ~ , 80 , , ~, , ;i 60 , a - '~ Sep-Pak extract ,, ,, ~ T4standard 20

O~~------~------~----~------r------r------~- 0.025 0.1 0.4 1.56 6.25 25 100 T4 concenbation (nglml)

26 Fig. 3. Displacement curves for T3 standard and serial dilution of whole body extract oftbreadfin larvae.

27 Tissue extract

1.25 5 20 80mg 100 A..._ • -"'4 " Sep-Pak extract, stripped 80 " "~ _ 60 \ ~ \ -o \ Sep-Pak extract m 40 \ h 20 T3standard o 0.025 0.1 0.4 1.56 6.25 25 100 T3 concentration (ng/ml)

28 Fig. 4. Displacement curves for cortisol standard and serial dilution of whole body extract ofthreafin larvae.

29 Tissue extract

0.78 3.12 12.5 mg 100 Sep-Pak extract,• stripped

80 ~ '\ '\ '\ '\ ~..... 60 '\ o \ Sep-Pak extract '\ '\ m 40 '\ '\ '\ '\ 20 A Cortisol standard

0.02 0.10 0.39 1.56 6.25 25.00 Cortisol concentration (ng/ml)

30 Fig. 5. Effect ofbroodstock diet on T4 concentration in fertilized threadfin eggs. Mean ± SEM (n=4) Different letters indicate significant differences at P:5 0.01, and

0.001 (t).

31 4

- 3 ~c -c .-0 j 2 c uCD c u0 1 ~

a 0 Raw Skretting Oceanic

32 Fig. 6. Effect ofbroodstock diet on T3 concentration in fertilized tbreadfin eggs. Mean ± SEM (n=4) Different letters indicate significant differences P :S 0.001.

33 8

- 6 ~c -c 0 :;::I 4 ~c CD CJ C 0 CJ 2 ~

O ...... __...... --"T--'--- Raw Skretting Oceanic

34 Fig. 7. Effect ofbroodstock diet on body length and survival of resultant threadfin larvae at day 25 post-hatch.

Mean ± SEM (n=4) ** Significantly different at P $ 0.01.

35 Length

18 .-. 16 - E 14 E .c--.... 12 g' 10 .!! 8 1! lIS "a 6 C .s 4 U) 2 0 Raw Skretting Oceanic

Survival

50

40

10

o .L.-_--'-_----'-_ Raw Skretting Oceanic

36 Fig. 8. Effect on KI supplementation ofbroodstock rearing water on T4 concentration in fertilized tbreadfin eggs. Mean

± SEM (n=4) ••• Significantly different from well seawater at P $ 0.001.

37 2.5

2.0 *** -~ Q C -c -0 1.5 ic CDu 1.0 c u0 ~ 0.5

Well seawater Well seawater + KI

38 Fig.9. Effect ofK! supplementation ofbroodstock rearing water on T3 concentration in fertilized threadfin eggs. Mean

± SEM (n=4) ••* Significantly different from well seawater at P $ 0.001.

39 10

- 8 ~c c -o 6

4 1c B ~ 2

o~------~--~~---- Well seawater Well seawater + KI

40 Fig. 10. Effect ofK! supplementation ofbroodstock rearing water on body length. body weight, and survival of resultant tbreadfin larvae at day 25 post-batch. (n=2) .. Significantly different at P ~ 0.01.

41 Length Weight Survival 16 70 ~ 40 E l' ~BO... .!12 EBO ~30 i10 ~ c -li!40 .!! 8 g} 20 30 'E 6 1 :;20 1 ...•c , 40 10 :Ii: 10 ~ 2 0 0 0 _+K1 _+K1 _+K1 Broods'- ckrearlng_ Broocfstock- /88l'Ing_ -188IIng--

42 Fig. 11. Development ofthreadfin larvae in ocean water.

The time of development was expressed as days post-hatch.

Day 0; unhatched embryo, 0.76 mm, Day 1; hatched larvae,

2.9 mm, Day 7; pre-flexion stage larvae, 3.2 mm, Day 14; pre-flexion stage larvae, 4.8 mm, Day 17; larvae at flexion stage, 5.6 mm, Day 23; post-flexion stage larvae, 12.3 mm,

Day 25; juvenile threadfin, 14.6 mm.

43 1mm . ··················: ...... , .... . Day 0 cr\"

~~~:::E.! Day 7 ' ... --.

Day 17

1.5mm

Day 23

Day 26

44 Fig. 12. Effect of rearing water on percent ofIarvae at flexion stage on days 14 and 17 post-batch.

45 100 _ Well seawater IZ2J Ocean water 80 -~ -GI ~ CIS 60 -GI ." J!1 III 40 c 0

--=u. 20

0..1..---- 14 17 Days post hatch

46 Fig. 13. Growth of tbreadfin larvae in ocean water and well seawater. Mean ± SEM (n=3) *, ** Significantly different from well seawater-raised larvae at corresponding time point at P:5 0.05 and O.oI, respectively.

47 16 Well seawater * 14 -<>-• Ocean water -E E 12 .c-.... DI 10 C CD -'E 8 ca "C C J!I 6 tn fl--** ...... - 4

2 3 5 7 9 11 14 17 20 23 25 Days post hatch

48 Fig. 14. Body length, body weight, and survival of threadfin larvae at day 25 post-hatch in ocean water and well seawater. Mean ± SEM (n=3) "', "'''' Significantly different from ocean water-reared larvae at P :5 0.05 and

0.01, respectively.

49 Length Weight Survival

15 • .. 8 80 • 114 'iii _8 .sIlO ~ ~ 13 l! oS! 40 2 1 14 "'2" 11 i20 rfII 10 __ Ocean_ 0 0 WelI_Ocean _

50 Fig. 15. Effects oflarval rearing water on developmental changes in whole body concentrations ofT", T3, and cortisol. Mean ± SEM (n=3) "', ••, **'" Significantly different from ocean water-raised larvae at P $ 0.05, P $

0.01, P $ 0.001, respectively. t Significantly different from values on day 1 post-hatch for ocean water larvae at P $

0.05.

51 3.5 ** A ** 3.0 1::::.-:= 1 t t ~2.5 l t i! 2.0 1.5 j * I I .!l 1.0 I 'i\ , \ J ~ I 0.5 -~'~-§:--2,-i 't' 0.0 01 3 5 7 9 11 13 15 17 19 21 2325 Days post hatch 0.7

0.6 B

-~ 0.5 S ~ 0.4 , i 0.3 .!l 0 0.2 i 0.1

0.0

01 3 5 7 9 11 13 15 17 19 21 23 25 Days post hab:h 40

35 C -~ l 30 "0 25 ~ 8 20 I f 15 I .!l 0 10 i 5 0 01 3 5 7 9 11 13 15 17 19 21 2325 Days post hab:h 52 Fig. 16. Effect ofKI supplementation to rearing water on percent of1arvae at flexion stage on days 13 and 15 post­ hatch.

53 100 _ Well seawater CZ2I Well seawater + KI 80 -'#. -CD

(II~ 60 -CD OJ S II) 40 c 0 i iL 20

0-'------13 15 Days post hatch

54 Fig. 17. Standard length of tbreadfin larvae reared in well seawater and Kl-supplemented well seawater. Cohort 1 from broodstock reared in untreated well seawater and fed a mw diet. Cohort 2 from broodstock reared in Kl­ supplemented well seawater and fed a mw diet.

55 Cohort 1

14

12 I~==+~I ...... 0 -E .0· S 10

iC .•..0.········.0· .!! 8 'E ~c 6 ~ 4

2 9 11 13 16 17 19 21 23 26 Days post hatch

Cohort 2

16

14 -E -E 12 10 .0· iC .!! 'E 8 ...... 0...... 0...... ~ c 6 ~ 4

2 9 11 13 16 17 19 21 23 26 Days post hatch

56 Fig. 18. Wet weight ofthreadfin larvae reared in well seawater and KI-supplemented well seawater. Cohort 1 from broodstock reared in untreated well seawater and fed a raw diet. Cohort 2 from broodstock reared in KI­ supplemented well seawater and fed a raw diet.

57 Cohort 1

70 60 I~~:=+~I 50 /0 m / - / .§.40 ;f 1: / ~30 / ; p' .. 20 ,/," ~ 10

0

9 11 13 15 17 19 21 23 25 Days post hatch

Cohort 2

70 p I 60 I I I 50 I -m /d .§.40 / / 1: /f ~ 30 / ; / 20 J:I I " 10 "

0 --

9 11 13 15 17 19 21 23 25 Days post hatch

58 Fig. 19. Standard length and wet weight ofthreadfin larvae

(Cohorts 1 and 2) reared in well seawater and KI­ supplemented well seawater. Mean:l: SEM. 16

...... Weliseawale' - 14 -0- WeUseawaIe,+KI ! 12 ~ 10 ; l! 8 ! 6 4

9 11 13 15 17 19 21 23 25 Days post hatch

70

60

50 CI -.5.40 l: / !}) 30 /{/ ; / ... 20 ~ ~ 10 "

0

9 11 13 15 17 19 21 23 25 Days post hatch

60 Fig.20. Effect ofK! supplementation oflarval rearing water on survival oftbread:fin larvae at day 25 post-hatch.

Mean±SEM.

61 35

30

25

-~ - 20

I::J 15 en 10

5

0-1....----....; Well seawater Well seawater + KI

62 Fig.2l. Effects ofKI supplementation of larval rearing water on developmental changes in whole body concentrations ofT4 and T3 (Cohort 1). Mean ± SEM (n=5)

*, **, *** Significantly different at P::5 0.05, P::5 0.01, P::5

0.001, respectively. Significantly different from values on day 1 or day 9 post-hatch for (t) well seawater larvae and

(l) KI treated larvae at P ::5 0.05.

63 Cohort 1

8 __ WeD seawater 7 :J: -0- Wen seawater + KI -6 t ~ aJ *** c -5 :J: ~ j! I ~4 tl .8 * .! 3 .c0 ~ 2 1

/ 0 / 01 3 5 9 11 13 15 17 19 21 23 25 Days post hatch

4.0

3.5 :J: -~ 3.0 c t -~ 2.5 >- .a"8 2.0 .! 0 1.5 ~ 1.0

/ 0.5 / 01 3 5 9 11 13 15 17 19 21 23 25 Days post hatch

64 Fig. 22. Effects of KI supplementation of larval rearing water on developmental changes in whole body concentrations ofT4 and T3 (Cohort 2). Mean ± SEM (n=5)

*, **, *** Significantly different at P:5 0.05, P :5 0.01, P :5

0.001, respectively. Significantly different from values on day 1 or day 9 post-hatch for (t) well seawater larvae and

(X) KI treated larvae at P :5 0.05.

65 Cohort 2

8 __ WeUseawatBr 7 -o--WeUseawatBr+Kl -& ~c -5 ~ :j: ~4 t .8 ** .!! 3 o j 2 1

o ~rr~.-~/~/-'--r-.--.-'r-.--r-'--r--- 01 3 5 9 11 13 15 17 19 21 23 25 Days post hatch

4.0

3.5 :j:

CD t -0, 3.0 c -C'I I- 2.5 ~ .8 2.0 .!! o j 1.5 1·0

01 3 5 9 11 13 15 17 19 21 23 25 Days post hatch

66 . CHAPTER IV

DISCUSSION

In the present study, environmental iodide concentrations were examined in relation to thyroid hormone content in fertilized eggs, larvai growth, survival, metamorphosis, and whole body concentrations of thyroid hormones and cortisol in Pacific threadfin.

Additionally, the relationship among broodstock diet and thyroid hormone content in the fertilized eggs and performance of resultant larvae was investigated.

Iodide deficiency inhibits thyroid hormone synthesis, causing the overproduction of thyroid stimulating hormone (TSH) (Eales, 1979). Recently, Mukhi and Patino (2007) reported that prolonged exposure ofzebrafish to perchlorate resulted in reduction in whole-body T 4 concentration in embryos, accompanied by significant morphometric differences. It has been shown that the seawater-injected well supplying Sea Life Park and the Oceanic Institute at Waimanalo, Hawai'i, has low levels ofiodide (less than

0.005 )JM) as compared with ocean water. Strong evidence indicates that the occurrence of goitre and hypothyroidism in white tip reef sharks held at Sea Life Park is related to low environmental iodide. After 2 weeks of transfer to a seawater lagoon at the Hawai'i

Institute of Marine Biology with higher levels ofiodide (0.15 JlM), thyroid status was stabilized with plasma 1H levels returning to levels comparable to those found in wild animals, and within 3 weeks there was significant reduction in the goitre diameter (Crow et ai., 1998).

67 In the present study, total iodine in well seawater (0.050 mg/L) was nearly equal to ocean water (0.051 mgIL), demonstrating that the concentration of total iodine is largely unaffected by the basanite lava rock, through which ocean water passes before recharging the seawater aquifer. However. iodide (f) concentration of well seawater (0.01 mg/L or

0.077 pM) was half of ocean water (0.02 mg/L or 0.154 pM), while iodate (I

(0.039 mgIL). Oxidation of iodide to iodate within the aquifer may account for this difference. Artificial seawater had reduced iodide levels after aerating and ozone treatment (Sherrill et al., 2004). Phytoplankton also has the ability to affect the chemical speciation of iodine. Cold water diatoms, Nitzschia spp. and Navicula spp., were shown to consume iodide during the stationary growth phase (Chance et al., 2007). The observed depression in iodide concentration and subsequent elevation of iodate concentration, in the well seawater likely results from oxidation of iodide to iodate, since the aquifer at Sea Life Park is known to have high ~ levels (Crowet al., 1998).

The presence of goitres in a large number of threadfin broodstock held at Oceanic

Institute, and the reported deficiency in iodide in the rearing water supports the hypothesis that certain broodstock threadfin at Oceanic Institute are hypothyroid. Of 14 broodstock mortalities reported to be attributed to goiters, 12 animals were receiving a raw feed which resulted in the production of eggs with low thyroid hormone content, and two broodfish were being fed a commercial marine broodstock feed (Skretting) shown to elevate thyroid hormone levels in spawned eggs (C. Laidley, unpublished data).

Verification of the broodfish's hypothyroid status, through measurement of plasma

68 thyroid hormone levels, was not undertaken, however, due to the high risk of disrupting the animals spawning cycle.

The presence of thyroid hormones in fish eggs is well documented (Kobuke et aI.,

1987; Tagawa and Hirano, 1987; Greenblatt et aI., 1989; Leatherland et aI., 1989a;

Tagawa et aI., 1990b). Ayson and Lam (1993) showed that thyroxine injection of female rabbitfish elevated both T4 and T3levels in maternal plasma and eggs, demonstrating transfer of both hormones from maternal circulation into eggs. Treatment offemale medaka broodstock with thiourea reduced plasma T4 and T3levels, with egg T4 and T3 levels subsequently falling to one-fourth of their initiaIlevels (Tagawa and Hirano,

1991). Thyroxine and T3 concentrations in embryos from goitred Coho salmon from

Lake Erie, were lower than in embryos at similar developmental stage from mildly goitred Lake Michigan and non-goitred British Columbia stocks (Leatherland et aI.,

1989b). It is, therefore, likely that hypothyroid tbreadfin females produce eggs with low thyroid hormone content.

In the current study, tbreadfin broodstock were held in seawater supplemented with

Kl, or were fed experimental diets with elevated levels of iodine to determine whether the treatments had an effect on thyroid hormone levels in spawned eggs. Broodstock reared in well seawater CODtaining a low concentration of iodide produced eggs with significantly lower T4 and T3 than was observed in eggs from the same broodstock held in iodide supplemented well seawater. Thyroid hormone levels were aIso elevated in eggs produced by broodstock reared in iodide-deficient water, but fed an iodide-rich feed

(Oceanic) or commerciaI marine broodstock feed (Skretting). These results support the hypothesis that spawning adults fed a raw diet of squid and smelt daily and shrimp once

69 weekly, had sub-optimal plasma thyroid hormones levels, and that KI supplementation to rearing water, or a diet rich in iodide, allowed for improved thyroid function. The increased concentration of thyroid hormones in fertilized eggs could mark a return to optimal hormone transfer from maternal plasma to egg, or possibly an abnormally high deposition. Hickman (1959) demonstrated a compensatory iodide trapping response by thyroid follicles of starry flounder held in iodine-supplemented water. The goitred thread:fin broodfish in the present study likely had elevated TSH secretion and hypertrophy of thyroid fullicles. An abrupt increase in iodide in the rearing water from the KI treatment may have provided broodstock anirrials with the required raw materials for thyroid hormone synthesis, presuming successful uptake of iodide at the gill and gut

(Eales, 1979) and an adequate supply of tyrosine. Nevertheless, there is minimal research that supports this claim, and Hickman (1959) reported no significant increase in circulating T4 following a marked increase in radioiodide accumulation in the starry flounder. It would be of value to measure thyroid hormone levels in eggs from wild moi.

No such measurements have been reported, however, and therefore, no estimation of what constitutes normal hormone levels can be made with confidence.

It has been demonstrated that the teleost ovary concentrates iodide from the blood

(Robertson and Chaney, 1953; Hmm and Reineke, 1964), and that a single intra­ peritoneal injection of either KI or NaI into rainbow trouJ significantly raised plasma T4 above levels in saline-injected controls (Eales et aI., 1986). It is possible that the deposition of thyroid hormones into eggs over a continuous spawning cycle may have depleted a significant amount of thyroid hormone that was not quickly replenished due to a lack of available iodide required for thyroid hormone synthesis. Iodide was not

70 detectable (~.01 mg/L) in tbreadfin broodstockrearing water, although well seawater supplying the tanks had 0.01 mg/L iodide. Oxidation ofiodide to iodate, within the tank during the seawater residence may have accounted for this difference. Threadfin broodstock tanks are heavily aerated and it has been demonstrated that aeration of seawater shifts the speciation of iodine from iodide to iodate (Sherrill et al., 2004).

No clear link was observed between thyroid hormone content in fertilized eggs and larval performance. In the broodstock diet trial, larvae hatched from eggs with elevated thyroid hormone levels (Skretting) grew larger than larvae from eggs with lower thyroid hormone content (Raw), while other larvae from eggs with elevated thyroid hormone concentration (Oceanic) failed to show significantly improved growth. Ayson and Lam

(1993) reported greater standard length in 7 -day-old rabbitfish larvae hatched from eggs spawned from T4-treated females. Tagawa and Hirano (1991) observed no significant differences in hatch rate, time to hatching, and survival under starvation ofMedaka between normal and thyroid hormone-deficient eggs. The authors suggested that a large fraction (>90"10) of thyroid hormones contained in eggs may not be required for early development. The observed differences in growth and survival in the present study may be related to differences in egg quality, such as fatty acid content, and may be independent of thyroid hormone concentration. Broodstock receiving KI treatment were held in indoor tanks for technical reasons and all other broodstock tanks were outdoors possibly leading to significant differences in the quality of spawned eggs. Variation in larval density in rearing tanks may also affect growth and survival. Low densities of reared larvae may lead to increased growth and possibly improved survival (A.

Moriwake, personal comm.).

71 In the ocean water larvaI rearing maI, the T4 concentration in newly fertilized thread:fin eggs (0.41 nglg) was similar to levels observed in fertilized eggs of Japanese flounder; T3 concentration (0.17 nglg) was much lower than that in flounder eggs (de

Jesus et aI., 1991). Tagawa et aI. (1990b) reported that the concentration ofT4 is generally higher than T3 in eggs and larvae from freshwater fish. while in the eggs and larvae of marine fish. T 3 is sometimes higher than T4. According to Mukhi and Patino

(2007) however, T 3 concentrations were significantly higher than T4 concentrations in zebrafish embryos in freshwater. The thyroid hormone balance in eggs probably reflects that of the maternal plasma, but the cause of higher T4levels than T3 in threadfin eggs in this 1ria1 remains unknown.

Thyroid hormones govern growth and development in many vertebrates, including teleost fish. and it is well documented that metamorphosis in flatfish and parr-smolt transformation in salmonids are regulated by thyroid hormones that undergo a well defined peak at the time of metamorphosis (Eales, 1979; Power et aI., 2001; Blanton and

Specker, 2007). In this study of thread:fin embryos, both T4 and T3 concentrations in eggs declined markedly before hatching, in accordance with observations in other species studied (Tagawa et aI., 1990b; Power et aI., 2001). Nevertheless, it remains to be determined whether thyroid hormones in egg yolk are available to the embryo, and ifso, how they function.

There were significant changes in whole body T4 concentrations throughout development of the thread:fin larvae. In ocean water-reared animals, T4 remained below

0.5 ng/g until day 15 post-hatch. at which time there was a distinct peak to 1.9 nglg. The sharp rise in T4 was then followed by a gradual decline through days 23 and 25. The

72 peak in T4 at day 15 post-hatch was closely correlated with larval transition from pre­ flexion stage to flexion stage, at which time the larvae begins to display juvenile characteristics, including pigmentation of the lower body, improved locomotion resulting from fin development, as well as some degree of benthic settling. A similar peak in tissue T4 has been observ.ed during metamorphic climax of Japanese flounder (Tagawa et al., 1990a; de Jesus et aI., 1991), conger eel (Yamano et aI., 1991) and black sea bream

(Tanaka et aI., 1991). For larvae reared in well seawater, whole body T4 gradually increased throughout the larvaI period reaching its highest level on day 25. Absence of a peak in the T4 profile in well-water larvae may indicate incomplete synchronization of development or metamorphosis within the cohort.

Nevertheless, the effects of environmental factors other than iodine aVailability on larval performance should not be dismissed. It is well established that external parameters such as water temperature, photoperiod, osmolality, and feeding regime directly influence fish growth (Boeuf and Payan, 2001; Varsamos et aI., 2005; Johnston,

2006). While the average water temperature of each rearing system was similar throughout the current study (26.7 and 26.8 for well seawater and ocean water, respectively), the ocean water system had greater daily fluctuation. The salinity (35.0%0) and pH (8.2) of the re-circulated ocean water were slightly higher than those in the well seawater (31.8%0, pH7.8) over the course of the study; however, it is unlikely that larval growth was significantly influenced by these differences (Boeuf and Payan, 2001;

Johnston, 2006). In the current study, chemical analysis of the rearing water was limited to iodine, iodide, iodate, total ammonia nitrogen, nitrate, and nitrite. Thus, it is possible

73 that compounds other than those monitored may have contributed to the observed treatment effects.

In the larval rearing trial for which well seawater was supplemented with Kl, there were significant differences in whole body T4 and T3 between treated and untreated animals during early development. Elevated T4levels in Kl-treated larvae on days 3 and

5 post-hatch may be associated with increased iodide availability. Similar increases in whole body T3levels, on days 3 and 5 post-hatch, may be linked to iodide availability or to increased T4. The elevation of both hormones in treated animals was most pronoUI1ced in larvae hatched from eggs with low thyroid hormone content suggesting that larvae potentially have the ability to compensate for a deficiency in matemaI thyroid hormone deposition into eggs.

Thyroid hormone treatment of eggs and larvae has been found to increase larval survival in several teleost species (Lam, 1980; Lam and Sharma, 1985; Reddy and Lam,

1992). Immersion of newly fertilized eggs of Pacific threadfin larvae in solutions ofT3 improved larval survival, growth, and development (Brown and Kim. 1995; Kim and

Brown, 1997). The effects of matemaI exposure to thyroid hormones on larval survival have been less consistent, enhancing survival in some species, while remaining ineffective in others (Brown et aI., 1989; Ayson and Lam, 1993; Hey et al., 1996;

Tachihara et al., 1997; Tachihara and Obara, 2003). Immersion of embryos and larvae with thyroid hormones or hormone injections into mature females may not be practical to large-scale hatchery production; therefore alternative approaches to synchronizing growth and metamorphosis are needed. In the event that seawater supplying a marine hatchery is deficient in iodide, directly supplementing the incoming seawater may allow larvae to

74 acquire adequate iodide. An alternative approach that may be suitable for delivery of

iodide to marine larvae is through enrichment of live feeds. Atlantic halibut larvae fed

wild zooplankton. rich in iodine, had elevated T4levels compared with larvae fed

enriched Artemia (Solbakken et al., 2002).

In the ocean water larval trial, growth of the threadfin larvae was close to uniform

during the first 2 weeks after hatching. Differences in the tota1length oflarvae increased

as the animals reached the flexion stage near day 15 post-hatch. At this same time the

larvae began to feed on Artemia nanplii in addition to rotifers, and shortly thereafter

transitioned to a dry crumble starter feed. Kim (1999) observed that threadfin larvae

undergo metamorphic change at 18 days post-hatch. with secondary development of tin

rays, elongation of the caudal region. significant increases in melanophores, and

activation of the gastric glands. This is in agreement with present observations on

developmental changes and peak in tissue T4 concentration. Hatched threadfin larvae

possess few melanophores and during the pre-flexion stage pigmentation is limited

mainly to the gut cavity. Shortly after entering the flexion stage, moi larvae showed

distinct increases in pigmentation in the head region. along the notochord including the

caudal peduncle, and at the base of tin rays. At this same time, reflective silvery

pigmentation. possibly from purine based iridophores (Fujii, 1993), appears at the base of the tin rays. As the larvae transition to juveniles vertical barring appears, one bar

posterior to the opercula near to the base of the pelvic fin, and a second at the base of the

anal fin.

Metamorphic change signifies the onset on cannibalistic behavior, as the flexion stage

larvae initiated antagonistic and predatory behavior within their cohort. In larviculture,

75 intra- and inter-cohort cannibalism of siblings is of primary concern (Hecht and Pienaar,

1993; Bams et aI., 2000). The survival of ocean water-reared larvae was significantly

greater than in well seawater-reared larvae, displaying a nearly two-fold difference at day

25 post-hatch. Larvae reared in ocean water not only developed more quickly than larvae

reared in well seawater, but varied less in size; by day 14 post-hatch 500/0 of ocean water­

reared larvae had reached flexion stage compared with 15% for well seawater-reared

larvae. Similar trends in developmental rates were seen in larvae reared in KI­

supplemented seawater. The synchronization of growth and development in the larvae

kept in ocean water may have reduced size variation within the cohort, thereby

minimizing sibling cannibalism and increasing survival at the end of the larval period.

Cortisol, the major corticosteroid secreted from the interrenal gland in teleosts, plays

important roles in stress response, and also in the regulation of carbohydrate metabolism

and hydromineral balance (WendeIaar-Bonga, 1997; Mommsen et aI., 1999). The

fertilized eggs of several teleost species are known to contain cortisol, the content of which declines rapidly during early development and increases again during metamorphic

climax (de Jesus et aI., 1993); (de Jesus and Hirano, 1992; Huang et aI., 1992; Deane and

Woo,2003). In experiment #3, fertilized eggs of Pacific tbreadfin contained small

amounts of cortisol (0.5 nglg), which increased significantly to 6-9 nglg at day 3 post­

hatch, and increased further to 15-30 nglg from days 5 to 25. Similarly, fertilized eggs of

pelagic mi1kfish and yellowfin bream revealed low cortisol levels, being undetectable

from hatching until the second or third day after hatching, increasing significantly

thereafter (Huang et aI., 1992). In the Japanese flounder, cortisol enhanced the

stimulating action of thyroid hormones on dorsal fin-ray resorption in vitro (de Jesus et 76 al., 1990; de Jesus et al., 1991). According to Brown and Kim (1995) and Kim and

Brown (1997), newly-hatched larvae ofPacitic threadtin exposed to T3 and cortisol by immersion for 1 h exhibited earlier development than untreated controls and larvae exposed to the individual hormones. Survival to 29 days of age was also improved in the hormone-treatment groups as compared with untreated controls, clearly indicating development-promoting interactions ofT3 and cortisol.

The increased cortisol content after hatching can only be explained by embryonic synthesis. Flik et al. (2002) observed that embryonic stage carp, sti11 protected by the egg membrane, are able to produce cortisol when the embryo is disturbed by manipulation of the egg with forceps. ACTH is produced very early during development of carp. Thus, the ACTHlinterrenal axis seems to be operational at early stages not only in carp but also in other species, including Pacific threadfin It is highly probable that the fluctuation in cortisol levels observed in the current study during days 5-25 may be due to stress cansed by some disturbance. For that reason, it is crucial to know when and how very young fish express their first stress responses (Flik and Wendelaar Bonga, 2001), and how one can avoid such responses or how they become exaggerated in laboratory and aquaculture settings. The fact that the fish is equipped with an endocrine stress axis some time before, but certainly upon hatching, clearly underlines the need to handle fertilized eggs and tish larvae with care.

Results from the present set of experiments suggest that threadtin larvae experience more distinct metamorphic changes than previously reported, and that developmental progress may be linked to changes in thyroid hormone levels. In ocean water-reared larvae, a distinct surge in whole body T4. and elevated T3 concentrations corresponded to 77 mpid morphological development including increased pigmentation, fin development and increased growth. The fact that subsequent rearing trials showing distinct peaks in T4 and marked increases in T3 as larvae underwent metamorphic changes supports this interpretation. Low levels of environmental iodide in broodstock rearing water, as well as a diet of frozen squid, lake smelt and shrimp appears to be associated with both the prevalence of goitred broodstock, and the production of fertilized eggs with low concentrations of thyroid hormones. Iodide supplementation ofbroodstock tank water using a simple infusion ofK! to rearing water, or feeding broodstock a commercial marine broodstock feed (Sk:retting) or a pelleted feed rich in iodide (Oceanic) resulted in a significant elevation of both T4 and T3 concentrations in fertilized eggs. The observed changes in thyroid hormone concentrations in threadfin eggs and larval performance may be specific to conditions at Oceanic Institute and are likely related to the well seawater supplying the broodstock tanks and hatchery having a low concentration of iodide. These findings may, however, be applicable to any marine aquaculture facility that is supplied with seawater deficient in iodide. Additional investigation is needed to confirm whether a relationship exists between marine teleost physiology and environmental iodide aVailability.

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82