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