500 G.L. JOURNAL CROW ETOF AL. EXPERIMENTAL ZOOLOGY 284:500–504 (1999) Serum T4 and Serum T3 Concentrations in Immature Captive Whitetip Reef Sharks, Triaenodon obesus GERALD L. CROW,1* BENNY RON,2 SHANNON ATKINSON,2 AND L.E.L. RASMUSSEN3 1 Waikiki Aquarium, University of Hawaii, Honolulu, Hawaii 96815 2 Hawaii Institute of Marine Biology, Kaneohe, Hawaii 96744 3 Oregon Graduate Institute, Beaverton, Oregon 97006 ABSTRACT Serum T3 (3,5,3′ triiodothyronine) and serum T4 (thyroxine) concentrations were repetitively assayed by radioimmunoassay over a three-year period in two male and two female immature captive whitetip reef sharks, Triaenodon obesus. These sharks were maintained at the Waikiki Aquarium, Honolulu, Hawaii, in an open system holding pool receiving 568 liters per minute of water from a saltwater well with an iodide concentration of 0.076 mg/liter. No significant male-female difference was observed for either serum T3 or serum T4. No seasonal pattern of serum T3 was detected (P = 0.07). Serum T3 concentrations ranged (mean ± SEM) from 0.52 to 0.83 ng/mL (0.67 ± 0.01; n = 64). A significant seasonal difference was observed for serum T4 (P < 0.001). Serum T4 concentration was higher in winter (October–January) with a mean (range ± SEM) of 6.58 ng/mL (1.48–8.77 ± 0.35; n = 24) and lower in summer (May–August) with a mean of 3.62 ng/mL (1.34–5.71 ± 0.22; n = 24). The thyroid hormone T4 has a seasonal rhythm even in immature sharks and may have an important role in physiology. J. Exp. Zool. 284:500–504, 1999. © 1999 Wiley-Liss, Inc. Thyroid hormones are believed to be involved in development, growth and reproduction (Norris, ’85) in teleost fishes, but currently, little is known about circulating concentrations of 3,5,3′-triiodothyronine (T3) and thyroxine (T4) in sharks (Leary et al., ’99). The thyroid gland in sharks is an encapsulated organ located in loose connective tissue between the ventral side of the coracohyal and the medial side of the coracomandibular muscles (Homna et al., ’87). Thyroid tissue is follicular and the principal hormone secreted in sharks is T4, with no detectable levels of T3 (Suzuki et al., ’75). Thyroidal T4 secretory activity is presumably adjusted to maintain a constant plasma T4 level according to a given physiological state (Eales and Brown, ’93). Serum “free” T4 feeds at both the pituitary and hypothalamic levels and inhibits thyroid stimulating hormone release (Eales and Brown, ’93). Thyroid function is regulated by the hypothalamo-pituitary-thyroid control of the supply of T4, and peripheral control of T4 conversion to T3 for tissue use (Leatherland, ’94; Eales, ’95). Circulating T4 is thought to be relatively inactive physiologically and is converted extrathyroidally to T3. For detailed reviews of thyroid measurement and regulation in teleost fish, see Eales and Brown (’93) and Leatherland (’94). The thyroid gland in mature Squalus acanthias © 1999 WILEY-LISS, INC. was reported to have a seasonally related increase in weight and histologic development (Woodhead, ’66) and was gender size-related in Raja porosa (Homna et al., ’87). However, no significant sexual difference was detected in the thyroid gland of Scyliorhinus torazame, Rhizoprionodon acutus, and Mustelus manazo (Homna et al., ’87). Immature S. acanthias showed no significant changes in thyroid weight or histologic development throughout the year (Woodhead, ’66). Lewis and Dodd (’76) reported no significant sexual difference in plasma T4 and plasma T3 concentrations of the small-spotted catshark, Scyliorhinus canicula. A significant seasonal difference was reported for T4 and T3 with peaks occurring in summer and autumn (Lewis and Dodd, ’76); however, this study did not report thyroid hormone concentrations. Stress of capture and captivity reduced T4 and T3 concentrations in sharks (Lewis and Dodd, ’76; Battersby et al., ’96). In addition, thyroid hormone concentrations may be influenced by age, reproductive Grant sponsor: NOAA; Grant numbers: NA36RG0507, -JC-98-09. B. Ron’s current address: Israel Oceanographic and Limnological Research LTD, Eilat, 88112, Israel. *Correspondence to: Gerald L. Crow, Waikiki Aquarium, 2777 Kalakaua Ave., Honolulu, HI 96815. E-mail: email@example.com T4 AND T3 CONCENTRATIONS IN WHITETIP REEF SHARKS state, overall animal health, food intake, dietary composition, water temperature, salinity, pH, aquatic iodide concentrations, time of day, and perhaps stress factors like population density. As part of our exploration of hormonal concentrations of the whitetip reef shark, Triaenodon obesus, we measured serum thyroid hormone concentrations. Repeated serial blood sampling of individual sharks may provide an insight into the hormonal regulation and physiology of this species and in this study, we examined possible seasonal changes in serum T4 and T3 concentrations. MATERIALS AND METHODS Four immature whitetip reef sharks, two males and two females, were studied from December 1988 through December 1991. The sharks were maintained in a multi-species indoor exhibit 6 × 3 × 1 m containing 18,000 liters at the Waikiki Aquarium, Honolulu, Hawaii. Water parameters obtained from a seawater well and from a Hawaii coastal lagoon are shown in Table 1. The exhibit had a highly variable light/dark cycle. The light period ranged from 9–14.5 hr and the dark from 9–15 hr. The sharks were fed, at an approximate 6–10% body weight per week, a ration of squid (Loligo sp.), capelin (Mallotus villosus), and occasionally herring (Clupea harengus). All sharks appeared to be adapted well to the exhibit (i.e., eating and interacting well with tank mates). The immature sharks at the start of the study ranged from 52 to 63 cm and grew to 74–97 cm in precaudal length at the end of the project. Blood samples (3 mL) were collected at approximately monthly intervals by caudal venipuncture (Stoskopf et al., ’84) using 20–21 gauge 1.5-inch TABLE 1. Water parameters at the Waikiki Aquarium and Hawaii coastal lagoon with nutrient and oxygen concentrations in mg/liter Parameter Temperature (°C) Salinity (ppt) pH Oxygen Calcuim NH4+ NO3– DON Total N PO43– Total P Iodide Iodate Total iodine Tank Lagoon 25–26.5 33–34 7.67–7.85 5.5–6.5 393 0.026 0.344 0.077 0.447 0.042 0.046 0.076 0.165 0.241 24–28 34–35 8.2–8.3 6.5–7.0 398 0.012 0.014 0.140 0.167 0.005 0.013 0.019 0.034 0.058 501 needles. To minimize, but not eliminate variation resultant from circadian rhythm factors, samples were taken at 06:30–09:00. All blood samples were obtained within 45 sec while the animal was gently restrained. The samples were placed on ice and allowed to clot for 1–3 hr, then centrifuged for 15 min at 1380g. The supernatant sample (serum) was stored at –20°C until thawed for analyses in September 1996. Serum T3 and T4 concentrations were measured in duplicate and determined by radioimmunoassay as described in Brown and Eales (’77) with modifications. Miniature sephadex columns with 0.45 g of G25 fine (Sigma, St. Louis, MO) were placed in 5 mL syringes. The columns were normalized to improve repeatability. All columns were run without samples and then only columns with counts per minute for the bound fraction within 5% of each other were used for sample analysis. Phosphate buffer (0.1 M sodium phosphate dibasic heptahydrate, 31.1 mM disodium ethylenediamine tetra-acetate in deionized-distilled water, pH adjusted to 7.5 with 10 N NaOH) was used in both the T3 and T4 assays. The T3 and T4 measurements were conducted in separate assays. 125 T3 and 125T4 were placed in 100 µL of 0.1 N NaOH and added to each column. The 125T3 and 125 T4 both had specific activities of 1080–1320 µCi/ µg (product no. NEX-110H and NEX-111H; Dupont, NEN, Boston, MA). Lyophilized antisera (T3 product no. T-2777 and T4 product no. T-2652; Sigma Chemical Company) was reconstituted and diluted 1:10 with deionized-distilled water. At the time of the assay, the T3 or T4 antisera was further diluted using phosphate buffer (750 µL/column) for a final dilution of 1:10,000. Twelve T3 and T4 standards were prepared in 0.1 N NaOH with concentrations ranging from 0 to 100 ng/mL. The precision of the measurement of thyroid hormones between and within assays was characterized by replicate measurements of sera pools in each assay. Intraassay precision, defined as the coefficient of variation of the T3 or T4 concentration of six duplicate measurements, was 5.7% and 6.4% respectively. Interassay variability was 9.3% (n = 6) and 10.2% (n = 6) for T3 and T4 respectively. Sensitivity of the assay, defined as the apparent T3 or T4 concentration two standard deviations below counts of maximum binding or as concentration at 95% B/Bo, was approximately 0.2 ng and 0.3 ng per tube, respectively. Although the number of sharks was small (two males and two females), very little thyroid hormone information is available on sharks. In addition, data 502 G.L. CROW ET AL. on individual sharks repetitively sampled over long periods of time are rarely obtained. The repetitive nature of the samples necessitated using either a one-way repeated measures analysis of variance (parametric) or Friedman’s repeated measures ANOVA on Ranks (nonparametric) tests depending on the results of the normality test, using a SAS package. The T3 and T4 concentrations for males and females were tested for a significant difference. Seasonality of T3 and T4 was tested by grouping data into summer (May–August) and winter (October–January) concentrations. RESULTS Serum T3 concentrations were not significantly different for male and female sharks (χ2 = 0.78, df = 1, P = 0.38). The T3 concentrations monitored in the two male and two female sharks ranged (mean ± SEM) from 0.52 to 0.83 ng/mL (0.67 ± 0.01; n = 64). No seasonality was observed in serum T3 concentrations (Fig. 1, χ2 = 3.24, df = 1, P = 0.07). Serum T4 concentrations were not significantly different for male and female sharks (F = 0.73, df = 1, P = 0.40). No long-term age-related trends were observed in serum T4 concentrations for the two male and two female sharks (Fig. 2). The T4 concentrations in the male and female sharks ranged from 1.34 to 9.24 ng/mL (4.92 ± 0.22; n = 75). A significant seasonal difference in serum T4 concentrations was detected in male and female sharks (Fig. 3, χ2 = 20.17, df = 1, P < 0.001). The T4 concentrations mean (range ± SEM) were significantly higher in winter (October–January) 6.58 ng/mL (1.48–8.77 ± 0.35; n = 24) and were lower in summer (May–August) 3.62 ng/mL (1.34–5.71 ± 0.22; n = 24). Fig. 2. Serum T4 concentrations of whitetip reef sharks, Triaenodon obesus (two males [A] and two females [B]) over the three-year study period (December 1988 to December 1991). Graph lines represent linear regression data for males (graph A), (shark a: slope = 0.065, r2 = 0.076, df = 13; shark b: slope = –0.042, r2 = 0.038, df = 14). Linear regression data for females (graph B) was (shark a: slope = 0.051, r2 = 0.14, df = 17; shark b: slope = –0.022, r2 = 0.013, df = 14). DISCUSSION Fig. 1. Mean monthly serum T3 concentrations in two male and two female whitetip reef sharks, Triaenodon obesus. In our study of four immature whitetip reef sharks repetitively sampled for three years, no significant sexual difference in circulating T3 and T4 concentrations was detected. These results were also reported by Lewis and Dodd (’76) for wild and experimental Scyliorhinus canicula and by T4 AND T3 CONCENTRATIONS IN WHITETIP REEF SHARKS Fig. 3. Mean monthly serum T4 concentrations in two male and two female whitetip reef sharks, Triaenodon obesus. Volkoff and Wourms (’99) for wild blacktip sharks (Carcharhinus limbatus) and Atlantic stingrays (Dasyatis sabina). Although the thyroid gland of immature sharks has not been reported to change in weight or histologic development throughout the year (Woodhead, ’66), our current data revealed a T4 seasonal component in captive immature whitetip reef sharks. In this species, serum T4 concentrations are significantly higher in winter (October–January) than in summer (May–August). A distinct seasonal change in thyroid activity with summer inactivity and increased winter activity was described for adult Squalus acanthias (Woodhead, ’66). This is in contrast to Lewis and Dodd (’76), who found that serum T4 and T3 levels peaked in summer and autumn. In the whitetip reef shark, no seasonality was observed in serum T3. Age and growth factors may influence thyroid hormone concentrations. Lewis and Dodd (’76) reported higher T3 and T4 concentrations in adult than immature sharks. Whitetip reef sharks mature at an age of 6–7 years, attaining a precaudal length (PCL) of at least 101 cm (Uchida et al., ’90). The largest shark at the end of our study was 97 cm PCL (4–5 years old) and still imma- 503 ture. The Figure 2 graph of serum T4 revealed no significant long-term increases over the study period. As a result, the changes in serum T4 concentrations appear to be correlated with season and not age related. It is generally believed that circulating thyroid hormone concentrations have daily and seasonal fluctuations. The thyroid gland is also regulated by the quantity and quality of the diet (Eales, ’88), and seasonal thyroid changes may reflect adaptive responses to metabolic events associated with increased feeding and water temperature (Leatherland, ’94). Plasma iodide levels in teleosts vary according to season, physiologic state, and iodide availability in food or water (Eales et al., ’86). In our study, the iodide concentration of the tank water was four times higher (0.076 versus 0.019 mg/liter) than the coastal lagoon water. In elasmobranchs, diffusional uptake of iodide occurs across the gills and stomach with excretion primarily at the kidney and rectal gland (Shuttleworth, ’88). Excess aquatic iodide could potentially alter thyroid hormone concentrations. However, in trout, Oncorhynchus kisutch, excess plasma iodide had no significant long-term effect on plasma T4 and T3 concentrations (Eales et al., ’86). This indicates an apparent insensitivity of the trout thyroidal system to prolonged elevation in iodide availability (Eales et al., ’86). The serum T3 and T4 concentrations from the whitetip reef sharks were similar to thyroid hormone values found in wild elasmobranchs (Volkoff and Wourms, ’99). This suggests that the excess aquatic iodide at the Waikiki Aquarium did not directly influence thyroid hormone concentrations and that the whitetip reef shark, like the trout, is relatively insensitive to excess iodide. However, water chemistry is a very important aspect of thyroid gland physiology and is not well studied (Crow et al., ’98). A difficulty in studying thyroid hormones results from total T4 and total T3 being noncovalently and reversibly bound to blood proteins (Eales and Brown, ’93). This binding results in only the “free” form being physiologically active (Leatherland, ’94). Both the T4 and T3 “free” form likely represent a very small percentage of the total circulating thyroid hormones and may range from 0.5 to 3.4% of the total concentration in teleost fish (Eales and Shostak, ’87). If the binding properties are unaltered, the free fraction will tend to increase in direct proportion to the total T4 fractions (Eales and Brown, ’93). Under these conditions the bound hormone can act as a source of readily available “free” hormones and the latter 504 G.L. CROW ET AL. is removed from the circulation by target tissues, or excreted (Leatherland, ’94; Leary et al., ’99). The thyroid gland and hormones appear to be tightly conserved throughout the vertebrates (Leatherland, ’94). This also includes the monodeiodinase enzyme system in the liver that forms T3 (Leary et al., ’99). This is the first report of a seasonal hormone cycle (serum T4) in immature sharks. Previous studies have reported no seasonal components for corticosterone, estradiol, and testosterone in immature sharks (Sumpter and Dodd, ’79; Rasmussen and Crow, ’93). More detailed studies are needed to determine the production of T4 and T3, the amount of free thyroid hormones available in elasmobranchs, and their role in elasmobranch physiology. ACKNOWLEDGMENTS We thank the Waikiki Aquarium and the live exhibits staff for support in all phases of this research. We also thank Gordon Grau of the Hawaii Institute of Marine Biology for providing laboratory space and support to run the thyroid hormone analysis. The paper greatly benefited from the comments and review of Drs. J. Eales and J. 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