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EXPERIMENTAL ZOOLOGY 284:500–504 (1999)
Serum T4 and Serum T3 Concentrations in Immature
Captive Whitetip Reef Sharks, Triaenodon obesus
Waikiki Aquarium, University of Hawaii, Honolulu, Hawaii 96815
Hawaii Institute of Marine Biology, Kaneohe, Hawaii 96744
Oregon Graduate Institute, Beaverton, Oregon 97006
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
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:
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.
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
Temperature (°C)
Salinity (ppt)
Total N
Total P
Total iodine
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.
T3 and 125T4 were placed in 100 µL of 0.1 N
NaOH and added to each column. The 125T3 and
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
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.
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).
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
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-
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
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.
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. Leatherland. This project complied with
the University of Hawaii’s Animal Care and Use
committee protocol no. 94-071. This publication
is funded in part by a grant/cooperative agreement
from the National Oceanic and Atmospheric Administration, project M/PM-2 which is sponsored
by the University of Hawaii Sea Grant College
Program, SOEST, under institutional grant NA36RG0507 from the NOAA Office of Sea Grant, Department of Commerce. The views expressed
herein are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies. UNIHI-SEA GRANT-JC-98-09.
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