AMER. ZOOL.

, 37:470-481 (1997)
Interaction of Cortisoi and Thyroid Hormone in the
Larval Development of Pacific Threadfin
1
BONG G. KIM AND CHRISTOPHER L. BROWN
2
Hawaii Institute of Marine Biology, School of Ocean and Earth Science and Technology,
University of Hawaii, Box 1346 Coconut Island, Kaneohe, Hawaii 96744 USA
SYNOPSIS. We have conducted three hatchery-scale experiments designed to ex-
amine the actions and interactions of cortisoi and thyroid hormones in the devel-
opment of a larval marine fish. Survival among controls varied significantly be-
tween the 3 replicate experiments. The threadfin (Polydactylus sexfilis) consistently
responds to 1 hr posthatch immersion in a combination of triiodothyronine and
cortisoi (T
3
+ F) with accelerated gut development and increased survival com-
pared with untreated controls (C). Survival among larvae treated with T, or F
separately was significantly improved over controls in one of the three experiments.
The frequency of spinal deformities was reduced by cortisoi treatment, alone or
in combination with T
3
. Growth did not vary with treatment, except that variance
in larval length was reduced in (T, + F) vs. C in all 3 experiments. A hormone-
induced increase in uniformity could lead to reduced cannibalism, which is a prob-
lem in the culture of threadnns. These results suggest interactive hormonal regu-
lation of developmental processes, working within the context of other biological
variables.
INTRODUCTION
A variety of bioactive or potentially
bioactive substances are transferred from
the maternal circulation into the yolk of
vertebrate eggs. These include hormones,
neurohormones, neurotransmitters, and
RNAs which encode growth factors and
other compounds (reviewed by Brown and
Nunez, 1994). The biological importance of
maternally-derived regulatory compounds
in development is still very much an incom-
plete picture.
In teleost fishes, the thyroid hormones
have received the most attention, in part be-
cause of a perceived potential for applica-
tions in fish culture (Higgs et al, 1982;
Lam, 1990). Thyroid hormones have been
known for decades to have stimulatory ef-
fects on developing larval and juvenile fish-
es (reviewed by Lam, 1985; Brown and
Bern, 1989). For example, thyroxine (T
4
)
reduced the time required for prehatching
development in the chum salmon, Onco-
1
From the Symposium Developmental Endocrinol-
Sy f Non-Mammalian Vertebrates presented at the
Annual Meeting of the Society for Integrative and
Comparative Biology, 26-30 December 1996, at Al-
buquerque, New Mexico.
2
E-mail: cbrown<a>iniki.soest.hawaii.edu.
rhynchus keta (Dales and Hoar, 1954). Cor-
tisoi accelerated hatching and thyroxine
both accelerated and synchronized hatching
in the steelhead trout, O. mykiss, (Yeoh,
1993).
Since the presence of thyroid hormones
was first reported in fish eggs (Kobuke et
al, 1987; Tagawa and Hirano, 1987), T
4
and triiodothyronine (T
3
) have been detect-
ed in the eggs of more than 30 species (Ta-
gawa et al, 1990; Brown and Nunez,
1994). Maternal deposition of thyroid hor-
mones in fish eggs occurs against a concen-
tration gradient, possibly by way of binding
to vitellogenin (Babin, 1992), although not
all data support this hypothesis (reviewed
by Specker and Sullivan, 1994). Not only
may thyroid hormones adhere to vitellogen-
in, they also have been shown to stimulate
vitellogenesis in the guppy, Poecilia reti-
culata (Lam and Loy, 1985) and to promote
the uptake of vitellogenin by trout oocytes
in vitro (O. mykiss; Shibata et al, 1993).
Thus a theoretical case can be made for a
mechanism by which circulating thyroid
hormones could promote their own trans-
port and sequestration into eggs.
Varied patterns of circulating thyroid
hormones characterize the time of ovarian
470

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

CORTISOL AND THYROID HORMONE IN LARVAL THREADFIN 471
development in female fishes, which appear
consistent with ovarian hormone uptake
(see Norberg et al., 1989). In general, the
T3/T4 ratio is higher in marine fish eggs
than in those of freshwater fishes (Tagawa
et al., 1990). Marine fish eggs are also usu-
ally smaller in diameter, produced in larger
numbers, they are more widely dispersed,
and embryonic development is more rapid
(for details, see Blaxter, 1988). Thus it is
possible that variations in the concentration
of T
3
in fish eggs may help to set an ap-
propriate rate of development in r-selected
marine larvae and in AT-selected freshwater
species. A cyclic increase in the T
3
/T
4
ratio
during oogenesis appears to favor the up-
take of T
3
by the eggs of Atlantic halibut
a marine species (Bjornsson et al, 1997).
Peripheral 5'monodeiodinase activity is in-
hibited by estradiol in freshwater rainbow
trout, presumably resulting in a reduction in
the T3/T4 ratio during oogenesis in this spe-
cies (Cyr et al, 1988). These reports invite
the association of differential hormone pro-
duction in marine and freshwater environ-
ments, which could account for the rela-
tively higher concentrations of T
3
in marine
fish eggs. However, not all reports fit this
generalization. Clarias batrachus, a fresh-
water catfish, exhibits a shift in the maternal
balance of circulating thyroid hormones
suggesting increased peripheral 5'monod-
eiodination during the time of yolk depo-
sition (Sinha and Singh, 1990). To our
knowledge, the hormone content of Clarias
spp. eggs has not been determined.
Our recent studies have focused on the
Pacific threadfin (Polydactylus sexfilis) as a
model for examination of the actions of
hormones in early development. This spe-
cies, known as "moi" in Hawaii, spawns in
groups of one to three females together with
several males. The small marine larvae of
this species are responsive to either yolk en-
richment with thyroid hormones, via mater-
nal injection, or brief posthatch hormone
immersions in a dose-dependent fashion
(Brown and Nunez, 1994). We have also
found that an apparent interaction of corti-
sol and T
3
has beneficial effects on devel-
oping larvae which is consistent with inter-
actions of these hormones in a number of
other vertebrate development models
(Brown and Kim, 1995). In that study, the
effects of these two hormones were tested
individually and in combination, during the
development of larval threadfins in an ex-
perimental marine hatchery. A positive ef-
fect on survival was seen in larvae receiv-
ing the combination treatment, possibly as
a consequence of a hormone-induced ad-
vancement of the initiation of digestive
function. The present study was designed to
examine further the interactive effects of
cortisol and T
3
on development of Pacific
threadfins. In the three large-scale experi-
ments reported herein, we have monitored
the growth, development, and survival of
threadfin larvae as affected by immersion
in cortisol, T
3
, or a combination of the two
hormones.
MATERIALS AND METHODS
Polydactylus sexfilis eggs were obtained
by natural spawning at the Oceanic Insti-
tute, Waimanalo, Hawaii, and transferred
within 10 hours to the Hawaii Institute of
Marine Biology, during the early embry-
onic stage. The experiment was repeated
three times to minimize or eliminate the
batch effect. Experiments 1 and 2 were con-
ducted using eggs spawned on 6 October
and 13 November 1994, respectively, and
Experiment 3 was conducted with eggs
spawned on 12 May 1996. All batches of
eggs were from the same broodfish kept in
an earthen pond fed on a mixture of frozen
squid, krill, and artificial pellets. Embryonic
development indicated that eggs were from
multiple females, and spawns occurred
within 2-4 hours in all occasions.
Similar handling of eggs and rearing con-
ditions were provided in all experiments.
Eggs were incubated indoors in a 30 liter
downwelling chamber suspended in a 250
liter tank. Filtered sea water was supplied
to the chamber from above at a rate of 120
liter/hr and drained through a bottom screen
(150 u,m mesh) into the tank which was
outfitted with an overflow drainage. More
than 70% of larvae hatched within 30 min
after the onset of hatching, and the time was
considered time 0 (DO). Because the ga-
metes used for these experiments came
from an unknown number of parents, we
were careful to mix them thoroughly before

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

472 B. G. KIM AND C. L. BROWN
distributing them into treatment groups.
Newly hatched larvae were transferred to
12 liter buckets for the one-hour hormone
immersion treatments, one bucket per treat-
ment. The four treatment groups were: 2.6
ppm of triiodothyronine (T
3
, T), 0.1 ppm of
cortisol (F), combination of both hormones
at same concentrations (TF), and untreated
seawater as a control (C).
Following hormone treatment, larvae
were stocked into rearing tanks (250 liter
fiberglass tank with a conical black bot-
tom), three tanks per treatment. The initial
stocking density was determined as 36-49
larvae/liter for Experiment 1, 19-26 larvae/
liter for Experiment 2, and 25-35 larvae/
liter for Experiment 3. Tanks were main-
tained as a flow-through system with a turn-
over rate of 700% tank volume per day (1.2
liter/min). The water level was maintained
using an inside standpipe screened with a
mesh; 120 urn during a rotifer period, 220
and 350 u.m during yolksac Artemia (nau-
plii) and one-day-old Artemia (metanauplii)
periods, respectively. Illumination was pro-
vided with fluorescent lights at a surface in-
tensity of 950 lux. (light: dark 14:10).
Rearing temperature (25.8-28.0 C), salin-
ity (32 ppt), and dissolved oxygen concen-
tration (6.2 0.3 ppm) were ambient, and
were similar between experiments. The ex-
periments were terminated at the onset of
metamorphosis, between D24 and D28. Ex-
periment 2 was terminated on D24, prior to
metamorphosis, due to high mortality. Early
in this experiment, water exchange was in-
terrupted for two days by a main water pipe
failure, and the Artemia feeding schedule
described below was not maintained cor-
rectly on days 16 and 17.
Rotifers {Brachionus plicatilis) were in-
troduced as first food upon initial mouth
opening, three days after hatching (D3). At
this time the rotifer density was maintained
at 20-25 rotifers/ml, and the green algae,
Nannochloropsis sp. was added at a con-
centration of approximately 5,000 cells/ml
to support the nutritional value of rotifers.
When larvae were capable of improved
searching and feeding success, starting on
D5, the rotifer density was reduced to 15-
20 rotifers/ml. Yolksac Artemia sp. nauplii
(0.35 nauplii/liter) were introduced starting
on D9 for 3 days along with rotifers. Yolk-
sac Artemia nauplii were the sole feed on
between Dl l and D13, at concentrations of
1-2.1 nauplii/liter. Selco enriched one-
day-old Artemia metanauplii were fed to
larvae beginning on D14 at a density of 2
3.5 metanauplii/liter. Feeding was conduct-
ed at a frequency of once every 2-3 hours
throughout the light phase.
Approximately 30-50 larvae from each
tank were sampled at 4- to 5-day intervals,
and larval growth was determined by mea-
suring standard lengths using a Nikon vi-
deomicroscopy system. Spontaneous growth
rate was calculated from a linear regression
after logarithmic transformation of standard
lengths, and the treatment effect was tested
using a one-way ANOVA comparison. Pos-
sible treatment-dependent effects on growth
were tested by comparing sizes on each
sampling day using a one-way ANOVA fol-
lowed by Fisher's PLSD comparison when
appropriate to identify differences (P <
0.05) between treatments.
To determine the onset of intestinal ab-
sorptive function, a stain that exhibits flu-
orescence specifically in the presence of ac-
tive mitochondria was used. Samples con-
sisting of 15 larvae from each treatment
group were collected every 6 hours starting
at mouth-opening until the gut content was
visible through the skin pigmentation, be-
tween 56 and 86 hours after hatching. The
sample larvae were immersed in DASPEI
(dimethylaminostyryl-ethylpyridiniumio-
dide) for 3 hours at a concentration of 6
ppm in 300ml glass beakers without aera-
tion. An inverted microscope fitted with an
epifluorescence attachment (TMD-EF; ex-
citation 470490 nm) was used to deter-
mine the relative amount of mitochondrial
activity in larval gut lining and other or-
gans. The degree of epifluorescence was
scored between 0 to 3 to compare the mi-
tochondrial activities among treatment
groups. Results for each sampling time
were evaluated using a one-way ANOVA
followed by Fisher's PLSD comparison.
Dead larvae were siphoned daily from
the bottom screen, and although we could
not quantify these larvae reliably, notes
were taken on the daily mortality pattern.
Upon termination of die experiment, sur-

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

CORTISOL AND THYROID HORMONE IN LARVAL THREADFIN
A
473
uu -
80
bU -
40
20 -
i l
,
[
b
i
b b

! i
! i
TF
FIG. 1. Survival of larval threadfin from hatching to metamorphosis after hornomal treatment at hatching; one
hour immersion with T
3
(T), cortisol (F), combination of T
3
and cortisol (TF), and no exogenous hormone (C).
A, B, and C represent the experiment 1, 2, and 3, respectively. Each bar shows the mean survival for a treatment
(n = 3 per treatment) with a standard error of the mean as a vertical line. Results of Fisher's PLSD comparison
were indicated at 5% confidence level for the experiments 1 and 2, and at 10% level for the experiment 3.
vival was calculated from the difference be-
tween the initial number of larvae stocked
and the final number remaining at the onset
of metamorphosis. Results were analyzed
using a one-way ANOVA followed by
Fisher's PLSD comparison. Two tanks (one
of each F and TF groups) from Experiment
1 were excluded in survival analysis due to
overflow from clogged screens.
RESULTS
Survival
Differences in survival were seen both
between experiments and between treat-
ments, with the extremes ranging from 11
to 61% survival from hatching to meta-
morphosis (Fig. 1). The best survival (36-
61% to 28 days) occurred in Experiment 1.
The relatively poor survival (11-28% to 24
days, pre-metamorphosis) in Experiment 2
may have been a consequence of the stress
from the static water during the early rear-
ing period. Despite the differences in sur-
vival between experiments, fairly consistent
treatment-dependent differences were ap-
parent. All treatments promoted survival
significantly during Experiment 1 (P <
0.001, MS = 329). Individual treatment de-

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

474 B. G. KIM AND C. L. BROWN
pendent differences (F vs. C, T vs. C) were
not significant in Experiments 1 (P = 0.09,
MS = 155) and 3 (P = 0.26, MS = 53).
The Fisher's PLSD test indicated, however,
that the difference between C and TF
groups was significant at 10% confidence
level (P = 0.02 for Experiment 2 and P =
0.08 for Experiment 3).
We found it impractical to quantify daily
survival because of the patch distribution of
larvae and their extremely delicate condi-
tion. Nevertheless, an episodic increase in
mortality was observed in three stages in
all tanks; hatching through the first feeding
(DOD5), immediately prior to notochord
flexion (D12D13), and post flexion prior
to metamorphosis (D20D22). Evidence of
a treatment-dependent difference in surviv-
al was first noticed during the late stage of
notochord flexion (D18). Larvae subjected
to the combination (TF) treatment had less
pronounced mortality, which was confirmed
at the end of the experiments. Less tank
variability in mortality patterns was also
observed among TF treated larvae. In Ex-
periment 3, higher mortality was evident
prior to notochord flexion (D12-D14).
Growth
The onset of metamorphosis was ob-
served when larvae reached 21 mm in stan-
dard length, or between D24 and D28 de-
pending on rearing temperature. Despite the
uniformity in size at hatching (1.59 mm
0.07, mean standard deviation), larvae
grew at different rates resulting in large size
variation at the onset of metamorphosis
(9.02 mm 1.93). A bimodal distribution
was observed in all groups of larvae. TF
treated larvae grew more uniformly with re-
gard to variance in total length (Fig. 2). The
pronounced variation of D5 larvae in the T
group in Experiment 1 may have been a
result of a high rate of skeletal deformities,
and consequently reduced body length
among some of the sampled larvae.
No difference in spontaneous growth in
length was observed among all groups of
larvae in any of the experiments (P = 0.68,
MS = 3.28 X 10
5
for Experiment 1; P =
0.11, MS = 7.09 X 10
5
for Exp 2; P =
0.63, MS = 6.13 X 10
5
for Exp 3). This
may be because the tank variability within
treatments overshadowed any possible
treatment effects. In addition, density-de-
pendent effects due to differential survival
rates probably also impacted growth rates.
While the treatment-dependent growth in
size was evident starting D12 (P < 0.001),
the effect was different between experi-
ments. The TF-treated larvae exhibited less
variance in length than controls in each of
the three experiments (Fig. 2).
Ontogenic development
Hormone treated larvae showed different
ontogenic development from untreated lar-
vae. Prior to mouth-opening, an advanced
eye development and more yolk absorption
were observed among larvae from T and
TF groups. Larvae from the Control group
retained most yolk and oil until beyond the
onset of feeding.
A treatment-dependent difference in in-
testinal mitochondrial activity was observed
following DASPEI immersion (Table 1).
Within 6 hours after mouth-opening (56 hr
at 26.2 0.8C), epifluorescence in the de-
veloping gut was apparent among larvae
from F and TF groups. Within 6 hr after the
initial epifluorescence (62hr), hindgut epi-
fluorescence was evident in all groups of
larvae, with C group the faintest and TF
group the strongest of all. Rapid ontogenic
development of the digestive tract was ob-
served among TF group larvae. At 62 hr,
epifluorescence in the urinary tract, kidney
and pancreas was observed in the TF lar-
vae, but only faintly in controls (Fig. 3),
and first peristaltic movement was observed
at 74 h. DASPEI immersion suggested that
TF treatment of larval threadfins at hatching
advanced the development of the digestive
system by approximately 12 hr (Table 1).
During immersion, larvae from C and F
groups were less active, and less capable of
avoiding capture by netting. Mortality from
immersion was less than 10%.
The onset of notochord flexion occurred
near D13 and D14, and the process took
approximately 3 days. Once flexion was ini-
tiated, about 30% of larvae reached flexion
within one day, and 55% of larvae under-
went the process within the following four
days (Fig. 4). Statistically no treatment-de-
pendent differences in flexion rate were

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

CORTISOL AND THYROID HORMONE IN LARVAL THREADFI N 475
B
9 14 18
days after hatching
22 25
FIG. 2. Size variation of larval threadfin from hatching to metamorphosis after hormonal treatment at hatching;
one hour immersion with T
3
(T), cortisol (F), combination of T
3
and cortisol (TF), and no exogenous hormone
(C). A, B, and C represent the experiment 1, 2, and 3, respectively. Each bar shows the mean variation for a
treatment (n = 3 per treatment) with a standard error of the mean as a vertical line.
TABLE 1. Hormonal treatment effect on DASPEI fluorescence of larval threadfin from 56 to 86 hr after hatching
at 26.2 0.8C.
Treatment
c
F
T
TF
O.O
0.8"
0.2
a
0.7"
56 hr
0.00
0.13
0.11
0.14
62 hr
0.7" 0. 14
1.8" 0.13
0.8" 0. 17
1.8" 0.11
68 hr
1.5 0.23
1.8 0.11
1.8 0.13
2.0 0.00
Age
74 hr
1.6* 0.19
1.9
a
0.08
1.8' 0.13
2.6" 0. 15
80 hr
1.8
a
0.18
2.7" 0.14
2.5
b
0.19
2.7" 0. 14
2.6
2.9
2.7
3.0
86 hr
0.19
0.08
0.14
0.00
One hour immersion with 2.6 ppm of T
3
(T), 0.1 ppm of cortisol (F), combination of T
3
and cortisol (TF),
and no exogenous hormone (C). Indexes indicate: 0, no fluorescence; 1, faint fluorescence in hindgut; 2, for
strong fluorescence in mid- and hindgut; 3, strong fluorescence in gut lining and other organs. Results of ANOVA
test for each sampling day indicated: 56 hr, P = 0.000 (MS = 1.632); 62 hr, P = 0.000 (MS = 4.410); 68 hr,
P = 0.114 (MS = 0.521); 74 hr, P = 0.000 (MS = 2.306); 80 hr, P = 0.001 (MS = 2.299); 86 hr, P = 0.077
(MS = 0.472). Results of Fisher's PLSD comparison were indicated at 5% confidence level. Superscript letters
(
a b
) indicate statistical differences.

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

476 B. G. KIM AND C. L. BROWN
FIG. 3. Hormonal treatment effect on DASPEI fluorescence of larval threadfin at 62h after hatching at 26.2
0.8 degree Celsius; one hour immersion with a combination of triiodothyronine and cortisol (TF) compared to
no exogenous hormone (C).
found (P > 0.10 in all cases), although the
average length of larvae at the time of flex-
ion suggested that C group larvae reached
the flexion stage at a larger larval size than
other treated groups. High mortality oc-
curred during Experiment 2, especially
among the Control group tanks, which was
represented in an increase in flexion rates
in four days.
The most common deformity evident
among larval threadfins was fused vertebrae
followed by scoliosis and lower jaw defor-
mities. The development of these skeletal
deformities was rather life stage specific;
much deformity was observed during pre-
flexion stage, though the survival of these
larvae to later stages is doubtful (Fig. 5).
The overall rate of deformities was highest
in Experiment 1. A treatment-dependent ef-
fect on the rate of deformity was also evi-
dent. Control and T
3
-treated larvae consis-
tently had the highest rates of deformities,
and significantly fewer occurred among lar-
vae treated with F and TF at D5, near the
completion of yolk and oil absorption.
DISCUSSION
A one-hour immersion in a combination
of triiodothyronine and cortisol significant-
ly improved survival, as we have reported
earlier (Brown and Kim, 1995). We find it
especially interesting that this effect per-
sisted despite major differences in survival
and rates of deformities among the three ex-
periments. Based on daily monitoring of
mortality, it appears that the net survival
effect may have been a result, at least in
part, of the amelioration of episodic mor-
tality events around D0-5 and D12-13.
These are both stages involving dietary
transitions, which is consistent with the idea
that the advancement of development as in-
dicated by DASPEI fluorescence may have
conferred some survival benefits.

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

CORTISOL AND THYROID HORMONE IN LARVAL THREADFIN 477
100
80 -
60 1
T3
I
i
40 -
20 -
standard lengt i
(mm)
(mm)
5 48 J S 14 b 5 1 b
.8,04,1 ,SGSb 8.61 C
3? li
! 8,28 c
D15
D19
TF
FIG. 4. Percent larvae that reached notochord flexion after hormonal treatment at hatching; one hour immersion
with T
3
(T), cortisol (F), combination of T
3
and cortisol (TF), and no exogenous hormone (C). A, B, and C
represent the experiment 1, 2, and 3, respectively. Each bar shows the mean percent larvae that underwent
notochord flexion for a treatment (n = 3 per treatment) with a standard error of the mean as a vertical line.
Differences in survival among controls in
the three experiments indicate that partially
controlled variables such as parental genet-
ics and nutrition may have an impact on
"egg quality" as manifested in survival.
Although the same broodstock animals
were used throughout the study, it was not
possible to identify the specific parentage of
each spawn. The increasing age of the
broodstock population may also have af-
fected egg quality; eggs used in experiment
3 were from females 1.5 years older than
they were in experiments 1 and 2. The time
of year of each of the three replicate ex-
periments differed, relative to the beginning
of the spawning season, which in this spe-
cies normally extends from May through
November in Hawaiian waters. Experi-
ments 1 and 2 used eggs produced late in
the season while experiment 3 used eggs
GANSER LIBRARY

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

478 B. G. KIM AND C. L. BROWN
10 15 20
post hatching (day)
30
FIG. 5. Cumulative deformity of larval threadfin from hatching to metamorphosis after hormonal treatment at
hatching; one hour immersion with T
3
(T), cortisol (F), combination of T
3
and cortisol (TF), and no exogenous
hormone (C). A, B, and C represent the experiment 1, 2, and 3, respectively.
from near the beginning of the season. Egg
size and free fatty acid composition decline
with successive spawns in the cod (Gadus
morhua; Marst0l et al., 1993). Egg size did
not change throughout our experiments
(0.86 0.01 mm) but we have not under-
taken chemical analyses.
Although efforts were made to maintain
consistency in environmental and nutrition-
al variables, subtle changes within the 19
months that elapsed between experiments 1
and 3 may have had some bearing on larval
survival. Different batches of broodstock
feed materials and slight weather variation
can not be discounted as having potential
effects on the viability of eggs produced in
captivity. Clearly the second experiment
was compromised by a break in a water
main which interrupted the flow of filtered
seawater to larval tanks, and by a brief
lapse in the feeding schedule. Despite the
physical and human variables unintention-
ally imposed on the experimental design in
the course of Experiment 2, we include
these data because they illustrate a point
about the consistency of the actions of a
combination of cortisol and triiodothyro-
nine, which had a substantial and beneficial
effect on survival which persisted under
these suboptimal circumstances. The dis-
tress endured by larvae in Experiment 2
may account for reduced overall survival

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

CORTISOL AND THYROID HORMONE IN LARVAL THREADFIN 479
among controls relative to the other exper-
iments (Fig. 1). In addition, Experiment 2
alone had more spinal deformities in con-
trols than in T
3
-treated larvae. If in fact the
effectiveness of T
3
was altered by compro-
mised water quality in Experiment 2, this
may help explain paradoxic larval re-
sponses to exogenous thyroid hormones
(reviewed by Brown and Nunez, 1994).
The effects of the individual hormones
were less consistent than those of cortisol
and T
3
administered together. In two of the
three experiments, the survival among T or
F groups did not differ from the controls,
although survival among these groups in-
creased by about 50% in Experiment 1. We
therefore conclude that these hormones in-
teract to promote one or more vital devel-
opmental processes, which convey survival
advantages even under compromised con-
ditions. When applied alone, the effects of
the individual hormones may be masked or
overridden by other variables.
Our DASPEI results (Table 1) indicate
that the development of the digestive and
urinary tracts was advanced to some extent
by cortisol treatment, and slightly more so
by a combination of cortisol and T
3
. A fun-
damental preadaptive role of thyroid hor-
mones in the preparation of larval intestinal
tissues for exploitation of new food sources
has been proposed (Specker, 1988). The
twelve-hour advancement of absorptive
function by the gastrointestinal tract implic-
it in our results (Table 1) coincided with the
intitial introduction of rotifers, and there-
fore may have had an impact on larval sur-
vival. We believe the importance of this
temporal advance is that it would reduce the
lapse between exhaustion of endogenous
energy, in the form of yolk and oil, and
initial utilization of exogenous energy from
first-feeding. We do not have any evidence
which would enable us to discriminate be-
tween possible direct or indirect actions of
these hormones on gut development, al-
though these hormones have been shown to
have direct peripheral interactions on de-
veloping target tissues in the Japanese
flounder, Paralychthys olivaceus (de Jesus
et al., 1990).
A clear difference in the effects of the
individual cortisol and T
3
treatments be-
came apparent in the quantification of skel-
etal deformities (Fig. 4). Cortisol-treated
groups were always among those larvae
with the lowest frequency of skeletal de-
formities, whether considering F or TF
groups. The T group displayed variable in-
cidence of skeletal deformities, but in both
Experiment 1 and Experiment 3, exhibited
the highest rate of deformities of all four
treatments. Exogenous thyroid hormones
reportedly often induce skeletal deformities
in fish (Higgs et al., 1982), however this
trend was reversed in both experiments 1
and 3 among the larvae exposed to both
cortisol and T
3
. The influence of cortisol ev-
idently precluded the increased rate of de-
formities seen in the T groups. At this point
we do not have sufficient data to explain
the mechanism of interaction of these hor-
mones. Our working hypothesis is that the
promotion of absorptive function by corti-
sol may have aided in the provision of nu-
trients needed for skeletal differentiation;
shortages of any number of substrate ma-
terials in T
3
-stimulated developing tissues
may result in scoliosis, opercular malfor-
mations, and other skeletal deformities.
This does not preclude the possibility of di-
rect peripheral interactions of the two hor-
mones in the developing vertebral and gill
tissues.
No consistent relationship of hormone
treatments with the timing of flexion
emerged from these experiments (Fig. 4).
Control larvae were larger than the TF lar-
vae at the time of flexion in Experiment 1,
and on D19 in Experiment 2. The inconsis-
tency of these data suggests that the hor-
mone treatments may have been secondary
to other factors in determining the timing
or size of larvae at notocord flexion. The
relatively large size and high frequency of
larvae at flexion in Experiments 1 and 2
could reflect a transient increase in growth
rate among the control larvae, resulting
from mortality-induced reduction of larval
density. A definitive explanation will re-
quire further study.
Survival effects were not accompanied
by any correlative effect on growth. The
treatment groups exhibiting improvements
in survival rate grew at essentially the same
rate as controls, although this is a notewor-

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

480 B. G. KIM AND C. L. BROWN
thy observation since mortality in control
tanks reduced larval densities, which
should favor more rapid growth. Those lar-
vae subjected to a combination of cortisol
and T
3
grew at somewhat more uniform
rates than controls (Fig. 2). The amount of
variance in length within a cohort is a crit-
ical determinant of cannibalism in Channa
striatus (Qin and Fast, in press). This rela-
tionship probably holds among most spe-
cies prone to cannibalism, including the
threadfins, which typically show cannibal-
istic behavior beginning at the time of
metamorphosis (~D25).
The use of thyroid hormones in potential
fisheries applications has been limited by
several complicating factors. Thyroid hor-
mone treatments are viewed as inconsistent
in their effectiveness, and replete with un-
toward side effects such as the promotion
of deformities and cannibalism. For exam-
ple, some beneficial effects of exposure of
walleye (JStizostedion vitreum) to thyroid
hormones may be overshadowed by ob-
served hormone-induced 8- to 10-fold in-
creases in the incidence of cannibalism
(Hey et al., 1996). It is possible that a fur-
ther examination of the interactive effects
of development-promoting hormones may
lead to combination treatments which pro-
mote survival without such side-effects.
ACKNOWLEDGMENTS
The authors wish to thank the organizers
of the symposium for the invitation to par-
ticipate. We also express thanks to Mr. W.
Douglas Crompton and to Mr. Scott Clem-
ent for assistance with larval care and sam-
pling. This research was sponsored in part
by a grant from the State of Hawaii De-
partment of Land and Natural Resources
Aquaculture Development Program. Fund-
ing for this work was also provided by pro-
ject R/AQ-57, which is sponsored by the
University of Hawaii Sea Grant College
Program, under Institutional Grant No.
NA36RGO5O7 from the National Oceanic
and Atmospheric Administration Office of
Sea Grant, Department of Commerce. This
is Sea Grant Publication number UNIHI-
SEAGRANT-JC-97-09.
REFERENCES
Babin, P. J. 1992. Binding of thyroxine and 3,5,3'
triiodothyronine to trout plasma lipoproteins. Am.
J. Physiol. 262 (Part l):E712-E720.
Bjornsson, B. T, O. Halldorsson, C. Haux, B. Norberg,
and C. L. Brown, (submitted in 1997) Photoperiod
manipulation of Atlantic halibut sexual matura-
tion: thyroid hormones and total plasma calcium
levels.
Blaxter, J. H. S. 1988. Pattern and variety in devel-
opment. In W. S. Hoar and D. J. Randall (eds),
Fish physiology. Vol. XI, Part A, Eggs and Lar-
vae, pp. 158. Academic Press, New York.
Brown, C. L. and H. A. Bern. 1989. Hormones in
early development, with special reference to te-
leost fishes. In M. P. Schreibman, and C. G. Sca-
nes (eds), Hormones in development, maturation,
and senescence of neuroendocrine systems. A
comparative approach, pp. 289306. Academic
Press, New York.
Brown, C. L., S. Doroshov, J. Nunez, C. Hadley, R.
S. Nishioka, and H. A. Bern. 1989. Maternal
triiodothyronine injections cause increases in
swimbladder inflation and survival rates in larval
striped bass, Morone saxatilis. I. Exp. Zool. 248:
168-176.
Brown, C. L. and B. G. Kim. 1995. Combined appli-
cation of cortisol and triiodothyronine in marine
finfish culture. Aquaculture. 135(l-3):79-86.
Brown, C. L. and J. M. Nunez. 1994. Hormone ac-
tions and applications in embryogenesis. In K. G.
Davey, R. E. Peter, and S. S. Tobe (eds), Per-
spectives in Comparative Endocrinology, pp.
333-339. National Research Council of Canada,
Ottawa.
Cyr, D. G., D. L. McLatchy, and J. G. Eales. 1988.
The influence of short-term 17 beta estradiol treat-
ment on plasma T3 levels and in vitro hepatic T4
5'monodeiodinase activity in immature rainbow
trout, Salmo gairdneri. Gen. Comp. Endocrinol.
69:431-438.
Dales, S. and W. S. Hoar. 1954. Effects of thyroxine
and thiourea on the early development of chum
salmon (Onchorhyncus keta). Can. J. Zool. 32:
244-251.
de Jesus, E. G., Y. Inui, and T. Hirano. 1990. Cortisol
enhances the stimulating action of thyroid hor-
mones on dorsal fin ray resorption of flounder lar-
vae in vitro. Gen. Comp. Endocrinol. 79:167173.
Hey, J., E. Farrar, B. T. Bristow, C. Stettner, and R. C.
Summerfelt. 1996. Thyroid hormones and their
influences on larval performance and incidence of
cannibalism in walleye Stizostedion vitreum. J.
World Aquacult. Soc. 27(l):40-51.
Higgs, D. A., U. H. Fagerlund, J. G. Eales, and J. R.
McBride. Kim, B. G. 1982. Application of thy-
roid and steroid hormones as anabolic agents in
fish culture. Comp. Biochem. Physiol. B. 73:143-
176.
Kobuke, L., J. L. Specker, and H. A. Bern. 1987. Thy-
roxine content in eggs and larvae of coho salmon,
Onchorhyncus kisutch. J. Exp. Zool. 242:89-94.
Lam, T. J. 1985. Role of thyroid hormones on larval
growth and development in fish. In Current trends

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

CORTISOL AND THYROID HORMONE IN LARVAL THREADHN 481
in comparative endocrinology. B. Lofts and W. N.
Holmes (eds), pp. 481485. Hong Kong Univer-
sity Press, Hong Kong.
Lam, T. J. 1990. Technology development in aqua-
culture: New ideas and approaches. In C. L. Ming,
and P. K. L. Ng (eds), Essays in Zoology, pp 357
370. Papers commerating the 40th anniversary of
the Department of Zoology, National University
of Singapore. National University of Singapore
Press, Singapore.
Lam, T. J. and G. L. Loy. 1985. Effect of L-thyroxine
on ovarian development and gestation in the vi-
vaparious guppy, Poecilia reticulata. Gen. Comp.
Endocrinol 60:324-330.
Marst0l, M. J., H. J. Fyhn, O. S. Kjesbu, and P. Solem-
dal. 1993. Free amino acid content as a potential
criterion of egg quality in Atlantic cod (Gadus
morhua). In B. T. Walther, and H. J. Fyhn (eds),
pp. 99-103. University of Bergen Press, Bergen,
Norway.
Norberg, B., B. Th., Bjornsson, C. L. Brown, U.-P.
Wichardt, L. J. Deftos, and C. Haux. 1989.
Changes in vitellogenin, sex steroids, calcitonin
and thyroid hormones related to sexual maturation
in female brown trout, Salmo trutta. General and
Comparative Endocrinology 75:316326.
Qin, J. and A. W. Fast. 1997. Size and feed dependent
cannibalism with juvenile snakehead, Channa
striatus. J. Fish Biol. In press.
Shibata, N., M. Yoshikuni, and Y. Nagahama. 1993.
Vitellogenin incorporation into oocytes of rain-
bow trout, Oncorhyncus mykiss, in vitro: Effects
of hormones on denuded oocytes. Dev. Growth
Differ. 35:115-121.
Sinha, N. and T. P. Singh. 1990. Extrathyroidal con-
version of T
4
to T
3
in a freshwater catfish, Clarius
batrachus, during prespawning and spawning
phases in its annual reproductive cycle. Indian J.
Experimental Biol. 28:680-682.
Specker, J. L. 1988. Preadaptive role of thyroid hor-
mones in larval and juvenile salmon: growth, the
gut, and evolutionary considerations. Amer. Zool.
28:337-350.
Specker, J. L. and C. V. Sullivan. 1994. Vitellogenesis
in fishes: Status and perspectives. In K. G, Davey,
R. E. Peter, and S. S. Tobe, (eds.), Perspectives in
Comparative Endocrinology, pp. 304315. Na-
tional Research Council of Canada, Ottawa.
Tagawa, M., M. Tanaka, S. Matsumoto, and T. Hirano.
1990. Thyroid hormones in eggs of various fresh-
water, marine, and diadromous teleosts and their
changes during development. Fish Physiol. Bio-
chem. 8(6):515-520.
Tagawa, M. and T. Hirano. 1987. Presence of thyrox-
ine in eggs and changes in its content during early
development of chum salmon, Oncorhyncus keta.
Gen. Comp. Endocrinol. 65:337345.
Yeoh, C.-G. 1993. The effects of hormones on devel-
opment of embryonic and post-embryonic salmo-
nids and hormone metabolism during these stages.
Master's Thesis, Oregon State University, Depart-
ment of fisheries and wildlife, Corvallis, OR.
Corresponding Editor: David Norris

b
y

g
u
e
s
t

o
n

M
a
y

3
1
,

2
0
1
4
h
t
t
p
:
/
/
i
c
b
.
o
x
f
o
r
d
j
o
u
r
n
a
l
s
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m