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171
Wright et al. 1990 for Atlantic salmon parr. Others have argued that such uncoupling is not always present, for instance Dickey et al. 1997 found no evidence for uncoupling
in larval striped bass. Regardless of the presence of an uncoupling, and assuming the rate of growth is neither extremely high nor low, increment width can be used to provide
estimates of recent growth of individual field-collected fish once the relationship between otolith growth increment width and somatic growth is established.
This paper will address aspects of growth in the fringed flounder Etropus crossotus Jordan and Gilbert, 1882, a small flatfish with a subtropical and tropical distribution.
Along the Atlantic coasts it has been described from Chesapeake Bay Virginia, USA, to the northern coasts of South America, but it is most abundant in the South Atlantic
Bight and the Gulf of Mexico, and very common in estuaries and shallow waters of South Carolina, USA Topp and Hoff, 1972; Martin and Drewry, 1978; Ogburn et al.,
1988; Reichert and van der Veer, 1991; Allen and Baltz, 1997; Reichert, 1998. The maximum reported total length of the fringed flounder is 16.9 cm, but individual fish are
rarely longer than 15 cm, and their wet weight is seldom more than 40 g Moe and Martin, 1965; Topp and Hoff, 1972; Reichert and van der Veer, 1991; Reichert, 1998.
Reichert 1998 described aspects of the age, growth, and reproduction of the species, showing that its maximum expected life span is about 14.5 months. The fringed flounder
can be found year round on mud and muddy sand in shallow coastal waters and estuaries at temperatures ranging from 11 to 318C and salinities from less than 5 to over 35 ppt
Topp and Hoff, 1972; Martin and Drewry, 1978; Reid, 1954; Ogburn et al., 1988; Reichert and van der Veer, 1991. The relatively small mouth of the fringed flounder
limits the size of their prey, predominantly small benthic and epibenthic crustaceans and polychaetes Reid, 1954; Topp and Hoff, 1972; Stickney et al., 1974; Reichert and van
der Veer, 1991. Spawning in South Carolina takes place from March through October, and the smallest size at which females can potentially reproduce is 7–7.5 cm SL with a
length at 50 maturity between 8.0 and 8.5 cm SL Reichert, 1998. Reichert and van
21
der Veer 1991 and Reichert 1998 estimated a growth rate of about 0.5 mm day at
24 to 288C for juveniles, but detailed information for growth under controlled, good quality conditions was not available. Using experimentally derived data, we investigated
1 the relationship between temperature and growth under defined conditions with no food limitation, 2 the validation of daily increment formation in the otoliths, and 3
the relationship between otolith growth and somatic growth. The data can be used to estimate natural rates of growth of individual fish collected in the field, and to model
growth of fringed flounder populations.
2. Materials and methods
2.1. Sample collection Juvenile fringed flounder were collected on June 13, 1996, from Town Creek and
Debidue Creek in North Inlet South Carolina, USA using a 1 m beamtrawl with a stretched mesh size of 1cm see Reichert, 1998. The seawater in the creeks was 278C
and had a salinity of 30 ppt. The standard length SL, 60.1 mm, total length TL, 60.1
172 M
.J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
mm and wet weight WW, 60.001 g of a subsample of the collected juvenile E .
crossotus were determined shortly after sampling. On the day of collection, live fish were transported to a walk-in experimental chamber with climate control in Columbia
SC. Here the fish were acclimated for 6 weeks in two 150 l seawater tanks 238C and S 5 29–30 ppt while being fed ad libitum with a mixture of live black worms
Lumbriculus variegatus, finely chopped grass shrimp Palaemonetes pugio, and live adult and juvenile brine shrimp Artemia grown on a Chlorella sp. suspension.
2.2. Experimental setup, otolith marking procedure, and otolith preparation The growth experiment was conducted at 14, 20, 24 and 298C each 60.58C in a
climate controlled experimental chamber. These temperatures were chosen to create a range of growth rates under ad libitum food conditions. Temperatures were randomly
assigned to eight tanks, two at each temperature. The tanks L 3 W 3 H 5 50 3 26.5 3 31 cm were equipped with an aquarium heater, a thermometer, a foam filter and air
supply, and filled with 33 1 of seawater 29–31 ppt. The bottom of each tank was covered with a 2 cm layer of cleaned fine sand from the same location the fish were
collected. The top of every tank was covered with clear Plexiglas and the four sides were covered with brown Styrofoam to provide insulation, prevent visual interaction between
tanks, and minimize disturbances. A ninth tank at 148C 60.58C with identical setup was used as a control to investigate the effect of handling and marking. Prior to the
beginning of the experiment, seawater was recirculated between all experimental tanks and a 150 1 storage aquarium equipped with a trickle wet dry filter to establish uniform
water quality in all tanks.
Six weeks after collection, 55 fish with a standard length between 23.2 and 53.0 mm mean 42.6 mm were marked by submerging them for 18 h in a solution of 75 ppm
Alizarin complexone in seawater 238C, 30 ppt. The marking procedure was tested by marking 10 fish 1 day after collection and again by marking 10 fish 8 days after
collection, or 109 and 100 days before the end of the experiment respectively. There was no mortality during the marking procedure or in the few days that followed. Immediately
after marking, the fish were measured with electronic calipers SL and TL, weighed WW, and uniquely externally marked by fin clipping for identification. Five fish were
placed in each of the experimental tanks. The remaining marked and unmarked fish were placed in the storage aquarium at 238C to replace fish that died during the experiment.
Over a period of several days the temperature levels were gradually adjusted to create the appropriate experimental temperatures in each tank. Temperature in all tanks was
stable at the nominal value within 10 days. Starting on the 18th day after marking, the SL, TL, and WW of each fish were determined five times at 12 day intervals, resulting in
four 12 day growth periods. The fish in the control tank were measured only at the beginning and at the end of the experiment. Three of these five fish were not marked
with Alizarin.
The oxygen concentration in the tanks was measured at least once a week and oxygen saturation levels rarely dropped below 90. The salinity in all tanks was monitored
daily and kept at 29–31 ppt. The ammonia level in all tanks was measured every other day Aquarium Systems-FasTest, and partial water changes were made when needed to
M .J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
173
keep the level under 0.1 ppm. Seawater for these changes came from the 150 l storage aquarium and was brought to the appropriate temperature before each change. The
light dark period during both the acclimation period and the growth experiment was L:D515:9, similar to the summer situation, and was switched on and off in two phases
to reduce light shock and to mimic sunrise and sunset. The fish were fed at least twice daily during the light period with pre-weighed portions of the food mixture described
above. The feeding pattern was irregular in frequency and time of day, and was predominantly based on the amount of left-over food and the willingness of the fish to
accept food. Observations showed that individuals frequently swam through the aquarium chasing adult brine shrimp, and that feeding activity slowed down during the
dawn and dusk period. No observations were made during the night. Unconsumed food was removed by pipette and weighed before each successive feeding. Fish that died
during the first three growth periods were replaced by individuals of about the same length to keep the fish densities in the experiment constant. Mortality during the growth
experiment varied Table 1. Within hours after beginning the experiment, two fish died in tank five 248C, both fish were immediately replaced with one unmarked fish and one
fish that was marked 100 days before termination of the experiment. Both were treated as ‘original fish’. All fish died in tank seven 298C during the first growth period and
were replaced at the beginning of the third growth period with five new fish, two were unmarked, two were marked at 100 days, and one was marked 109 days before
termination of the experiment. In tank three 208C all original fish, as well as their replacements, died in the third growth period for unknown reasons and were not
replaced.
At the end of the experiment, both sagittal otoliths of each fish were removed, cleaned, stored dry and coded. Odd numbers represented the otoliths from the right
blind side, even numbers those from the left side of the fish. The left sagitae were prepared for microstucture analysis following standard techniques see Secor et al.,
1991; Stevenson and Campana, 1992; Reichert, 1998. The embedded otoliths were
Table 1 Mortality and replacement of juvenile E
. crossotus during the growth experiment. T, temperature 8C; n, number of fish present at the beginning of the indicated growth period; M, mortality: number of fish that died
during the period Growth period
1 2
3 4
Tank [ T
n M
n M
n M
n M
1 14
5 5
5 5
2 14
5 5
5 5
4 3
20 5
5 3
5 5
– –
4 20
5 5
5 5
1 5
24 5
2 5
5 5
6 24
5 5
1 5
2 5
7 29
5 5
– –
5 5
8 29
5 5
1 5
5 9
14 5
5 5
5
174 M
.J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
sectioned and polished along the transverse plane to a thickness of a few mm with the primordium visible. If the polishing inadvertently resulted in the destruction of the
otolith, the other sagitta was prepared. The preparations were examined under a compound microscope with a 1003 dry objective lens. Polarized light and a blue filter
were used to enhance the visibility of increments. Although the theoretical resolution of the microscope setup was 0.3 mm, a test indicated that the actual resolution was 0.5 mm.
The Alizarin mark was detected using UV light at 540–585 nm for excitation and a 610–680 nm emission filter.
2.3. Somatic growth and temperature The growth response of juvenile fringed flounder at the experimental temperatures
was investigated using the somatic growth data from ‘original’ fish and those added to tank seven at the beginning of the third growth period. Somatic growth was calculated as
the net daily increase in SL of the fish over any of the 12 day growth periods. Uneven mortality in the tanks resulted in an unbalanced statistical design. We analyzed the data
using a mixed model nested ANOVA with repeated measures. The fixed effects were temperature T , growth period, and the temperature by growth period interaction. Tank
was treated as a random effect nested in temperature. Fish were treated as a repeated measures factor with responses recorded for each period in which the fish was alive.
Compound symmetry, ARl and unstructured correlation structures were considered for the repeated measures; Akaike Information Criterion AIC was used to select an
unstructured correlation structure as the most appropriate SAS Institute, 1999. Before analysis we removed two data points tank three, 208C, period 2 from the data set.
These were from two original fish that survived the second growth period, but had very low or negative growth rates and died early in the third growth period. All post hoc
power analyses were done for
a 5 0.05. The gross growth efficiency E was calculated as E 5 G I Brody, 1945, with G as
the somatic growth in total fish WW increase per tank, and I the food intake in net WW of food eaten per tank, using only those periods in which all fish survived.
2.4. Daily increment validation and the relationship between otolith growth and somatic growth
The deposition of daily increments was validated by counting increments outside the fluorescent mark in the otoliths of 23 fish see Fig. 5A for an example of the daily
increments. The otoliths of 20 fish were marked at 66 days, three at 100 days, and one at 109 days prior to the termination of the experiment. The increments were counted in
two designated areas located on the dorsal and the ventral side of the sulcal groove of the otolith see Fig. 2 in Reichert, 1998. The number of increments was counted four
times in each area totaling eight counts in each otolith. If the coefficient of variation CV was more than 10, four additional counts were made. This procedure was
repeated until the CV was ,10 or 16 counts were made. In those otoliths where the increments were not consistently visible in the designated area, increments in other
regions were counted. Otoliths were randomly selected for each counting, and all counts
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175
were made by the senior author. A t-test was used to compare the mean count in each otolith with the expected value based on the number of days the fish lived after being
marked. The relationship between otolith growth and somatic growth was investigated in fish
that survived the complete experiment from marking through termination. In this part of the analysis we were interested in the relationship between somatic growth and otolith
growth only, irrespective of how the somatic growth was achieved. We estimated the
2
otolith growth by measuring the surface area SA in mm and width W in mm outside the mark with the aid of image analysis software. SA was measured once in each otolith.
W is the mean of six linear measurements, three on each side of the sulcal grove in the designated area, from the Alizarin mark to the edge of the otolith and perpendicular to
the increments. The mean daily increment width IW in mm was estimated by dividing W by 66 four 12 day growth periods plus the 18 day post-mark period. In five fish, W
was determined in both the left and the right sagittal otolith to examine variability in width measurements within fish.
We also investigated a method that can be used in unmarked, field-collected fish by measuring the width of the 24 most recently deposited increments in 22 fish that
survived the last two growth periods. Three measurements were made on each side of the sulcal groove, perpendicular to the increments and the IW was compared with the SG
of the fish in the last two growth periods.
3. Results
