M .J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
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
3.1. Somatic growth and temperature Based on all measurements of all fish, the standard length SL was 81.7 of the total
2
length TL n 5 176, adj.r 5 99.9 and the overall SL wet weight relationship was
26 3.363
2
WW54.52310 3SL
n 5 176, adj.r 5 97.8. The mean condition factor
23
CF5WW?SL at the beginning of the first growth period was 0.0174 n 5 46,
s.e.50.0032 and increased during the successive growth periods at all temperatures except 148C Fig. 1. At the end of the growth experiment, only the mean CF of fish
growing between 24 and 148C were significantly different Bonferroni 95 corrected multiple range test.
The effect of handling and marking was investigated by two separate ANOVAs analyzing the daily somatic growth SG of fish in tanks one, two, and the control tank
nine. In tanks one and nine the SG over the four growth periods was used for the analysis, for tank two we used only the first three growth periods because four fish died
in the fourth period. A Bonferroni multiple comparison test indicated no significant differences in somatic growth rates between the three tanks or between marked and
unmarked fish Table 2. We assumed that neither marking nor handling significantly affected growth during the experiment. Most fish resumed feeding within minutes after
being returned to the tanks after being measured and weighed. Earlier growth experiments also indicated that the marking procedure did not affect growth of juvenile
flounder Reichert, unpublished data.
176 M
.J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
23
Fig. 1. Change in mean condition factor CF5W?SL during the growth experiment with juvenile E
. crossotus. The numbers 0, 12, 24, 36 and 48 refer to the days the fish were measured and weighed, with 0
indicating the start of the first of four 12 day growth periods. The error bars are 61 standard error. Data for the two tanks at each temperature were pooled. The dotted horizontal line indicates the CF of field-collected fish of
the same size range data from Reichert, 1998.
The repeated measures analysis of growth data from the eight tanks revealed that the main effect of temperature T was moderately significant P 5 0.0376, Table 3 and
depended greatly on period P , 0.0001. The variability of tank nested within
2
temperature was moderate s 5 0.0050 compared to the within-tank variation be-
T
tween 0.0102 and 0.0046. Examination of the data indicated that somatic growth of the fish in tank four 208C in period four, tanks five and six 248C in period two, and tank
eight 298C in period one was lower than the SG in all other tanks and periods at the
Table 2 Daily somatic growth used to investigate the effect of handling and marking of juvenile E
. crossotus. n is the number of fish used in the analysis. A Bonferroni multiple comparison test yielded no significant differences
21
between tanks F 5 2.88, P 5 0.095 and post hoc power 0.25 for mm SL day
and F 5 0.74,
0.05[2,12] 0.05[2,12]
21
P 5 0.497 and post hoc power 0.10 for mg WW day , or between marked and unmarked fish F
5
0.05[1,13] – 1
0.090, P 5 0.766 and post hoc power 0.05 mm SL day and F
5 1.73, P 5 0.211 and post hoc power
0.05[1,13] – 1
0.06 for mg WW day
21 21
Main n
mm SL day mg WW day
effect Mean
S.D. Mean
S.D. Tank
Tank 1 5
0.07 0.027
8.91 2.50
Tank 2 5
0.08 0.015
13.36 6.23
Tank 9 5
0.12 0.053
13.16 9.08
Mark Mark
12 0.09
0.027 12.87
4.41 No mark
3 0.08
0.044 7.57
6.52
M .J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
177 Table 3
21
ANOVA for the fixed effects of the daily somatic growth mm SL day in experiments with juvenile E
. crossotus at 14, 20, 24 and 298C during four subsequent 12 day growth periods
Effect Num.
Den. F-
P- df
df ratio
value Temperature
3 4
7.84 0.038
Period 3
99 1.25
0.295 TempPeriod
9 99
10.71 ,0.000
same temperature Fig. 2. In tank five two fish died in the first growth period and in tank six three fish died late in the second and early in the third growth period. In tanks
four and eight the reason for lower growth in the first growth period was unclear. The initial size was a significant negative effect on SG only in tank four 208C P 5
0.001. The lack of a consistent effect of initial size on growth indicated that, within the size range of the juveniles used, SG at each T was independent of the size of the fish.
Since we were interested in the relationship between T and SG under good quality growth conditions we omitted the data from period four at 208C, period two at 248C and
period one at 298C based on the above analysis. Data from all fish and all periods at each T were then pooled. The somatic growth increased with temperature reaching a
maximum at 24 and 298C and showed similar patterns whether expressed as mm SL
21 21
21
day or g WW d
Fig. 3. The maximum observed growth rate was 0.7 mm SL d
21
or 0.136 g WW d , both at 298C. A Bonferroni multiple comparison yielded a
Fig. 2. Mean daily somatic growth SG of juvenile E . crossotus in each of the four 12 day growth periods
I–IV at 14, 20, 24 and 298C. Data from both tanks per temperature were combined. The error bars are 61 standard error. The asterisk indicates a significant difference from all other values at that temperature.
178 M
.J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
Fig. 3. Mean daily increase in length left axis, s and wet weight right axis, 3 of juvenile E . crossotus at
the four experimental temperatures. The error bars indicate 61 standard error. d, The maximum observed values.
significant difference between SG for 14 versus 208C only. The gross growth efficiency E increased with T to 248C, with a subsequent decrease at 298C Fig. 4.
3.2. Validation of daily increment deposition and check formation The Alizarin complexone left a clear fluorescent mark that could be followed
consistently throughout all otolith preparations. For the validation of the daily increment deposition, eight replicate counts were enough to generate a mean value with a
coefficient of variation CV of 10 in 11 of the 24 examined otoliths Table 4. Increasing the counts lowered the CV to 10 in only two more otoliths. In six of the
remaining otoliths, the increments on one side of the sulcus two on the dorsal and four on the ventral side were inconsistent and difficult to distinguish, resulting in variable
counts CV 14–26. Subsequently, the other side was used for the analysis and in all otoliths eight counts on that side yielded a CV 10. A t-test revealed that in only five
otoliths was the mean number of counted increments significantly different from the expected value; in all cases they were lower Table 4.
Some otoliths showed indications of stress checks; discontinuities in the appearance of the micro-increments Fig. 5B. These checks were visible as darker and more
pronounced opaque zones and could possibly be associated with the measurement events. The first occurred on the 18th day after marking, and the number of daily
increments between the subsequent stress checks was 12, coinciding with the number of days in each of the four growth periods.
3.3. Somatic growth and otolith growth A regression analysis indicated a moderately strong linear relationship between the
M .J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188
179
Fig. 4. Mean gross growth efficiency E of juvenile E . crossotus at the four experimental temperatures. E was
calculated as daily somatic growth of all fish per tank divided by the daily food intake per tank. Data from only those growth periods in which no fish died were used and data from the two tanks per temperature were
pooled. The vertical bars indicate 61 standard error.
2
increase in otolith surface area outside the mark SA in mm and daily somatic growth
21 2
SG in mm SL day 50.06910.9193SA, F
5 55.3, adj.r 5 74, Fig. 6A,
1,19
suggesting a positive relationship between somatic growth and otolith growth. We assumed that either otolith could be used for the analysis since linear measurements of
otolith growth outside the mark W in both otoliths of five fish indicated that W was not consistently higher or lower in the left or right otolith, and that there were no significant
differences in the mean W of the two sagittal otoliths of each fish P-values ranged from 0.471 to 0.606, while the post hoc power ranged from 0.10 to 0.19, Table 5.
To describe the relationship between SG and increment width IW, we first compared the data based on the linear measurements from the Alizarin mark to the edge 66 days,
d in Fig. 6B with the data based on the last 24 daily increments 24 days, 3 in Fig. 6B. A visual inspection of the regression lines suggested differences between the two
data sets when somatic growth was low. An ANCOVA yielded no significant differences between the slopes P 5 0.750 or the intercepts P 5 0.998 of two linear regression
21
lines for SG0.2 mm SL day , a value selected on the basis of the visual inspection.
21
There were, however, significant differences for SG,0.2 mm SL day . With equal
slopes P 5 0.327, the IW based on 66 days intercept IW50.42 mm was significantly lower P 5 0.001 than the IW based on 24 days intercept IW50.62 mm. This
indicated that, at low growth rates, the daily increments might have been too narrow to be distinguished, resulting in an underestimation of their true number. This would in turn
cause an overestimation of the average width of the daily increments. Data based on 24 counts for SG,0.2 were therefore omitted from further analyses. This analysis yielded a
significant linear relationship between IW and SG SG5 20.00410.2823IW, F
5
1,33
180 M
.J.M. Reichert et al. J. Exp. Mar. Biol. Ecol. 254 2000 169 –188 Table 4
Validation of daily increment deposition in E . crossotos otoliths. E, expected number of increments; C, counted
number of increments; P , 0.05, P , 0.01, counts significantly different from expected value; S.D., standard deviation; CV, coefficient of variation; I, additional counts did not decrease the CV; II, counts
were based on one side of the otolith only see text Otolith
E C
S.D. CV
n A2
66 33.2
8.3 24.9
16 A3
66 37.5
9.9 26.5
16 A5
66 62.8
6.1 9.7
8 A5
66 34.4
11.1 32.1
16 A10
66 38.8
5.9 15.2
16 D3
66 66.8
7.2 10.8
16 67.3
4.1 6.0
8 II
D8 66
63.9 6.4
10.0 8
El 66
70.2 14.2
20.3 16
I E3
66 63.6
8.7 13.8
16 65.3
4.6 7.1
8 II
E9 66
70.6 6.9
9.7 8
F1 66
69.1 4.7
6.8 8
F4 66
66.1 6.6
10.0 8
F6 66
66.4 5.1
7.6 8
F8 66
67.6 6.2
9.2 8
G1 66
68.8 6.2
9.0 8
G3 66
65.4 4.0
6.1 8
H2 66
51.8 13.0
25.9 12
63.8 3.3
5.2 8
II H3
66 49.9
5.5 19.1
16 68.9
9.5 7.1
8 II
H6 66
57.6 13.8
24.0 16
69.4 6.8
9.8 8
II H7
66 64.3
6.3 9.8
12 C5
100 99.4
9.8 9.8
8 C7
100 101.7
11.3 11.1
16 100.4
5.8 5.8
8 II
E6 100
95.9 6.3
6.5 16
C3 109
108.8 7.8
7.1 8
197. Changes in IW accounted for 85 of the variability in SG. A comparison of regression models yielded a logarithmic relationship SG50.28910.170 lnIW, F
5
1,22 2
98.1, adj.r 5 82 for the lower part of the data SG,0.35 and a reciprocal model for
2
the upper part of the data SG.0.18, SG51 6.34–2.623IW, F 5 63.0, adj.r 5
1,24 2
72. The r values for the linear models using the same data were respectively 5 and 2 lower.
4. Discussion
