Somatic growth and otolith growth in juveniles of a small subtropical flatfish, the fringed flounder, Etropus crossotus

Journal of Experimental Marine Biology and Ecology254 (2000) 169–188

www.elsevier.nl / locate / jembe

Somatic growth and otolith growth in juveniles of a smallsubtropical flatfish, the fringed flounder, Etropus crossotus

a , a,b,c a,b,c*Marcel J.M. Reichert , John M. Dean , Robert J. Feller ,dJohn M. Grego

aBelle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina,Columbia, SC 29208, USA

bMarine Science Program, University of South Carolina, Columbia, SC 29208, USAcDepartment of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA

dDepartment of Statistics, University of South Carolina, Columbia, SC 29208, USA

Received 16 August 1999; received in revised form 21 July 2000; accepted 31 July 2000

Abstract

A growth experiment was conducted with juvenile fringed flounder (Etropus crossotus) fromNorth Inlet (South Carolina, USA) to provide information on the growth of a small, shortlivedflatfish with a subtropical and tropical distribution. The fringed flounder has a maximum life spanof 1.5 y and its long spawning period from March through October complicates the determinationof growth rates based on length frequency data. Otoliths of juveniles with a standard length (SL)23.1–53.0 mm were marked with Alizarin complexone and the fish were held in the laboratory for66 days at 14, 20, 24 and 298C while being fed ad libitum. The mean somatic growth increased

21 21with temperature from 0.1 mm SL day at 148C to 0.4 mm SL day at both 24 and 298C. The1maximum observed somatic growth rate was 0.7 mm SL day at 298C. The number of

micro-increments formed in otoliths was not significantly different from the expected value,validating formation of one increment per day. The significant relationship between incrementwidth and somatic growth rate can be used to estimate somatic growth rates of individual wild fishbased on daily increment information in their otoliths. 2000 Elsevier Science B.V. All rightsreserved.

Keywords: Flatfish; Growth; Otoliths; Temperature

*Corresponding author. Tel.: 11-803-777-3135; fax: 11-803-777-3935.E-mail address: [email protected] (M.J.M. Reichert).

0022-0981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 00 )00277-X

170 M.J.M. Reichert et al. / J. Exp. Mar. Biol. Ecol. 254 (2000) 169 –188

1. Introduction

There has been considerable discussion of the role of food in determining maximumgrowth rates and survival of juvenile flatfishes in nursery areas (Miller et al., 1991; Vander Veer and Witte, 1993; Gibson, 1994). It is generally accepted that among the manyfactors governing growth of fish, the most important are the quality and quantity of foodas the driving force, temperature as a rate controlling factor, and the size of fish as anallometric scaling factor (Brett and Groves, 1979). Abundant food, favorable tempera-tures, and shelter from predation are key factors in allowing juvenile fish to growquickly to a less vulnerable or adult size in nursery areas (e.g., Gibson, 1994). Themajority of the available information on flatfishes is based on studies of relativelylonglived, large, temperate species. Information on growth of shortlived, small subtropi-cal and tropical flatfish species is limited and almost exclusively based on field-collectedmaterial (e.g., Topp and Hoff, 1972; Reichert and van der Veer, 1991; Van der Veer et al.,1994; Joyeux et al., 1995). Experimental data on growth under well-defined conditions islacking.

Many subtropical and tropical (flat)fish species have an extended spawning period(Topp and Hoff, 1972; McEvoy and McEvoy, 1992; Reichert, 1998). When reproductionis spread out over several months, the increase in mean size of the population over timecannot be used to estimate growth rates. Since the discovery of daily structures inotoliths, the microstructure of otoliths has provided a tool to study both growth rates andlife history characters in fish (e.g., Pannella, 1971; Stevenson and Campana, 1992; Secoret al., 1995). Various studies have shown that resorption of otolith material does not takeplace, even under periods of low or even negative growth (Simkiss, 1974; Campana,1983; Neilson and Geen, 1985; Jones, 1992, p. 2). The resulting permanent records ofdaily increments in otoliths allow detailed determinations of the age of individual fish,permitting estimates of recruitment, mortality rates, and related parameters in fishpopulations. Despite wide acceptance of the use of increments in age and growth studiesin fish, and the fact that ‘‘the deposition of daily increments appears to be a universalphenomenon under perhaps all but the most severe conditions’’ (Jones, 1992), anappropriate validation is still essential for a correct interpretation of the otolithmicrostructure (Geffen, 1992).

A strong correlation between otolith growth and somatic growth in fish has lead to theuse of otoliths for estimating growth rates of fish (e.g., Brothers and McFarland, 1981;Neilson and Geen, 1985; Volk et al., 1984; Bradford and Geen, 1987; Dickey et al.,1997). A linear relationship between otolith growth and somatic growth has beendescribed for larval plaice (Karakiri and von Westenhagen, 1989), juvenile winterflounder (Jearld et al., 1992), juvenile greenback flounder (Jenkins et al., 1993), andlarval and early juvenile striped bass (Dickey et al., 1997), among others. An uncouplingof a tight relationship between otolith growth and somatic growth has been demonstratedmostly at the lower, and sometimes the extreme upperend of the fish’s growth spectrumin many studies. Such uncoupling results in slower growing fish having relatively largerheavier otoliths, e.g. Geffen (1982) for Clupea harengus and Scophthalmus maximus,Mosegaard et al. (1988) for arctic charr, Bradford and Geen (1987) for chinook salmon,Secor et al. (1989) for Morone saxatilis, Pagrus major and Leiostomus xanthurus, and

M.J.M. Reichert et al. / J. Exp. Mar. Biol. Ecol. 254 (2000) 169 –188 171

Wright et al. (1990) for Atlantic salmon parr. Others have argued that such uncoupling isnot always present, for instance Dickey et al. (1997) found no evidence for uncouplingin larval striped bass. Regardless of the presence of an uncoupling, and assuming therate of growth is neither extremely high nor low, increment width can be used to provideestimates of (recent) growth of individual field-collected fish once the relationshipbetween otolith growth (increment width) and somatic growth is established.

This paper will address aspects of growth in the fringed flounder (Etropus crossotusJordan 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 AtlanticBight and the Gulf of Mexico, and very common in estuaries and shallow waters ofSouth 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). Themaximum reported total length of the fringed flounder is 16.9 cm, but individual fish arerarely longer than 15 cm, and their wet weight is seldom more than 40 g (Moe andMartin, 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 floundercan be found year round on mud and muddy sand in shallow coastal waters and estuariesat 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 flounderlimits the size of their prey, predominantly small benthic and epibenthic crustaceans andpolychaetes (Reid, 1954; Topp and Hoff, 1972; Stickney et al., 1974; Reichert and vander 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 alength at 50% maturity between 8.0 and 8.5 cm SL (Reichert, 1998). Reichert and van

21der Veer (1991) and Reichert (1998) estimated a growth rate of about 0.5 mm day at24 to 288C for juveniles, but detailed information for growth under controlled, goodquality conditions was not available. Using experimentally derived data, we investigated(1) the relationship between temperature and growth under defined conditions with nofood 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 toestimate natural rates of growth of individual fish collected in the field, and to modelgrowth of fringed flounder populations.

2. Materials and methods

2.1. Sample collection

Juvenile fringed flounder were collected on June 13, 1996, from Town Creek andDebidue Creek in North Inlet (South Carolina, USA) using a 1 m beamtrawl with astretched mesh size of 1cm (see Reichert, 1998). The seawater in the creeks was 278Cand had a salinity of 30 ppt. The standard length (SL, 60.1 mm), total length (TL, 60.1


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 fishwere 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 andS 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 liveadult 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 aclimate controlled experimental chamber. These temperatures were chosen to create arange of growth rates under ad libitum food conditions. Temperatures were randomlyassigned 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 airsupply, and filled with 33 1 of seawater (29–31 ppt). The bottom of each tank wascovered with a 2 cm layer of cleaned fine sand from the same location the fish werecollected. The top of every tank was covered with clear Plexiglas and the four sides werecovered with brown Styrofoam to provide insulation, prevent visual interaction betweentanks, and minimize disturbances. A ninth tank at 148C (60.58C) with identical setupwas used as a control to investigate the effect of handling and marking. Prior to thebeginning of the experiment, seawater was recirculated between all experimental tanksand a 150 1 storage aquarium equipped with a trickle wet /dry filter to establish uniformwater 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 ppmAlizarin complexone in seawater (238C, 30 ppt). The marking procedure was tested bymarking 10 fish 1 day after collection and again by marking 10 fish 8 days aftercollection, or 109 and 100 days before the end of the experiment respectively. There wasno mortality during the marking procedure or in the few days that followed. Immediatelyafter marking, the fish were measured with electronic calipers (SL and TL), weighed(WW), and uniquely externally marked by fin clipping for identification. Five fish wereplaced in each of the experimental tanks. The remaining marked and unmarked fish wereplaced 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 createthe appropriate experimental temperatures in each tank. Temperature in all tanks wasstable at the nominal value within 10 days. Starting on the 18th day after marking, theSL, TL, and WW of each fish were determined five times at 12 day intervals, resulting infour 12 day growth periods. The fish in the control tank were measured only at thebeginning and at the end of the experiment. Three of these five fish were not markedwith Alizarin.

The oxygen concentration in the tanks was measured at least once a week and oxygensaturation levels rarely dropped below 90%. The salinity in all tanks was monitoreddaily and kept at 29–31 ppt. The ammonia level in all tanks was measured every otherday (Aquarium Systems-FasTest), and partial water changes were made when needed to


keep the level under 0.1 ppm. Seawater for these changes came from the 150 l storageaquarium and was brought to the appropriate temperature before each change. Thelight /dark period during both the acclimation period and the growth experiment wasL:D515:9, similar to the summer situation, and was switched on and off in two phasesto reduce light shock and to mimic sunrise and sunset. The fish were fed at least twicedaily during the light period with pre-weighed portions of the food mixture describedabove. The feeding pattern was irregular in frequency and time of day, and waspredominantly based on the amount of left-over food and the willingness of the fish toaccept food. Observations showed that individuals frequently swam through theaquarium chasing adult brine shrimp, and that feeding activity slowed down during thedawn and dusk period. No observations were made during the night. Unconsumed foodwas removed by pipette and weighed before each successive feeding. Fish that diedduring the first three growth periods were replaced by individuals of about the samelength to keep the fish densities in the experiment constant. Mortality during the growthexperiment varied (Table 1). Within hours after beginning the experiment, two fish diedin tank five (248C), both fish were immediately replaced with one unmarked fish and onefish that was marked 100 days before termination of the experiment. Both were treatedas ‘original fish’. All fish died in tank seven (298C) during the first growth period andwere replaced at the beginning of the third growth period with five new fish, two wereunmarked, two were marked at 100 days, and one was marked 109 days beforetermination of the experiment. In tank three (208C) all original fish, as well as theirreplacements, died in the third growth period for unknown reasons and were notreplaced.

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 wereprepared for microstucture analysis following standard techniques (see Secor et al.,1991; Stevenson and Campana, 1992; Reichert, 1998). The embedded otoliths were

Table 1Mortality 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 diedduring the period

Growth period

1 2 3 4

Tank [ T n M n M n M n M

1 14 5 0 5 0 5 0 5 02 14 5 0 5 0 5 0 5 43 20 5 0 5 3 5 5 – –4 20 5 0 5 0 5 0 5 15 24 5 2 5 0 5 0 5 06 24 5 0 5 1 5 2 5 07 29 5 5 – – 5 0 5 08 29 5 0 5 1 5 0 5 09 14 5 0 5 0 5 0 5 0


sectioned and polished along the transverse plane to a thickness of a few mm with theprimordium visible. If the polishing inadvertently resulted in the destruction of theotolith, the other sagitta was prepared. The preparations were examined under acompound microscope with a 1003 dry objective lens. Polarized light and a blue filterwere used to enhance the visibility of increments. Although the theoretical resolution ofthe 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 a610–680 nm emission filter.

2.3. Somatic growth and temperature

The growth response of juvenile fringed flounder at the experimental temperatureswas investigated using the somatic growth data from ‘original’ fish and those added totank seven at the beginning of the third growth period. Somatic growth was calculated asthe net daily increase in SL of the fish over any of the 12 day growth periods. Unevenmortality in the tanks resulted in an unbalanced statistical design. We analyzed the datausing a mixed model nested ANOVA with repeated measures. The fixed effects weretemperature (T ), growth period, and the temperature by growth period interaction. Tankwas treated as a random effect nested in temperature. Fish were treated as a repeatedmeasures factor with responses recorded for each period in which the fish was alive.Compound symmetry, AR(l) and unstructured correlation structures were considered forthe repeated measures; Akaike Information Criterion (AIC) was used to select anunstructured correlation structure as the most appropriate (SAS Institute, 1999). Beforeanalysis 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 verylow or negative growth rates and died early in the third growth period. All post hocpower analyses were done for a 5 0.05.

The gross growth efficiency (E) was calculated as E 5 G /I (Brody, 1945), with G asthe somatic growth in total fish WW increase per tank, and I the food intake in net WWof food eaten per tank, using only those periods in which all fish survived.

2.4. Daily increment validation and the relationship between otolith growth andsomatic growth

The deposition of daily increments was validated by counting increments outside thefluorescent mark in the otoliths of 23 fish (see Fig. 5A for an example of the dailyincrements). The otoliths of 20 fish were marked at 66 days, three at 100 days, and oneat 109 days prior to the termination of the experiment. The increments were counted intwo designated areas located on the dorsal and the ventral side of the sulcal groove ofthe otolith (see Fig. 2 in Reichert, 1998). The number of increments was counted fourtimes 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 wasrepeated until the CV was ,10% or 16 counts were made. In those otoliths where theincrements were not consistently visible in the designated area, increments in otherregions were counted. Otoliths were randomly selected for each counting, and all counts


were made by the senior author. A t-test was used to compare the mean count in eachotolith with the expected value based on the number of days the fish lived after beingmarked.

The relationship between otolith growth and somatic growth was investigated in fishthat survived the complete experiment from marking through termination. In this part ofthe analysis we were interested in the relationship between somatic growth and otolithgrowth only, irrespective of how the somatic growth was achieved. We estimated the

2otolith growth by measuring the surface area (SA in mm ) and width (W in mm) outsidethe 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 thedesignated area, from the Alizarin mark to the edge of the otolith and perpendicular tothe increments. The mean daily increment width (IW in mm) was estimated by dividingW by 66 (four 12 day growth periods plus the 18 day post-mark period). In five fish, Wwas determined in both the left and the right sagittal otolith to examine variability inwidth measurements within fish.

We also investigated a method that can be used in unmarked, field-collected fish bymeasuring the width of the 24 most recently deposited increments in 22 fish thatsurvived the last two growth periods. Three measurements were made on each side ofthe sulcal groove, perpendicular to the increments and the IW was compared with the SGof the fish in the last two growth periods.

3. Results


Based on all measurements of all fish, the standard length (SL) was 81.7% of the total2length (TL) (n 5 176, adj.r 5 99.9%) and the overall SL/wet weight relationship was

26 3.363 2WW5(4.52310 )3SL (n 5 176, adj.r 5 97.8%). The mean condition factor23(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 temperaturesexcept 148C (Fig. 1). At the end of the growth experiment, only the mean CF of fishgrowing between 24 and 148C were significantly different (Bonferroni (95%) correctedmultiple range test).

The effect of handling and marking was investigated by two separate ANOVAsanalyzing the daily somatic growth (SG) of fish in tanks one, two, and the control tanknine. In tanks one and nine the SG over the four growth periods was used for theanalysis, for tank two we used only the first three growth periods because four fish diedin the fourth period. A Bonferroni multiple comparison test indicated no significantdifferences in somatic growth rates between the three tanks or between marked andunmarked fish (Table 2). We assumed that neither marking nor handling significantlyaffected growth during the experiment. Most fish resumed feeding within minutes afterbeing returned to the tanks after being measured and weighed. Earlier growthexperiments also indicated that the marking procedure did not affect growth of juvenileflounder (Reichert, unpublished data).


23Fig. 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 0indicating the start of the first of four 12 day growth periods. The error bars are 61 standard error. Data for thetwo tanks at each temperature were pooled. The dotted horizontal line indicates the CF of field-collected fish ofthe same size range (data from Reichert, 1998).

The repeated measures analysis of growth data from the eight tanks revealed that themain effect of temperature (T ) was moderately significant (P 5 0.0376, Table 3) anddepended greatly on period (P , 0.0001). The variability of tank nested within

2temperature 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 thefish in tank four (208C) in period four, tanks five and six (248C) in period two, and tankeight (298C) in period one was lower than the SG in all other tanks and periods at the

Table 2Daily somatic growth used to investigate the effect of handling and marking of juvenile E. crossotus. n is thenumber of fish used in the analysis. A Bonferroni multiple comparison test yielded no significant differences

21between 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]21P 5 0.497 and post hoc power 0.10 for mg WW day ), or between marked and unmarked fish (F 50.05[1,13]

–10.090, 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 power0.05[1,13]–10.06 for mg WW day )

21 21Main n mm SL day mg WW dayeffect

Mean S.D. Mean S.D.

Tank Tank 1 5 0.07 0.027 8.91 2.50Tank 2 5 0.08 0.015 13.36 6.23Tank 9 5 0.12 0.053 13.16 9.08

Mark Mark 12 0.09 0.027 12.87 4.41No mark 3 0.08 0.044 7.57 6.52


Table 321ANOVA 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 0.038Period 3 99 1.25 0.295Temp*Period 9 99 10.71 ,0.000

same temperature (Fig. 2). In tank five two fish died in the first growth period and intank six three fish died late in the second and early in the third growth period. In tanksfour and eight the reason for lower growth in the first growth period was unclear. Theinitial 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 thesize 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 qualitygrowth conditions we omitted the data from period four at 208C, period two at 248C andperiod one at 298C based on the above analysis. Data from all fish and all periods at eachT were then pooled. The somatic growth increased with temperature reaching amaximum at 24 and 298C and showed similar patterns whether expressed as mm SL

21 21 21day or g WW d (Fig. 3). The maximum observed growth rate was 0.7 mm SL d21or 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 61standard error. The asterisk indicates a significant difference from all other values at that temperature.


Fig. 3. Mean daily increase in length (left axis, s) and wet weight (right axis, 3) of juvenile E. crossotus atthe four experimental temperatures. The error bars indicate 61 standard error. (d,*) The maximum observedvalues.

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 followedconsistently throughout all otolith preparations. For the validation of the daily incrementdeposition, eight replicate counts were enough to generate a mean value with acoefficient 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 theremaining otoliths, the increments on one side of the sulcus (two on the dorsal and fouron the ventral side) were inconsistent and difficult to distinguish, resulting in variablecounts (CV 14–26%). Subsequently, the other side was used for the analysis and in allotoliths eight counts on that side yielded a CV #10%. A t-test revealed that in only fiveotoliths was the mean number of counted increments significantly different from theexpected value; in all cases they were lower (Table 4).

Some otoliths showed indications of stress checks; discontinuities in the appearance ofthe micro-increments (Fig. 5B). These checks were visible as darker and morepronounced opaque zones and could possibly be associated with the measurementevents. The first occurred on the 18th day after marking, and the number of dailyincrements between the subsequent stress checks was 12, coinciding with the number ofdays 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


Fig. 4. Mean gross growth efficiency (E) of juvenile E. crossotus at the four experimental temperatures. E wascalculated as daily somatic growth of all fish per tank divided by the daily food intake per tank. Data from onlythose growth periods in which no fish died were used and data from the two tanks per temperature werepooled. The vertical bars indicate 61 standard error.

2increase in otolith surface area outside the mark (SA in mm ) and daily somatic growth21 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. Weassumed that either otolith could be used for the analysis since linear measurements ofotolith growth outside the mark (W ) in both otoliths of five fish indicated that W was notconsistently higher or lower in the left or right otolith, and that there were no significantdifferences in the mean W of the two sagittal otoliths of each fish (P-values ranged from0.471 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 comparedthe 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 twodata sets when somatic growth was low. An ANCOVA yielded no significant differencesbetween the slopes (P 5 0.750) or the intercepts (P 5 0.998) of two linear regression

21lines for SG$0.2 mm SL day , a value selected on the basis of the visual inspection.21There 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 significantlylower (P 5 0.001) than the IW based on 24 days (intercept IW50.62 mm). Thisindicated that, at low growth rates, the daily increments might have been too narrow tobe distinguished, resulting in an underestimation of their true number. This would in turncause an overestimation of the average width of the daily increments. Data based on 24counts for SG,0.2 were therefore omitted from further analyses. This analysis yielded asignificant linear relationship between IW and SG (SG520.00410.2823IW, F 51,33


Table 4Validation of daily increment deposition in E. crossotos otoliths. E, expected number of increments; C, countednumber 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), countswere 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 16A3 66 37.5 ** 9.9 26.5 16A5 66 62.8 6.1 9.7 8A5 66 34.4 ** 11.1 32.1 16A10 66 38.8 ** 5.9 15.2 16D3 66 66.8 7.2 10.8 16

67.3 4.1 6.0 8 (II)D8 66 63.9 6.4 10.0 8El 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 8F1 66 69.1 4.7 6.8 8F4 66 66.1 6.6 10.0 8F6 66 66.4 5.1 7.6 8F8 66 67.6 6.2 9.2 8G1 66 68.8 6.2 9.0 8G3 66 65.4 4.0 6.1 8H2 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 12C5 100 99.4 9.8 9.8 8C7 100 101.7 11.3 11.1 16

100.4 5.8 5.8 8 (II)E6 100 95.9 * 6.3 6.5 16C3 109 108.8 7.8 7.1 8

197). Changes in IW accounted for 85% of the variability in SG. A comparison ofregression models yielded a logarithmic relationship (SG50.28910.170 ln(IW), F 51,22

298.1, adj.r 5 82%) for the lower part of the data (SG,0.35) and a reciprocal model for2the upper part of the data (SG.0.18, SG51/(6.34–2.623IW), F 5 63.0, adj.r 51,24

272%). The r values for the linear models using the same data were respectively 5 and2% lower.

4. Discussion


The growth rates, condition factor and growth efficiency indicated that the experimen-


Fig. 5. Details of a sagittal otolith of a juvenile E. crossotus polished along the transverse plane shown under acombination of UV and visible light. The black bars indicate 10 mm. (A) Example of the Alizarin complexonemark and daily increments using a 1003 dry objective. The orientation is such that the core is located towardsthe upper right corner, the edge of the otolith towards the lower left corner. (B) Example of four stress checks(indicated as ‘measurement marks’) using a 403 objective. The edge of the otolith is visible on the left side.Some of the daily increments are visible between the checks.


Fig. 6. Regression analyses of the relationship between daily somatic growth (SG) and otolith growth. Thedotted lines are the 95% confidence limits. (A) Relationship between the increase in otolith surface outside the

2 2mark (SA in mm ) and SG. SG50.06910.9193SA, F 5 55.3, adj.r 5 74%. (B) Relationship between SG1,19

and increment width (IW). (d) Data based on measurements from the Alizarin mark to the edge (66 days);(3) data based on the measurements of the last 24 daily increments. SG520.00410.2823IW, F 5 197,1,33

2adj.r 5 85%. The horizontal lines indicate 61 standard error in the increment widths.


Table 5Variability in width measurements in both sagittal otoliths of five juvenile S. crossotus. Odd otolith number,right side; even otolith number, left side of the fish; W, width (mm) measured from the Alizarin mark to theedge of the otolith in the designated area (see text); n, number of measurements (one side of ES was polishedaway resulting in three measurements only); S.E., standard error

Otolith W n S.E.[

Al 27.2 6 2.88A2 24.6 6 1.88A7 26.9 6 4.07A8 29.0 6 3.64E5 85.1 3 3.99E6 88.1 6 2.16F3 78.0 6 3.33F4 72.3 6 6.69F7 82.4 6 3.48F8 88.7 6 9.35

tal growth conditions were generally good. At the beginning of the first growth period,the condition factor (CF) of the experimental fish at all four temperatures was below thatof field-collected fish. The condition factor (CF) increased during the growth experimentat all temperatures to values not different from or exceeding those determined shortlyafter the fish were caught. By the end of the growth experiment the CF increased most at248C, suggesting near optimal growth conditions, also supported by high growth rates atthat temperature. The increase in growth efficiency with the growth rate suggested thatoptimal food conversion took place near the optimum growth temperature. Growthefficiency data for E. crossotus are not available in the literature, but a growth efficiencybetween 7 and 17% is within the range given for other fish species (e.g., Volk et al.,1984; Fonds et al., 1992). The lack of a significant relationship between fish size andsomatic growth in juveniles between 25 and 70 mm SL was supported by informationfrom field-collected fish of the same size range (Reichert and van der Veer, 1991;Reichert, 1998). The high variability in the growth data at 298C might have been causedby the fact that this is close to the potential lethal temperature of 318C for this species(Topp and Hoff, 1972; Reichert, unpublished data). The low growth rate in the firstgrowth period at 298C might have resulted from incomplete acclimation. The analysisindicated that acclimation was completed before the first growth period at the othertemperatures. The growth rates observed at the various temperatures were similar to thefew values provided in other studies. Growth experiments with juvenile E. crossotus by

21Reichert and van der Veer (1991) yielded a mean growth rate of 0.5 mm SL day at21 21both 24 and 298C, with maximum values of 0.7 mm day at 248C and 0.8 mm day at

298C. The growth curve based on otolith data of field-collected fringed flounder by21Reichert (1998) indicated a mean growth rate of 0.5 mm day for juveniles, but the

effect of temperature was not taken into account in constructing that curve.A maximum growth rate in the range of 24 to 298C is consistent with the fact that

juveniles are most abundant between April and September, when mean water tempera-tures are between 22 and 308C (Reichert, 1998; Ogburn et al., 1988; Ogburn, personal


communication). Additional growth experiments with fish of a different size rangeshould provide information on size-dependent temperature preferences as reported byFonds and Saksena (1977) and Fonds et al. (1992) for plaice and sole. Juveniles of thesetwo species have a higher optimum temperature for growth than larger fish, which growbetter at lower temperatures. In both plaice and sole the young adults migrate offshorewhere they find optimum growth conditions at lower temperatures. While juvenile E.crossotus are abundant in southeastern estuaries, the large adults (.8 cm SL) are rarelyfound there, but are commonly caught offshore (Reichert and van der Veer, 1991; Allenand Baltz, 1997; Reichert, 1998; Boylan, SCDNR, personal communication). AlthoughFonds et al. (1992) worked on northern temperate species, a similar migration patternmight be reflected in a similar size-dependent growth response to temperature.

4.2. Daily increments validation and check formation

The formation of daily increments was validated for E. crossotus. In five fish with lowgrowth rates the number of counted daily increments was less than the expected valueand the daily increments might have been too narrow to be consistently distinguishedusing a light microscope. The overall variability in the counts of the daily incrementssuggested that four replicate counts in each of the two designated areas are sufficient toestimate the fish’s age.

The formation of checks in otoliths following environmental or physiological eventshave also been described by several other authors (e.g., Campana, 1983; Volk et al.,1984, 1999; Berghahn and Karakiri, 1990; Geffen and Nash, 1995; Berghahn, 2000). Inour experimental work with E. crossotus, stress checks were not present or clearlyvisible in all otoliths, possibly because of careful handling to reduce stress. Campana(1992) and Geffen (1992) provide an excellent overview of the many factors affectingthe deposition and appearance of daily increments: feeding frequency, temperature, andlight can influence deposition, appearance, and width of daily increments. Recently, Volket al. (1999) described the use of temperature cycles to induce stress checks in otolithsof juvenile salmonids and reviewed its use in large-scale marking efforts. Less carefulhandling or a temporary change in oxygen level or temperature might create a moredistinct stress mark that can be used in lieu of chemical marking.

4.3. Somatic growth and increment width

Campana and Jones (1992) discussed important requirements for using otolithincrement information to analyze fish growth retrospectively. In asymmetric otoliths therelationship between otolith size and fish size must be related to a standard transect onthe otolith along which increment widths can be measured. Once this relationship isknown, then size at prior age can be back-calculated from the otolith data alone. Amethod of counting and measuring the width of the most recently deposited increments(in our case 24) to estimate somatic growth over that period provided results similar to


the experimental method using the Alizarin mark in the otolith. The standardizedmeasurements used in this study revealed a highly significant linear relationship betweenincrement widths (IW) and somatic growth rates, indicating that increment widths canbe used to estimate recent somatic growth. A linear relationship has also been reportedfor other species, e.g. Jenkins et al. (1993) for juvenile greenback flounder, and Dickeyet al. (1997) for juvenile striped bass. There is, however, reason for some caution. Alinear relationship between IW and SG with an intercept close to the origin implies thatthere was no evidence for a loss of a linear relationship (uncoupling) between SG andIW. The deviation from a linear relationship at the lower and higher ends of the somaticgrowth rate spectrum suggested that an uncoupling between somatic growth andincrement width might be present. The exact nature of the relationship between otolithgrowth and fish growth near maximal end minimal growth rates is not entirely clearusing the currently available data. The minimum increment width in the area wedesignated for our measurements was about 0.4 mm, but the analysis suggests thatincrement widths less than 0.6 mm should be used with caution to estimate daily somaticgrowth. The data also indicated that somatic growth might be somewhat underestimatedwhen the increment width becomes greater than l.5 mm and may be better described by areciprocal function. Detailed studies of the lower and upper end of the growth ratespectrum may reveal a relationship between SG and IW that is more accuratelydescribed with a reciprocal natural log or polynomial function.

The validation of daily increment deposition and the relationship between incrementwidth and rate of somatic growth can be used to estimate recent growth of field-collectedjuvenile E. crossotus. Using the known growth response to temperature, a comparisoncan be made between the potential growth and realized growth based on recenttemperature profiles in the natural environment. Possible discrepancies can be traced topossible growth-limiting factors in the environment such as food and the physicalenvironment. These experimental data fill a void in the information on growth ofsmaller, shortlived subtropical flatfishes. Our work is currently used to estimate growthrates of individual fish collected in the field, to investigate the role of food indetermining growth rates of field populations, and to model both individual andpopulation growth of the fringed flounder.

Acknowledgements

William Driggers, John Seigle, Brice Gill, Michael Cameron, Stephen Sabatino,Andrew Bauman and Jason Flake are gratefully acknowledged for their assistance in thefield and the laboratory. We thank Dr. R. Drent, Dr. H.van der Veer, and two reviewersfor their comments on an earlier draft of the manuscript. This study was carried out withsupport from the Belle W. Baruch Institute for Marine Biology and Coastal Research atthe University of South Carolina, and is part of the International Research Project onFlatfish Ecology, a cooperative project between the Netherlands Institute for SeaResearch, North Carolina State University, Louisiana State University, and the Baruch


Institute. This is Contribution No. 1240 of the Belle W. Baruch Institute for MarineBiology and Coastal Research. [AU]

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Documents

Somatic growth and otolith growth in juveniles of a small subtropical flatfish, the fringed flounder, Etropus crossotus