Changes in isotopic composition of red drum (Sciaenops ocellatus

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Changes in isotopic composition of red drum (Sciaenops ocellatus) larvae in response to dietary shifts: potential applications to settlement studies Sharon Z. Herzka and G. Joan Holt

Abstract: The stable isotope composition of larval fish tissues may serve as a chemical tracer of recent settlement due to food web differences among planktonic and demersal habitats. We present the background for the utilization of δ13C and δ15N to trace settlement of red drum (Sciaenops ocellatus), an estuarine-dependent species. The effect of ontogeny and temperature on the relative contribution of growth and metabolic turnover to changes in isotopic composition was examined by simulating dietary shifts in the laboratory. Fractionation was examined as a function of size and the effect of food deprivation was evaluated. Published growth rates were used to estimate the time period within which the isotopic composition of a new food source should be reflected in larval tissues. In response to dietary shifts, larvae exhibited quick changes in δ13C and δ15N in a pattern closely resembling predictions based on growth alone. Fractionation values were about +1‰ for δ13C and +1.6‰ for δ15N. There was no effect of 4 days of food deprivation on δ13C and δ15N. Given the fast growth rates reported for newly settled red drum, their isotopic composition should exhibit a shift within 1–2 days and stabilize about 10 days following settlement. Résumé : La composition en isotopes stables des tissus des larves de poisson peut servir de traceur chimique de l’implantation récente dans la zone benthique étant donné les écarts trophiques existant entre les habitats planctonique et benthique. Nous donnons le contexte de l’utilisation des rapports δ13C et δ15N pour suivre l’implantation du tambour (Sciaenops ocellatus), une espèce estuarienne. Les effets de l’ontogénie et de la température sur les apports relatifs de la croissance et de la transformation métabolique sur la composition isotopique ont été examinés en provoquant des changements du régime alimentaire en laboratoire. Le fractionnement a été étudié en fonction de la taille et de l’absence de nourriture. Les taux de croissance publiés ont été appliqués à l’estimation du temps nécessaire pour que la composition isotopique d’une nouvelle source de nourriture se reflète dans les tissus des larves. En réponse au changement de leur régime alimentaire, les larves ont présenté des variations rapides des δ13C et δ15N dont l’allure correspondait de près aux prévisions fondées sur la croissance seule. Les valeurs du fractionnement étaient environ de +1‰ pour le δ13C et de +1,6‰ pour le δ15N. Une privation de nourriture de 4 jours n’a pas eu d’effet sur ces rapports. Étant donné les taux de croissance élevés signalés pour les tambours récemment implantés sur les fonds, leur composition isotopique devrait présenter un décalage de 1 à 2 jours et se stabiliser environ 10 jours plus tard. [Traduit par la Rédaction]

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Introduction Recruitment variability is often attributed to processes acting upon the early life of marine fish species. Settlement to demersal nursery habitat has been identified as an important component of recruitment and is influenced by a host of physical and biological processes acting on the pre- and post-settlement stages (Boehlert and Mundy 1988). Consequently, the relationship between planktonic larval supply and settlement to nursery habitat must be examined on a variety of carefully chosen spatial and temporal scales. The logistic difficulties and high cost of such encompassing studies have resulted in trade-offs between the simultaneous study Received May 4, 1999. Accepted August 20, 1999. J15129 S.Z. Herzka1 and G.J. Holt. Marine Science Institute, University of Texas at Austin, 750 Channelview Drive, Port Aransas, TX 78373, U.S.A. 1

Author to whom all correspondence should be addressed. e-mail: [email protected]

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of large-scale spatial and fine-scale temporal patterns of early life settlement dynamics (Doherty and Williams 1988). Studies addressing settlement dynamics have relied on a variety of techniques, including length–frequency analysis, visual sensing, and the identification of dietary shifts or specific developmental stages associated with settlement (e.g., Leis 1991; Grover et al. 1998). Otolith settlement marks have been used to estimate time since settlement on an individual basis (Victor 1991). In addition, there is recent evidence that otolith microchemistry may prove useful for tracing movement of larval and juvenile fishes (Thorrold et al. 1997). The utilization of these techniques has allowed for considerable progress in the study of the early life dynamics of fishes. Unfortunately, these techniques are not suitable for some marine fish species, particularly when seeking to address questions pertaining to the fine-scale temporal dynamics of settlement. The resolution of length–frequency distributions is limited by the sampling interval (Leis 1991), and it can be difficult to distinguish among individuals that have settled at different times. In addition, visual techniques are not suit© 2000 NRC Canada


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able for turbid areas or cryptic larvae, and not all species exhibit an otolith settlement mark or pronounced developmental stage that can be associated with the settlement transition. Hence, the identification of additional chemical or morphological characters that could be used to estimate the time since settlement of fish larvae on an individual basis would be beneficial. Animal tissues reflect the isotopic composition of their food sources in a predictable manner (Peterson and Fry 1987). Variations in food web structure and available food sources among habitats can result in differences in the isotopic composition of resident organisms. Stable isotope ratios have been used to identify the change in habitat of estuarine larval and juvenile fishes (Deegan et al. 1990), trace migration patterns of juvenile shrimp and fish (Fry 1983; Hesslein et al. 1991), and distinguish among populations of adult fishes (Roelke and Cifuentes 1997). Since rapidly growing animals quickly reflect the isotopic composition of a new diet (Fry and Arnold 1982), the isotopic composition of larval fish tissues may change after settlement as a result of a dietary shift. Hence, stable isotope ratios may serve as tracers of recent arrival to nursery habitat. The utilization of stable isotope ratios as tracers of settlement requires estimates of the rate of change in the isotopic composition of larval fish tissues following a dietary shift. For growing animals, the isotopic composition of newly added biomass will reflect that of the current food source (Fry and Arnold 1982). Simultaneously, metabolic turnover results in the breakdown and replacement of existing body tissues (Tieszen et al. 1983). As a result, the rate of isotopic change is a function of both growth and metabolic turnover. We present estimates of the rate of change in δ13C and δ15N of red drum (Sciaenops ocellatus) larvae subjected to dietary shifts under laboratory conditions. Red drum settle from coastal waters to estuarine nursery habitat early in their life cycle (Holt et al. 1983; Rooker et al. 1998). Although abundance pulses of recent recruits have been observed, the fine-scale temporal resolution of settlement patterns is limited. Red drum settle at a range of sizes (4–8 mm standard length (SL); Rooker et al. 1998), and the use of specific developmental stages to trace settlement is not suitable. In addition, since red drum larvae lack a clearly defined otolith settled mark (S.A. Holt, Marine Science Institute, University of Texas at Austin, 750 Channelview Drive, Port Aransas, TX 78373, U.S.A., and J.R. Rooker, Texas A&M University, 5007 Avenue U, Galveston, TX 77551, U.S.A., personal communication), it is currently not possible to estimate the time since settlement for individuals. Lastly, movement of recently settled larvae within the nursery habitat cannot be differentiated from settlement. The data presented provide the foundation for using stable isotope ratios to examine the temporal dynamics of settlement of red drum and other marine fish species that exhibit a distinct habitat and (or) dietary change as a result of settlement. Our objectives were to (i) determine the influence of ontogenetic state and temperature on the contribution of growth and metabolic turnover to the rate of δ13C and δ15N change following a dietary shift, (ii) evaluate size-dependent fractionation, short-term food deprivation, and the size of the carbon and nitrogen pool throughout ontogeny, and (iii) derive a realistic estimate of the time within which the isotopic

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composition of a recently settled red drum larva should change based on published growth rates for postsettlement red drum.

Materials and methods Red drum larval culture Red drum eggs were obtained from adults spawning under controlled temperature and photoperiod at the Marine Science Institute, University of Texas at Austin (Arnold 1988). Eggs hatch after 24 h and larvae commence feeding on day 3 posthatch after yolk-sac reserves are almost depleted (Holt et al. 1981a). Larvae were reared at initial densities of 2000 fish in 150-L conical tanks (Holt 1993) and kept on a 12 h light : 12 h dark photoperiod in a temperaturecontrolled laboratory. All experiments were completed at salinities of 30–35‰.

Dietary shift experiments

The change in δ 13C and δ 15N of red drum larvae in response to a diet of differing isotopic composition was evaluated for firstfeeding (2.6 mm SL) and settlement-size (4–8 mm SL) fish. The experiment at first feeding was completed at 28°C, within the temperature for optimum growth of early red drum larvae (Holt et al. 1981b). Isotopic shifts for settlement-size red drum were evaluated at 24 and 28°C, within the 19–31°C encountered by populations in nursery habitat (Rooker and Holt 1997). The isotopic compositions of food sources and larvae used in the dietary switch experiments described below are listed in Table 1. The initial δ 13C and δ 15N of larvae prior to the initiation of feeding (day 3) was determined by collecting 15 larvae from each of two experimental tanks. Larvae were subsequently fed rotifers (Brachionus plicatilis) grown on an algal monoculture (Nannochloropsis oculata) (Holt 1992). Collections were completed for the next 7 days. Due to their small size (mean SL = 2.6 mm on day 3 to 4.7 mm on day 10), larvae collected from any one tank were later pooled for stable isotope ratio analysis (5–15 individuals, n = 2 samples per day). Following collection, larvae were held in filtered seawater for 2–4 h to allow for gut clearance (G.J. Holt, unpublished data). Red drum were then anesthetized with a minute quantity of MS 222 and SL measurements were obtained using a Wilde stereomicroscope and Summa Sketch digitizing table. Larvae were stored in clean glass autosampler tubes and dried at 60°C for 24–48 h. Isotopic shifts for settlement-size red drum were examined by feeding larvae rotifers grown on N. oculata from the initiation of feeding until the target size (about 6–7 mm SL) was achieved; the median size was 6.4 mm SL for the 28°C experiment (day 13) and 6.6 mm SL for the 24°C experiment (day 23). Larvae were switched to freshly hatched Artemia fransiscana (San Francisco Bay Brand, Supreme 99, Salt Lake Brine Shrimp, Newark, Calif.) nauplii fed daily. Twenty larvae were collected prior to the dietary switch and every 1 or 2 days for the following 10 days and processed as described previously. Due to the large size variability of cultured red drum and the need to examine δ 13C and δ 15N shifts as a function of accurate growth measurements, the four larvae most closely approximating the median SL for each sampling date were selected and stable isotope ratio analyses were completed on those individual larvae.

Fractionation and food deprivation Fractionation can be defined as the difference in isotopic ratios between an animal and its food source (Peterson and Fry 1987). We hypothesized that fractionation exhibited by fish larvae could be size dependent due to the potential effect of ontogenetic development on assimilatory capacity and metabolism. Hence, we examined fractionation as a function of size. © 2000 NRC Canada



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Table 1. δ13C and δ15N values (mean ± SD) of food sources and red drum larvae used during dietary switch and fractionation experiments and DWs of the larvae immediately prior to the dietary shift (Winitial). Dietary switch at first feeding (28°C) Rotifer culture Red drum larvae (initial) Red drum larvae (final) Dietary switch at settlement size (28°C) Rotifer culture Artemia nauplii Red drum larvae (initial) Red drum larvae (final) Dietary switch at settlement size (24°C) Rotifer culture Artemia nauplii Red drum larvae (initial) Red drum larvae (final) Fractionation Rotifer culture Artificial diet

Winitial (mg)

δ13C (‰)

δ15N (‰)

n

0.024±0.0004

–23.7±1.3 –18.2±0.0 –23.9

–2.3±1.5 12.0±0.3 0.9

3 2a —

–23.7±1.3 –22.1±0.1 –22.2±0.2 –20.5

–2.3±1.5 10.7±0.2 1.3±0.2 12.2

3 2 4b —

–26.2±0.2 –22.1±0.1 –24.3±0.3 –20.9

–3.8±1.2 10.7±0.2 0.4±0.7 14.1

3 2 4b —

–23.7±1.3 –22.7±0.2

–2.3±1.5 6.8±0.2

2 2

0.74±0.12

0.89±0.84

Note: Stable isotope ratios of the larvae following equilibration on the final food source (δf) represent asymptotic values derived through nonlinear curve fitting. a Stable isotope ratio isotopic analysis completed on pooled samples of larvae (15). b Stable isotope ratio isotopic analysis completed on individual larvae.

Because red drum cannot be raised on artificial diets alone and the variability in isotopic ratios of the rotifer culture (Table 1) was within the range of values typically observed as fractionation factors, red drum were weaned from rotifers to an artificial diet (Fry Feed Kyowa, Kyowa Hakko Kogyo Co., Japan) by day 8 posthatch (Holt 1993). A single batch of the diet was ground into different sizes ( 0.05) (Fig. 5). Because of the low proportion of the variance explained by the regressions (r2 < 0.1 in all cases) and the narrow range in percent carbon values observed for different sizes, no attempt was made to correct for the size of the carbon or nitrogen pool in modeling rates of isotopic change, which are therefore expressed as a function of DW. The SL (millimetres) – DW (milligrams) relationship developed by curve fitting a power function to the data yielded © 2000 NRC Canada



142 Fig. 3. Linear regression and 95% confidence intervals of (a) δ13C and (b) δ15N fractionation factors calculated for individual red drum larvae fed an artificial diet of constant isotopic composition. Fractionation factors were calculated as δanimal – δdiet. The broken line represents no fractionation. Solid circles denote data included in regressions; open circles represent data that were not included in regressions because larvae had not equilibrated on the artificial diet.

the following relationship: W = 0.0019SL3.3 (r2 = 0.98, n = 340). The relationship was used to estimate the rate of isotopic change for natural populations as described in the Materials and methods section. Contribution of growth and metabolic turnover to the rate of isotopic change Curve-fitted values for the exponent of metabolic decay (c) approximated –1 for both ontogenetic states and rearing conditions, with the exception of δ13C turnover for settlementsize larvae reared at 28°C, for which the value of c was about –2 (Table 2). However, statistical comparison of c values from (i) first-feeding and settlement-size larvae reared at 28°C and (ii) settlement-size red drum reared at 24 and 28°C yielded no significant differences between parameter estimates (p > 0.05). In addition, none of the parameter results were found to be significantly different from –1 (Table 2). Regression analysis of measured versus δ values predicted using the simple dilution model indicated that in most cases, growth explained >90% of the observed variability, although only 56% of the variability observed for settlement-size red drum reared at 28°C was attributable to growth (Table 2). Statistical analysis of the exponent of metabolic decay values indicated that isotopic shifts can be adequately de-

Can. J. Fish Aquat. Sci. Vol. 57, 2000 Fig. 4. δ13C and δ15N of individual settlement-size red drum larvae subjected to a 4-day food deprivation period. Day 0 represents the isotopic composition of larvae prior to food deprivation. The solid lines and p values represent linear regression results.

scribed using a simple dilution (growth only) model. Hence, the percent initial carbon or nitrogen comprising the total pool during growth was calculated using c = –1 and expressed as a function of relative biomass increase (WR) (Fig. 6). As expected, the initial addition of biomass following a dietary shift should lead to rapid changes in the isotopic composition of larval tissues (Fig. 6). After a sixfold increase in biomass (WR = 6), the initial carbon or nitrogen would account for only 20% of the total. Turnover rates under natural conditions An exponent of metabolic decay (c) of –1 was used to model the rate of dilution of initial carbon or nitrogen following a theoretical dietary shift occurring as a result of settlement to nursery habitat. Model results indicate that the addition of new carbon or nitrogen should occur quickly following a dietary shift; as a result, the δ13C and δ15N of newly settled red drum should change rapidly (Fig. 7). One day after settlement, only 75% of the existing carbon or nitrogen would correspond to that deposited prior to the settlement event. After 10 days, only about 20% of the initial carbon or nitrogen would comprise the initial pool at settlement. The variability in growth rates reported by Rooker and Holt (1997) resulted in about a 10% difference in contribution of carbon or nitrogen present at the time of settlement to the total pools as a function of time (Fig. 7).

Discussion The assumptions underlying the interpretation of isotopic data have often been unsubstantiated, resulting in ambiguous conclusions (Gannes et al. 1997). To utilize stable isotope ratios to trace recent settlement of larval fishes, two assump© 2000 NRC Canada



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Fig. 5. Linear regression results of percent carbon and nitrogen of red drum larvae as a function of SL and DW (n = 340). Values were derived concomitant to stable isotope ratio analysis of larvae collected in fractionation and dietary switch experiments.

Table 2. Exponents of metabolic decay (c) derived through nonlinear curve fitting of Fry and Arnold’s (1982) equation to δ13C and δ15N of red drum during dietary switch experiments. Carbon First feeding (28°C) Settlement size (28°C) Settlement size (24°C)

Nitrogen

c

SE

r2

p

c

SE

r2

p

–0.96 –1.96 –1.12

0.35 0.89 0.32

0.93 0.56 0.91

ns ns ns

–0.99 –1.13 –0.94

0.29 0.25 0.09

0.96 0.93 0.99

ns ns ns

Note: When c = –1, isotopic changes are solely a function of growth (simple dilution model), whereas c less than –1 is indicative of the added effect of metabolic turnover. Standard error values refer to c estimates. Coefficients of determination (r2) refer to the regression of measured δ values versus those predicted with a simple dilution model and represent the fraction of the observed variability that is explained by growth alone. Values of p were derived using a Student’s t test to examine for significant differences between c and –1. ns, not significant.

tions must be met. First, fractionation must be independent of size so that observed patterns in δ13C and δ15N of pre- and post-settlement larvae can be attributed to dietary food sources and not to size-related fractionation. For teleosts, some researchers have reported trends in fractionation as a function of size (e.g., Rounick and Hicks 1985; SholtoDouglas et al. 1991; Hobson and Welch 1995), while others have not found a consistent relationship (Fry and Parker 1979; Fry 1983). Many have relied on field collections (but see Rounick and Hicks 1985), and as noted by the authors, the observed trends could have been confounded by seasonal effects, size-dependent feeding, and fluctuations in the composition of available foods. Variations in tissue lipid content may lead to size-dependent fractionation (DeNiro and Epstein 1977), although the contribution of isotopically lighter lipids to the δ13C values of whole-fish tissues has been found to be negligible in some cases (Fry and Parker 1979; Hesslein et al. 1993). Second, in the case of food deprivation, the isotopic composition of larval tissues must not change substantially over the time scale in which isotopic changes in response to a dietary shift are expected to occur. Since fish larvae that are

not feeding must rely on resorption of existing tissues to sustain metabolic demands, it was hypothesized that catabolism could result in selective respiration of the lighter carbon isotope (DeNiro and Epstein 1978) or selective excretion of the lighter nitrogen isotope (Hobson et al. 1993). Both processes could result in a shift in isotopic composition over time, confounding the identification of dietary switches following settlement. Fractionation was examined as a function of size by weaning red drum larvae onto an artificial diet of constant isotopic composition. Despite a slight but significant effect of size on δ13C fractionation, our results indicate that a value of +1‰ can be considered representative for red drum larvae (4–15 mm SL). Due to an incomplete shift in δ15N (see Results section), the range of larval sizes used to estimate fractionation was limited to 9–15 mm SL. Nevertheless, no effect of size was detected within that size range and fractionation was +1.6‰. Fractionation can also be estimated through comparison of the isotopic values of food sources utilized and larvae collected during dietary switch experiments. Fractionation factors ranged between 0.2 and 1.9‰ for carbon and between © 2000 NRC Canada



144 Fig. 6. Estimated dilution of the initial carbon or nitrogen pool by the addition of new biomass during growth. A simple dilution model was utilized. The arrow indicates dietary shift.

1.5 and 4.2‰ for nitrogen (Table 1). These values, and those derived from larvae fed the artificial diet, are within literature values for larval fishes reared in the laboratory (δ13C fractionation of +2‰ for red drum larvae; Anderson et al. 1988) and collected in the wild (Thayer et al. 1983; Hobson and Welch 1995). They are also within the range of fractionation factors reported for juvenile and adult teleosts (Rounick and Hicks 1985; Sholto-Douglas et al. 1991; Hesslein et al. 1993). Nevertheless, at least part of the variability observed in fractionation values calculated from data collected during dietary switch experiments can arise from variability in the isotopic composition of the rotifer culture, the small number of fish analyzed, and the parameter estimate error associated with δfinal. Therefore, we believe that the calculation of fractionation factors based on larger numbers of fish fed a diet of constant isotopic composition is more accurate. During the 4-day food deprivation period examined, there was no indication of changes in the δ13C and δ15N of settlementsize red drum. In a similar experiment with larval krill (Euphausia superba), Frazer et al. (1997) found limited effects of starvation on δ13C and δ15N. Since we did not measure individual weight loss, which would represent a measure of the extent of starvation, it is possible that catabolic processes had not proceeded for a sufficiently long period to result in a shift in isotopic composition. Nevertheless, the lack of a measurable effect of food deprivation on δ values of red drum larvae suggests that it is improbable that its effects could be confounded with a dietary shift in the wild, particularly due to the rapid growth rates exhibited by larvae under natural conditions (Rooker and Holt 1997). In addition, gut content analysis of postsettlement red drum collected in Texas seagrass meadows revealed a low incidence of empty guts in this species, suggesting that long-term food deprivation is not prevalent (Soto et al. 1998). Contribution of growth and metabolic turnover to isotopic change Curve-fitted values for the exponent of metabolic decay were about –1 in five out of six dietary switch experiments

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(except for δ13C in settlement-size red drum reared at 28°C). Such a value indicates the absence of an effect of metabolic turnover on isotopic composition and implies that patterns of isotopic change are a result of the dilution of an initial carbon or nitrogen pool by the addition of newly deposited biomass. Although metabolic turnover appeared to accelerate the pattern of δ13C change in settlement-size fish reared at 28°C, the exponent of metabolic decay was not significantly different from –1. This result may be associated with the narrow range in δ13C values (1.5‰) (Table 1) between the initial and final diet during that dietary switch experiment and consequent large standard error associated with the parameter estimate (Table 2). Nevertheless, due to the overwhelming effect of growth on the isotopic composition of the larvae following dietary shifts, we suggest that, for red drum, patterns of isotopic change in response to dietary shifts can be adequately predicted based on growth rate estimates. Although studies addressing rates of isotopic change in growing organisms are few, those completed on poikilotherms support this study’s finding that biomass gain plays the dominant role. Hesslein et al. (1993) utilized cultured broad whitefish (Coregonus nasus) juveniles (initial size 21– 210 mm) to examine δ13C, δ15N, and δ34S in response to a dietary shift and attributed 90% of the observed isotopic changes to growth. Fry and Arnold (1982) examined δ13C shifts in juvenile brown shrimp (Penaeus aztecus) and found that biomass gain was the primary cause of change in isotopic composition, although the added effect of metabolic turnover was also detected. Likewise, Frazer er al. (1997) examined δ13C and δ15N of larval krill and found an effect of metabolism turnover only at the highest of two rearing temperatures. The high contribution of metabolic turnover to isotopic changes in studies with mammals and birds (Tieszen et al. 1983; Hobson and Clark 1992) compared with our own and with those of Fry and Arnold (1982), Hesslein et al. (1993), and Frazer et al. (1997) is probably attributable to the higher basal metabolism of endotherms, which is considerably higher than for poikilotherms such as fish and crustaceans. Nevertheless, the absence of a detectable effect of nitrogen metabolic turnover in larval fishes was unanticipated because nitrogen cycling is a complex process that is intimately linked with growth (Houlihan et al. 1995a), and the rate of protein degradation in marine fish larvae is known to be high (Houlihan et al. 1995b). However, in this study and others, metabolic turnover as related to isotopic shifts refers specifically to (i) breakdown and (ii) replacement of existing body tissues. Hence, metabolic turnover would only accelerate the rate of isotopic change relative to the simple dilution model if the replacement of existing body tissues were accomplished with proteins synthesized from the “new” food source (de novo synthesis). On the other hand, if protein resynthesis from degraded products were high, it is possible that little of the degraded nitrogen pool would be replaced through de novo synthesis. Unfortunately, there is limited information on the mechanisms of protein degradation and fate of degraded products in marine fish larvae (Houlihan et al. 1995b). As recently noted by Gannes et al. (1997), the use of stable isotope ratios in ecological studies would be © 2000 NRC Canada



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Fig. 7. Estimated dilution of the initial carbon and nitrogen pool expressed as a function of time after settlement for a 7-mm SL red drum larvae. Estimates were derived using instantaneous growth coefficients (g) reported by Rooker and Holt (1997) for postsettlement red drum and an exponent of metabolic decay of –1. Dilution as a function of growth is expressed as the percent contribution of carbon or nitrogen present at the time of settlement to the total and as a function of initial and final isotopic composition.

greatly advanced if the relationships between an animal’s diet and the fate of assimilated components and synthesized products were elucidated and the biochemical origin of fractionation determined. Isotopic change in natural populations Based on the absence of an effect of metabolic turnover, it can be estimated that red drum larvae should reflect the isotopic composition of a new food (5 mm SL or about 400 µg DW). Hence, the utilization of stable isotope analysis to identify new settlers could be accomplished individually.

Conclusions The study of the fine-scale temporal dynamics of settlement requires the identification of chemically or morphologically based characters that can be used to identify new settlers over short time intervals. Given adequate differences in food webs among habitats, new settlers should be distinguishable from previous arrivals to nursery habitat over a time scale of days. Comparison of δ13C and δ15N values of planktonic larvae with those of postsettlement larvae could be used to estimate the proportion of new settlers on a sizeclass and date-specific basis. Furthermore, the time since settlement could be estimated using growth rate estimates (Fry and Arnold 1982). The utilization of stable isotope ratios as tracers of individual recruitment to nursery areas could allow for increased temporal resolution of settlement patterns, therefore contributing to the understanding of the early life dynamics of marine fish species.

Acknowledgments We thank two anonymous reviewers for their thorough comments and suggestions, which greatly improved this manuscript. E. Ingall, L. Clark, and F. Goulet-Miller provided expert and willing assistance during the completion of isotopic analyses. Our work benefitted considerably from conversations with C.R. Arnold, L.A. Fuiman, S.A. Holt, E. Ingall, G.A. Jackson, J.P. Lazo, and J.R. Rooker. We thank J. Muñoz for help with phytoplankton and rotifer cultures and P. Pickering for advice on red drum rearing. C. Faulk, J.P. Lazo, and M. Smith provided insightful comments on a draft of this manuscript. This is contribution No. 1121 of the Marine Science Institute, University of Texas at Austin.

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