Isotopic Turnover in Aquatic Predators: Quantifying the Exploitation of Migratory Prey

Isotopic turnover in aquatic predators: quantifying the exploitation of migratory prey

Stephen E. MacAvoy, Stephen A. Macko, and Greg C. Garman

Abstract : In the tidal freshwaters of Virginia, U.S.A., the blue catfish ( Ictalurus furcatus ), an introduced piscivore, derives a significant proportion of its nutrition from spawning anadromous fish (genus Alosa , including blueback her - ring ( A. aestivalis ), American shad ( A. sapidissima ), and alewife ( A. pseudoharengus )). Because the Alosa are not con - tinually available to I. furcatus , there is an isotopic turnover, defined as change in isotope composition due to growth and metabolic tissue replacement, in I. furcatus tissues associated with the diet switch from freshwater to anadromous fishes. However, isotopic turnover rates for ictalurid fish are unknown. This study determined the maximum isotopic turnover rate of channel catfish ( Ictalurus punctatus ) tissues and compared this maximum rate with that of I. furcatus captured in the field over the 3-month Alosa spawning run. Maximum turnover rates for 13 C were 0.014 and 0.017‰ per day in muscle and blood. For 34 S, rates were 0.017 and 0.020‰ per day in muscle and blood, respectively. Isotopic turnover of muscle carbon reflected growth rate, but sulfur did not match growth as well. Ictalurus furcatus captured in the field showed no enrichment during the Alosa spawning run owing to slow turnover and variable diet. In aquatic ecosystems that have migrating prey, exploitation by predators may be underestimated using isotopes because of slow tissue turnover.

Résumé : Dans les eaux douces intertidales de la Virginie, aux États-Unis, Ictalurus furcatus , un poisson piscivore in- troduit, retire une proportion significative de son alimentation des poissons anadromes du genre Alosa , dont le l’Alose d’été ( A. aestivalis ), l’Alose savoureuse ( A. sapidissima ) et le Gaspareau ( A. pseudoharengus ), qui viennent y frayer. Parce que les Alosa ne sont pas toujours disponibles à I. furcatus , il se produit un virement isotopique dans les tissus de la barbue, c’est à dire un changement dans la composition isotopique reliée à la croissance et au renouvellement métabolique des tissus consécutif à un remplacement dans le régime des poissons d’eau douce par des poissons anadromes. Cependant, les taux de renouvellements isotopiques chez les poissons ictaluridés sont inconnus. Noua avons déterminé le taux maximal de renouvellement isotopique dans les tissus de la Barbue de rivière ( Ictalurus punctatus ) et l’avons comparé avec le taux maximum observé chez les I. furcatus capturés en nature durant les 3 mois de la montaison de reproduction des Alosa . Les taux maximaux de renouvellement du 13 C sont respectivement de 0,014 et de 0,017‰ par jour dans le muscle et dans le sang, alors que les taux maximaux de 34 S sont respectivement de 0,017 et de 0,020‰ par jour dans les mêmes tissus. Le taux de renouvellement isotopique du carbone musculaire reflète le taux de croissance, mais celui du soufre ne suit pas la croissance d’aussi près. Les I. furcatus capturés en nature ne font preuve d’aucun enrichissement durant la montaison des Alosa à cause du faible taux de renouvellement et du régime alimentaire variable. Dans les écosystèmes aquatiques où il y a des proies migratrices, l’utilisation des isotopes peut sous-estimer l’exploitation par les prédateurs à cause du faible taux de renouvellement des tissus.

[Traduit par la Rédaction] MacAvoy et al. 10

Introduction be relatively constant over time. Therefore, the isotopic signa - ture of the consumer organisms is assumed to reflect the Stable isotopes are useful in the study of food webs be - available prey items at any given time. Often, this assumption cause they can often be used to differentiate among nutrient may be valid; however, it may be violated in ecosystems that sources contributing to consumers’ diets. The carbon, nitro - occasionally experience migrations of animals, such as birds, gen, and sulfur isotope ratios of prey animals are assumed to mammals, and fish. The migratory animals may have unique carbon, nitrogen, or sulfur isotopic signatures relative to prey Received August 14, 2000. Accepted January 26, 2001. items in the system they enter. In such a system, the predators Published on the NRC Research Press Web site on XXX XX, that consume the migrating prey should have their own isoto - 2001. pic signatures shift toward that of the prey species (Kline et J15917 al. 1998; Persson and Hansson 1999). However, the isotopic S.E. MacAvoy 1,2 and S.A. Macko. Department of turnover rate of most animals, especially large vertebrates, is Environmental Sciences, University of Virginia, unknown (Gannes et al. 1997). Charlottesville, VA 22903, U.S.A. Isotopic turnover is defined in this paper as the isotopic G.C. Garman. Center for Environmental Studies, Virginia change due to growth and metabolic tissue replacement as - Commonwealth University, Richmond, VA 23284, U.S.A. sociated with a change in diet. Isotopic turnover studies on 1Corresponding author (e-mail: [email protected]). brine shrimp (Fry and Arnold 1982) and krill (Frazer et al. 2Present address: Department of Marine Sciences, University 1997) have shown high rates of isotopic turnover owing to of Georgia, Athens, GA 30601, U.S.A. the quick growth of these invertebrates. Herzka and Holt

(2000) also found quick turnover rates in larval red drum bolic tissue replacement components in order to address the (Sciaenops ocellatus ) owing to growth. Hesslein et al. relative importance of these two components (Hesslein et al. (1993) found slow turnover rates in broad whitefish 1993). Ideally, documenting isotopic turnover allows tempo - (Coregonus nasus ). In their study, Hesslein et al. (1993) de - ral interpretation of the percentage of marine carbon, sulfur, termined that more than a year was required for C. nasus and nitrogen consumed by I. furcatus during the course of muscle tissue 13 C and 34 S to completely reflect consumed the Alosa spawning run and would augment estimates of food, and they hypothesized that in slow-growing wild pop - I. furcatus exploitation of marine material derived from iso - ulations, it could take longer. Because of the uncertainties tope mixing equations (MacAvoy et al. 2000). about tissue turnover rates, it becomes difficult to determine isotopically the extent to which a migrating population is Materials and methods being preyed upon by local predators. Additionally, if the predator is migratory, then its role in the ecosystem it moves Turnover determination through would be difficult to determine isotopically. Deter - Channel catfish ( Ictalurus punctatus ) were used in this experi - mination of isotopic turnover rate is also important for un - ment as a surrogate for I. furcatus because they could be obtained derstanding the temporal relationship between the isotopic locally with minimal transport and handling stress and the growth signature of a predator and that of its prey. Some period of rates of the two ictalurids are similar (Conder and Hoffarth 1962; time may be required for a predator to reflect the isotope Webster et al. 1992, 1995). On 7 April 1999, 36 I. punctatus were signature of a new prey item. obtained from Hale Farm, Oilville, Va. Seven fish were sacrificed Many coastal river systems worldwide experience a sea - to determine initial isotopic values. All fish were transported to the University of Virginia and, once there, were measured (total length sonal influx of spawning anadromous fish. The large influx in inches), tagged, and placed in a Living Stream model LSW-900 may be an important nutrient source to freshwater ecosys - (190 gal; Frigid Units, Inc., Toledo, Ohio). The catfish were fed a tems, particularly if the species are semelparous (Kline et al. marine fish diet (100% tuna meat) of known isotopic composition 1993; Ben-David et al. 1998; MacAvoy et al. 1998). The beginning on 8 April 1999 and ending on 17 June 1999; over the anadromous fish are enriched in 13 C, 15 N, and 34 S relative to 90 days of the study, food was added so that it was always visible freshwater animals and plants because they have spent the (Fry and Arnold 1982). Ninety days is the approximate length of majority of their lives in the marine environment and have the Alosa spawning run in Virginia and was chosen as the optimal acquired its relatively enriched isotopic signal. Therefore, length for the turnover experiment because it would encompass the the nutrients (in the form of their biomass) that they deliver time that Alosa adults and I. furcatus share the tidal freshwater. to freshwater are detectable using stable isotope techniques Fish were placed on ice prior to sampling. For all fish, muscle and skin were sampled. Most fish had blood samples taken (in some as shown in Alaska and the Pacific Northwest by Kline et al. cases, blood could not be obtained), and catfish with barbels had (1990, 1993) and Ben-David et al. (1998). those sampled as well. In the coastal rivers of the eastern United States, the dom- As a control group, 19 I. punctatus obtained from Hale Farm inant anadromous fishes are Alosa (including blueback her- were kept in the Living Stream for 60 days and were fed the com- ring ( A. aestivalis ), American shad ( A. sapidissima ), and mercial fish food (Big Strike floating fish food, SSC-338220) they alewife ( A. pseudoharengus )), and they have been found to had been raised on. This food had 32.0% crude protein and 4.0% be substantially enriched in 34 S and 13 C relative to freshwa - fat (Southern States Cooperative Inc., Richmond, Va.). The fish ter species (Garman and Macko 1998; MacAvoy et al. 1998). had their total length and fork length measured prior to being Enriched 13 C and 34 S values associated with Alosa have placed in the Living Stream. been detected in the introduced piscivorous blue catfish Water temperature in the Living Stream was raised slowly from 11 to 19°C over a 60-day period during both the experimental and (Ictalurus furcatus ) (MacAvoy et al. 2000); however, the the control procedures. This was done to approximate the water changing isotope signature that should occur in a predator temperature change in the tidal freshwater areas of Virginia during consuming seasonally available isotopically distinct prey has the spring months. not been examined. The purpose of this study was to determine the isotopic Isotopic analysis turnover rate of ictalurid catfish in a controlled laboratory Samples of dorsal muscle, blood, skin, and barbel were dried at experiment and compare it with isotope data collected from 60°C for 3 days and homogenized with a mortar and pestle in prep - I. furcatus in the field. It was hypothesized that I. furcatus at aration for analysis. A Carlo Erba elemental analyzer coupled to a the beginning of the Alosa spawning run should reflect the Micromass Optima isotope ratio mass spectrometer (Micromass, 13 15 34 isotope values of the resident freshwater fish. Then, as the Manchester, U.K.) was used to obtain C, N, and S values (Fry et al. 1992; Giesemann et al. 1994). The 13 C and 15 N were spawning run progressed and Alosa became a regular com - 34 ponent of the I. furcatus diet, the isotope signature of the obtained concurrently and S was determined during separate analysis runs. I. furcatus should increasingly resemble that of Alosa . The isotope compositions are reported relative to standard mate - The maximum isotope turnover could be defined as the re - rial and follow the same procedure for all stable isotopic measure - sult of a 100% diet change from foods with a freshwater iso - ments as follows: tope signature with foods with a marine isotope signature. x xy xy We hypothesized that the maximum isotope turnover rate in (1) E = ([( E / E)sample ( E / E) standard ]1 ) 1000 the laboratory could be compared with turnover rates ob - where E is the element analyzed (carbon, nitrogen, or sulfur), x is served in the field and thereby estimate the amount of ma - the molecular weight of the heavier isotope, and y is the molecular rine material being consumed at any point in time (see Fig. 1 weight of the lighter isotope ( x = 13, 15, and 34 and y = 12, 14, for an illustration of this point). The isotope change ob - and 32 for carbon, nitrogen, and sulfur, respectively). The standard served in the laboratory was divided into growth and meta - materials with which the samples are compared are Pee Dee

Fig. 1. Example of theoretical maximum turnover rate, resulting from a 100% diet switch from freshwater to marine foods (top line), versus a hypothetical turnover rate that may be observed in the field (bottom line). The field rate would probably be less than the max - imum rate because the predator would not be forced to undertake a 100% diet switch. The figure illustrates that any isotopic turnover observed in the field should be interpreted relative to a measured maximum turnover rate. This is necessary in order to understand the extent that a predator is feeding upon a migratory population at any given time.

Beleminte for carbon, air N 2 for nitrogen, and Canyon Diablo The observed isotopic turnover can be better predicted if knowl- Troilite for sulfur. The reproducibility of all measurements was edge of what contributes to it exists. typically 0.3‰ or better. The turnover model used was based on that of Hesslein et al. (1993), which contained both a growth and a metabolic tissue re- Normalization of turnover rates placement component. Growth can be described by the following The rate of turnover observed for the different isotopes is rela- equation: tive to the separation of the isotope endmembers. The greater the (3) k = (ln( W /W )/ t) separation, the greater is the observed change over time. Therefore, s 0 when turnover rates are compared between field and laboratory sit- where k is the specific growth rate constant, t is days, Ws is the uations or different field situations, the degree of separation of iso- weight of the fish at any given time, and W0 is the initial fish topic endmembers should be addressed. weight (Webster et al. 1992). Field measurements, where predators are not expected to switch In an equation where metabolism ( m) and growth ( k) contribute prey completely, should display a fraction of the maximum turn - to isotope turnover, the following equation holds true: over rate, obtained from a 100% diet switch. If, hypothetically, a laboratory a turnover rate of 0.017‰ per day was observed and the (4) d C/d t = –( k + m)( C – Cn) endmembers were 7‰ apart, then if in the field there was a 14‰ separation, the expected shift would be 0.034‰ per day. where d C/d t is change in isotope value over time, C is the isotope Subsequently, the expected maximum in the field situation can value of the fish when sampled, Cn is the expected isotopic value be found by the following equation: for a fish in equilibrium with its new diet, and k is the specific growth rate constant (from eq. 3). (2) y = x([Fa – Fb]/[La – Lb]) Let ( C – Cn) = C ; then, integrate eq. 4: where y is the maximum isotope turnover rate for ictalurids caught (5) ln C = –( k + m)t in the field (unknown), x is the turnover rate measured in the labora - and tory for a particular tissue, [Fa – Fb] is the absolute value of the dif - ference between isotopic endmembers in the field, and [La – Lb] is (6) C = e–( k+m)t the absolute value of the difference between isotopic endmembers in the laboratory. In this study, the isotope endmembers in the field where is the difference between the initial isotope value of the were approximately equivalent to laboratory endmembers (Table 1). fish ( C0) and the expected isotopic value for a fish in equilibrium with its new diet ( Cn), which is a constant. Therefore, eq. 6 be - Turnover modeling comes The turnover model was divided into two components, growth –( k+m)t (7) C = Cn + ( C0 – Cn)e . and metabolic tissue replacement. This was done in order to iden - tify whether metabolic tissue replacement was important relative to To solve eq. 7, values for k must be determined. Webster et al. growth. It is important to know this because isotope turnover in (1995) observed that, generally, the more fish in a catfish feed, the mammals may have a substantial metabolic component (Hobson larger the k. They observed a k of 0.00218 fish per day in juvenile and Clark 1992). However, it was hypothesized that growth would I. furcatus consuming the most fish protein of those in their treat - be the major contributor to isotopic change because the metabolism ments (14% menhaden); therefore, this value was used as a first ap - of the poikilothermic catfish is low relative to that of mammals. proximation of k; however, a range of k values derived from the

Table 1. Equating the maximum turnover measured in the laboratory to the theoretical maximum possible in the field. Laboratory rate Field rate (‰perday) La Lb Fa Fb (‰ per day) 13 C Muscle 0.014 –23.1 –16.7 –25.6 –18.6 0.015 Blood 0.017 –23.6 –16.7 –25.6 –18.6 0.018 34 S Muscle 0.017 3.7 15.9 4.8 18.4 0.019 Blood 0.020 5.3 15.9 4.8 18.4 0.026 Note: La, initial isotope value for I. punctatus muscle; Lb, isotope signal of the laboratory food; Fa, freshwater isotope signal of the blood; Fb, marine isotope signal of the tidal freshwater (see eq. 2). Based on the relative difference between the isotopic endmembers, the maximum expected turnover rate in the field is calculated. experimental data were also used (see below). The metabolic tissue Results replacement component of the turnover ( m) was to be arrived at by fitting predicted values (using eq. 7) to the measured data and min - Carbon isotopes imizing the mean difference between the two by estimating various 13 values for m (Hesslein et al. 1993). From the laboratory study, regressions of C versus time Measuring the total length of the fish was problematic because indicated turnover rates of 0.014‰ per day in muscle tissue of fin abrasion. For some of the experimental fish where total and 0.017‰ per day in blood (Fig. 2). Both regressions were length could not be determined, fork length was measured instead. statistically significant ( p = 0.0001 and 0.0004, respectively) Total length is highly correlated with fork length ( r2 = 0.97) for the (Table 2). The mean initial 13 C values of the I. punctatus I. punctatus , and a simple regression of fork length (FL) with total muscle and blood were –22.9 and –23.4‰, respectively. The length (TL) yielded the equation 13 C of the marine diet was –16.6 ± 0.5‰. Over the course of the 84-day experiment, the isotope signatures of the (8) TL = 1.04(FL) + 0.63. I. punctatus muscle and blood became enriched by 1.3 and 1.5‰, respectively (Fig. 2). Skin tissue showed larger vari- Therefore, an estimated change in total length was obtained from ability, and regressions of 13 C versus time were not signifi- some experimental fish, and from those values, weight was esti- cant. Barbels were sampled from only a few I. punctatus mated using the following equation: because most of them were missing the tissue. The regression of barbel versus time was significant ( p < 0.001, r2 = (9) Y = 0.152299(TL) – 0.013302(TL) 2 0.87); however, owing to the limited samples available, only + 0.000706(TL) 3 – 0.5834 the regressions of muscle and blood were considered for the turnover models. Among the controls, the 13 C values of all where Y is weight in pounds and TL is in inches (Kelly 1969). tissues did not change significantly ( p > 0.05). Skin tissue 13 From these weights, experimental values of k (ln( Ws/W0)/ t) were C was variable among controls as it was among fish in the estimated. The k values calculated for the fish in both the experimental group. If the observed turnover rates were to experimental and the control groups ranged between 0.0037 and remain reasonably consistent over time, it would take ap - 0.009, which were in the range observed in other ictalurid growth proximately 400 days for a complete 13 C turnover of blood studies (Conder and Hoffarth 1962; Webster et al. 1992). tissue and over 450 days for muscle 13 C to turn over.

Field collections Sulfur isotopes In the spring (between March and May) of 1997 and 1998, coin - As hypothesized, there was a significant enrichment in 34 S cident with the anadromous Alosa spawning run, 22 I. furcatus among laboratory-held I. punctatus muscle tissue over time ( 38 cm total length) were captured from the upper tidal freshwater 34 reaches of the Rappahannock River in Virginia (64–113 km up - (p = 0.05) (Table 2). The mean initial muscle S among 34 stream from where the Rappahannock meets the Chesapeake Bay) I. punctatus was 3.5 ± 0.7‰ ( n = 5). The S of the marine by boat electrofishing. Resident freshwater fish and spawning ana - diet was 16.0 ± 2.1‰. Over the course of the 84-day experi - dromous Alosa were also collected to provide freshwater and marine ment, the muscle 34 S values increased by 1.5‰ (Fig. 3). examples of fish that the piscivorous I. furcatus may have been con - The regressions of blood, skin, and barbel tissue versus time suming. Samples of dorsal muscle were either taken in the field or were not significant. There was considerable scatter in the obtained from whole fish in the laboratory. All fish were prepared observed 34 S values, and, although significant in the case of for isotope analysis and analyzed using the method described above. muscle tissue, the r2 values for the regressions for all tissues Kruskal–Wallis nonparametric procedures were used to test for were very low. Some of the variability observed in the 34 S differences in isotopic values among the three groups of fish cap - values may be due to the variability in 34 S of the food that tured in the field (native resident freshwater, anadromous fish, and I. furcatus ) ( = 0.05). The Dunn procedure was used to examine they were fed (standard deviation of 2.1‰, see above). differences between groups (Rosner 1990). Simple regressions Slopes of 0.02 and 0.017 were measured for blood and mus - 34 were used to examine isotope change over time. Statview SE + cle S versus time, respectively, which were similar to the Graphics (Abacus Concepts, Inc.) and Microsoft Excel version 5.0 slopes observed with 13 C. This indicates that carbon is re - (Microsoft, Inc.) were used for statistical tests. placed faster than sulfur in I. punctatus because the degree

Fig. 2. Simple regressions showing I. punctatus tissue 13 C turnover rate from the laboratory study. ( a) Muscle; ( b) blood. The rate is the observed change in stable isotope value starting from when the diet was switched (day 0) from commercial catfish food (see Mate - rials and methods) to a marine food (tuna). The initial 13 C value of the I. punctatus muscle and blood was –22.9 and –23.4‰, respec - tively. The marine food that the I. punctatus were being fed had a 13 C of –16.6 ± 0.5‰.

Table 2. Regression analysies of isotope differences in endmember isotope values. Among the con - change versus time. trols, 34 S values of all tissues did not change significantly (p > 0.05). At the observed turnover rates, it would take well ‰ per day p over 450 days for the 34 S of the I. punctatus blood and Muscle tunover muscle tissue to completely turn over. 13 C 0.014 0.0001 34 S 0.017 0.05 Nitrogen isotopes 15 N 0.014 0.0026 There was considerable scatter in the nitrogen data among Blood turnover I. punctatus in the laboratory study. However, the muscle 13 C 0.017 0.0004 (p = 0.0026, r2 = 0.41), blood ( p = 0.0074, r2 = 0.46), and 34 S ns ns barbel ( p = 0.009, r2 = 0.64) tissues showed statistically sig - 15 N 0.018 0.0074 nificant enrichment over time. The turnover rate measured Note: ns, not significant. for 15 N was equal to that measured for 13 C in muscle (0.014‰ per day) and blood (0.018‰ per day). However, the 15 N isotopic endmembers were farther apart than the of separation between the experimental marine feed and 13 C endmembers. Therefore, the actual isotopic turnover baseline values is roughly 6‰ for 13 C and 12‰ for 34 S. If was slower than that of 13 C (as with 34 S above). The 15 N the turnover rate was the same for the two elements, then the of the marine diet was 13.1 ± 0.9‰. The mean initial muscle regression slopes would only be equal once normalized for 15 N among the laboratory-held I. punctatus was 6.2 ±

Fig. 3. Simple regressions showing I. punctatus muscle tissue 34 S turnover rate from the laboratory study. The rate is the observed change in stable isotope value starting from when the diet was switched (day 0) from commercial catfish food (see Materials and methods) to a marine food (tuna). The initial 34 S value of the I. punctatus muscle tissue was 3.5 ± 0.7‰. The marine food that the I. punctatus were being fed had a 34 S of 16.0 ± 2.1‰.

Fig. 4. Simple regressions showing I. punctatus muscle tissue 15 N turnover rate from the laboratory study. The rate is the observed change in stable isotope value starting from when the diet was switched (day 0) from commercial catfish food (see Materials and methods) to a marine food (tuna). The initial 15 N of the I. punctatus muscle tissue was 6.2 ± 0.8‰. The marine food that the I. punctatus were being fed had a 15 N of 13.1 ± 0.9‰.

0.8‰. The blood and skin tissues fell within the same 15 N 13 C and 34 S among the I. furcatus , their isotopic signatures range as did muscle (Fig. 4). The barbel tissue 15 N turnover were significantly enriched ( p < 0.05) relative to freshwater rate was roughly equivalent to that of 13 C muscle and blood fish captured within the same stream (the Rappahannock) (measured rate = 0.035‰ per day). As with the 13 C mea - (Fig. 6). The I. furcatus 15 N values were also significantly surements, the skin 15 N values exhibited a high degree of higher ( p < 0.05) than resident fish values (Fig. 6). Relative to variability. However, the regression of 15 N versus time had anadromous fish captured in the Rappahannock, the a poor, but statistically significant, r2 (0.22). I. furcatus and resident freshwater fishes were significantly depleted ( p < 0.05) in 13 C and 34 S (–21.7 ± 1.8 and 10.6 ± Field data 2.1‰ and –25.6 ± 2.3 and 4.8 ± 1.6‰, respectively) (Fig. 6). Regressions of field 13 C, 34 S, and 15 N showed no statis - tically significant enrichment of I. furcatus muscle tissue dur - ing the anadromous Alosa spawning run ( p > 0.05) (Fig. 5). Although there is no obvious isotopic enrichment over time in

Fig. 5. Isotopic signatures of I. furcatus (>37 cm total length) caught in the Rappahannock River, Virginia, between March and June 1997–1998. ( a) 13 C; ( b) 34 S; ( c) 15 N. Days from initiation indicates the time from when the Alosa spawning run begins during the last week of February. The isotopic signatures of the Alosa were –18.6 ± 1.4‰ for 13 C, 18.4 ± 1.1‰ for 34 S, and 12.9 ±0.6‰ for 15 N.

Discussion from –22.9 to –23.7‰. However, eq. 7 assumes that the ini - tial isotope value of the I. punctatus would all be equal at Observed versus modeled turnover: isotope change due time 0. Because this was not the case, there was some vari - to growth ability associated with the violation of this assumption. Based on eq. 7, the expected 13 C change in muscle tissue Various values of k were used in eq. 7 ranging between due to growth alone was compared with that actually mea - 0.00218 and 0.009. The low value (0.00218) was measured sured in the laboratory (Table 3). Using the specific growth in I. furcatus fed a diet with 14% fish meal (Webster et al. rate k measured by Webster et al. (1995) for juvenile 1995). The highest growth rate observed in this study was I. furcatus (0.00218), there was fairly good agreement be - 0.009 and reflected both the greater proportion of fish pro - tween the predicted isotope change (calculated from eq. 7) tein used in the feed and the competitive feeding among the and the actual isotope change ( r2 = 0.72). One source of vari - I. punctatus . Using the range of k values changed the slope ability was that the I. punctatus obtained from Hale Farms did of the predicted values versus the measured values. How - not all have identical 13 C values. Their 13 C values ranged ever, there was not a large improvement in the r2 for the

Fig. 6. 34 S versus 13 C for fish captured in the tidal freshwater of the Rappahannock River, Virginia. Anadromous fish ( ×) include A. pseduoharengus and one A. aestivalis. Native resident freshwater fish (+) include Hyboganthus regios , Perca flavescens , and Erimizon oblongus . The apex predator I. furcatus (᭝) is significantly enriched ( p = 0.05) in 13 C and 34 S relative to other freshwater fishes, indicating the importance of anadromous Alosa in their diet. Figure adapted from MacAvoy et al. (2000).

regressions using the various k values. In general, the larger tory (range 13–17‰, mean ± SD = 15.9 ± 2.1‰). Nitrogen values of k + m resulted in large mean differences between isotope turnover was not modeled against what would be ex - predicted and modeled results. The k + m value that yielded pected due to growth because of the variability among indi - the best r2 (0.74) was 0.004. This value was within the mea - vidual I. punctatus and the high fractionation expected for sured range of the k values, which accounted only for growth. nitrogen isotopes relative to carbon and sulfur isotopes. Therefore, relative to growth, metabolic tissue replacement There is a 3–4‰ enrichment of 15 N in a predator’s muscle was probably minimal. It would come to be important only tissue relative to its prey, and when combined with the ini - when growth rate would not account for the observed isotope tial variability of the 15 N in the I. punctatus , carbon and turnover, such as in the case of small mammals and birds sulfur isotope signatures probably are a superior method for (Tieszen et al. 1983; Hobson and Clark 1992). documenting turnover of tissue due to growth. The observed 34 S values were also compared with pre - As expected, the isotope turnover of the I. punctatus tis - dicted values using eq. 7. The modeled 34 S isotopic change sue was slow relative to that of Japanese quail (Hobson and did not fit the laboratory data as well as the modeled 13 C, Clark 1992), gerbils (Tieszen et al. 1983), brown shrimp and the r2 values for k + m values of 0.00218 and 0.004 (Fry and Arnold 1982), and krill (Frazer et al. 1997). In the were only 0.27 and 0.28, respectively. The poor correlation cases of the birds and gerbils, their growth rates and metabo- was due to two factors: first, the variability in initial 34 S lism were orders of magnitude higher than those of the values among the farm-raised I. punctatus (as noted for 13 C I. punctatus in this study. The krill quickly assume the iso - above) and, second, a large variation in the 34 S of the ma - tope composition of their food because they have a high rine fish that were used to feed the I. punctatus in the labora - growth rate (Frazer et al. 1997). When krill are starved or

Table 3. Measured versus calculated isotope turnover. 13 C calculated (‰) 34 S calculated (‰) Time (days) 13 C measured (‰) k + m = 0.00218 k + m = 0.004 34 S measured (‰) k + m = 0.00218 k + m = 0.004) 1 –23.1±0.3 –22.98 –22.87 3.7±0.7 3.70 3.72 6 –22.5 –22.90 –22.73 5.7 3.83 3.96 8 –22.7 –22.87 –22.67 9 –23.0 –22.86 –22.64 3.7 3.91 4.10 10 –22.3±0.2 –22.84 –22.61 4.8 3.93 4.15 30 –22.2 –22.53 –22.07 45 –22.0 –21.31 –21.70 4.0 4.81 5.68 65 5.9 5.29 6.47 75 –22.0±0.4 –21.88 –21.01 4.1±0.8 5.51 6.84 78 –21.8±0.2 –21.84 –20.94 6.0±2.8 5.58 6.95 84 –21.9 –21.76 –20.82 –( k+m)t Calculated values were arrived at using eq. 7 ( C = Cn + ( C0 – Cn)e ), where C is the signature at time of sampling, Cn is the signature of a fish in 13 34 equilibrium with the new food, C0 is the initial signature of the fish, and k + m is growth rate per day plus metabolic turnover. Initial C and S values of the I. punctatus muscle were –22.9 and 3.5‰, respectively. The marine food (tuna muscle) that they were fed had a 13 C of –16.6‰ and a 34 S of 16.0‰. kept at low temperatures, they show no turnover of 13 C. In First, some I. furcatus heavily utilize the anadromous re - brown shrimp, Fry and Arnold (1982) measured half-lives of source when it is available and others clearly consume only 13 C turnover to be between 4 and 19 days depending on freshwater material (MacAvoy et al. 2000). Therefore, since their diet. Herzka and Holt (2000) found rapid turnover of the population as a whole does not consume Alosa at the 13 C and 15 N in red drum larvae, which generally matched same rate or with the same frequency, there is considerable the very high growth rate of these very young fish (doubling variation in their isotope signature at any given time. Second, of biomass occurred in 1 or 2 days). Hesslein et al. (1993) is at the maximum rate, isotopic turnover will be between 1 and the only study that we are aware of that examined juvenile 2‰ for 34 S and 13 C, respectively, over a 90-day period. The fish (broad whitefish ( Coregonus nasus )), and they found maximum turnover rate is so slow that it made observing a very slow turnover rates, comparable with those measured in significant enrichment among the I. furcatus population as a this study. They attributed most of the observed isotope whole during the 3 months of the Alosa spawning run un- change to growth. However, they estimated that metabolic likely. Isotopic variability among individual I. furcatus masks turnover might have accounted for 0.0013‰ per day for any enrichment trend. The more enriched isotope signatures 34 S and 0.0018‰ per day for 13 C. It should be noted that of the I. furcatus were likely a result of years of feeding upon Hesslein et al. (1993) estimated the k of C. nasus to be be- the migratory Alosa populations. Using isotope mixing equa- tween 0.03 and 0.07 in their study, which is somewhat tions, MacAvoy et al. (2000) estimated that approximately higher than that observed among ictalurids in this study. 40% of the biomass carbon and sulfur in adult I. furcatus was Factors contributing to the slow isotopic turnover of fish are, derived from marine sources. first, the slow metabolism of poikilotherms and, second, It is important to note that in systems that experience mi - slow growth rates. In addition, ictalurid catfish may reduce grations of prey species, the consumption of these prey items feeding during the winter months as their metabolism slows, by predators may be underestimated using isotopes if predator therefore reducing growth rate. tissue turnover is slow. This may be particularly true if the prey species is not a regular migrant. Conversely, the impact Application of laboratory results to field data or influence of a migrating predator on resident prey popula- Isotopic turnover rates measured in the laboratory could tions may be underestimated if isotopic turnover in the preda - not be uniformly applied to the accumulated field data due tor is slow. It follows that the often-assumed equilibrium in to the variability of the I. furcatus isotopic signatures. Deter - isotopic signature between predator and prey may be violated mining a maximum isotopic turnover rate in the laboratory if predator tissues have slow isotope turnover. would have yielded fine-scale information about the impor - tance of marine nutrients during the Alosa run instead of Acknowledgments coarse-scale approximations arrived at by proportionation equations. However, the field data did not support the hy - We would like to thank Dr. Stephen McIninch of Virginia pothesis that I. furcatus isotope values would be depleted at Commonwealth University for his efforts in the field. We the beginning of the Alosa run and would become steadily thank Nicole Cortese, Snoopy Dawson, Dr. Samantha Joye, enriched over the course of the spring as Alosa are con - Stefania Korontzi, Dr. Tom O’Donnell, Dr. Vaughan Turekian, sumed. However, it should be noted that there was a limited Peter Yanik, three referees, and an anonymous Associate Edi- number of captures of large (>38 cm) I. furcatus in the first tor for their helpful suggestions. This project was funded in several weeks of the study. part by a Moore Award granted to S.E. MacAvoy by the De - The fact that no significant enrichment trend with time was partment of Environmental Sciences, University of Virginia. seen in the isotope values of I. furcatus in the field, although there was a clear isotopic enrichment associated with marine influences in these same I. furcatus , led to two conclusions.

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