Anadromous fish as marine nutrient vectors.

Abstract--The tidal freshwater of Virginia supports anadromous herring (Alosa spp.) spawning runs in the spring; however, their importance as nutrient delivery vectors to the freshwater fish food web remains unknown. The stable isotope signatures of fishes from 21 species and four different guilds (predators, carnivores, generalists, and planktivores) were examined in this study to test the hypothesis that marine derived nutrients (MDNs) brought by anadromous fish would be traced into the guilds that incorporated them. Spawning anadromous fish were [sup.13]C and [sup.34]S-enriched ([delta][sup.13]C and [delta][sup.34]S of approximately 18[per thousand]. and 17.7[per thousand], respectively) relative to resident freshwater fish. Of the guilds examined, only predators showed [sup.13]C and [sup.34]S-enrichment similar to the anadromous fish; however, some generalist catfish also showed enriched signatures. Specific fatty acid [delta][sup.13]C signatures for gizzard shad (Dorosoma cepedianum), blue catfish (Ictalurus furcatus), and alewife (Alosa pseudoharengus), show a 10[per thousand] range among fishes, clearly reflecting isotopically distinct dietary sources. The [delta][sup.13]C and [delta][sup.34]S distribution and range among the freshwater fishes suggest that both autochthonous and allochthonous (terrestrial C3 photosynthetic production and MDN) nutrient sources are important to the tidal freshwater fish community.

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Streams in which anadromous fish spawn are often nutrient poor and the spawning anadromous fish may be an important source of nutrients to them (Kline et al., 1993; Wipfli et al., 2003). Sometimes spawning anadromous fish even fertilize near-stream terrestrial environments (Ben-David et al., 1998; Koyama et al., 2005). The spawning fish are frequently semelparous and deliver marine derived nutrients (MDN) to the freshwater as moribund biomass, excreted ammonium ion (N[H.sub.4].sup.+]), or through gamete release (Cederholm et al., 1989; Browder and Garman, 1994; Wipfli et al., 2003). Several studies in Alaska and the Pacific Northwest of North America have demonstrated the importance of marine nutrients brought to freshwater streams by anadromous salmonids (Bilby et al., 2003; Kline et al., 1993; Francis et al., 2006). In the Gulf of Mexico, migrating Gulf menhaden (Brevoortia patronus) transported estuarine nutrients into inshore environments (Deegan, 1993), and returning salmon contributed to the productivity of Lake Ontario tributaries (Rand et al., 2002). However, less work has been done on the East Coast of the United States where coastal development has been much more intense and the dominant anadromous species (Alosa spp.; herring CA. aestivalis), American shad (A. sapidissima), and alewife (A. pseudoharengus)) are often not highly abundant (Deegan, 1993; Garman and Macko, 1998). Although the Alosa spp. on the east coast tend towards an iteroparous life cycle rather than a semelparous one, they do experience heavy postspawning mortality (alewife postspawning mortality has been measured as 41% (Havey, 1961) and between 39% and 57% (Durbin et al., 1979)). Because tidal freshwater streams receive nutrients from marine and freshwater primary productivity at different times, the incorporation of these nutrients by consumers may be different depending on feeding guilds. Fish found in the same area in a stream may derive nutrition from local or translocated productivity. In nutrient poor systems, such as East Coast United States tidal freshwater areas, it is important to understand nutrient sources to different feeding guilds (e.g., predators, carnivores, generalists, and planktivores).

For more than 20 years now, carbon and nitrogen stable isotopes (reported as a ratio of heavy to light isotopes and given [delta] notation with units of [per thousand], see Materials and methods section for more detail) have been used to determine the importance of MDN in freshwater systems, and to characterize the trophic structure within those systems (Kline, et al., 1993; Vander-Zanden et al., 1999). For example, carbon and nitrogen isotopes have shown that anadromous Pacific salmon (Oncorhynchus spp.) were a significant source of allochthonous nitrogen to coastal streams where spawning occurs (Kline et al., 1993).

Hesslein et al. (1991) used sulfur isotopes to differentiate freshwater migratory and non-migratory fishes in the Mackenzie River Basin, Canada. On the East Coast of the United States, anadromous river herring (Alosa spp.) retain their marine isotope signal after spending part of the spring spawning in freshwater, and that some freshwater piscivores are [sup.34]S and [sup.13]C-enriched after preferentially consuming migrating Alosa spp. during the spawning run (Garman and Macko, 1998; MacAvoy et al., 2000).

An additional tool for determining origins and transformations of organic material from different sources is the stable isotope ratio of specific compounds. Isolating a specific compound, or class of compounds, then measuring the isotope ratio on those compounds, may offer a more robust technique to trace biologically significant compounds (such as fatty or amino acids) than would be possible from bulk isotope analysis alone. For example, examining the carbon isotopic composition of fatty acids from an animal, particularly essential fatty acids, allows the direct determination of dietary sources that contribute to the fatty acid pool of that animal (Stott et al., 1997). Although bulk isotope analysis can be an effective nutrient tracer in systems with isotopically distinct nutrient sources (Peterson et al., 1985), the isotopes of specific fatty acids may provide more confidence in identifying sources (Canuel et al., 1997).

Carnivorous heterotrophs are unable to synthesize fatty acids longer than 18-carbons, nor can they desaturate carbon-carbon bonds between the ninth and terminal methyl carbon, therefore, these essential fatty acids must be obtained from diet (Olsen 1999). Because essential fatty acids are not influenced by subsequent metabolism within a eukaryotic heterotroph, they retain their original isotope composition (Stott et al., 1997). Fatty acids synthesized by marine plankton and incorporated into marine fish would be highly enriched in [sup.13]C relative to those produced by freshwater primary producers or C3 photosynthesis. Additionally, short chain fatty acids, used as precursors in the biosynthesis of unsaturated or longer chain saturated fatty acids, should be [sup.13]C enriched in relation to biosynthesized fatty acid products (Murphy and Abrajano, 1994). In this study, the fatty acid nomenclature used is carbon number:number of double bonds. For example, 18:2 is an 18-carbon fatty acid with two points of unsaturation. The desaturation of 16:0 to 16:1 and 18:0 to 18:1-18:2 occurs by a systematic fractionation of roughly 2[per thousand] per desaturation (DeNiro and Epstein, 1977; Monson and Hayes, 1982). Also, studies have shown that the elongation of fatty acids by de novo synthesis results in a 2[per thousand] per 2-carbon acetyl group addition. These fractionations allowed the identification of fatty acids that were directly incorporated from symbiotic bacterial sources in mussels as opposed to those obtained through de novo synthesis (Murphy and Abrajano, 1994).

In this study we compared the [delta][sup.15]N, [delta][sup.13]C, [delta][sup.34]S of bulk tissues, plus the [delta][sup.13]C of specific fatty acids among four guilds of fish plus anadromous Alosa spp. in a tidal freshwater stream on the East Coast of the United States. Our objective was to determine if anadromous fish, captured more than 40 km from the salt-wedge, were isotopically distinct from freshwater residents, and to determine if freshwater guilds showed the incorporation of marine allochthonous organic material.

Materials and methods

Field collections by boat electrofisher were made in the tributaries and main-stem of the Rappahannock River, VA (within a 40-mile area between Fredericksburg and Tappahannock, VA) during March and May 1997 and 1998 (Fig. 1). The Rappahannock River is tidal in this region (tidal range: 0.1 to 1 meter) and shares many physicochemical characteristics with other tidal freshwater rivers in the region (Garman and Nielsen, 1992). Fishes were collected and placed on ice in the field, transported back to the laboratory, and muscle tissue samples were taken, which were then dried for later analysis. Analysis of the sulfur and compound specific fatty acid samples took several years and were completed by 2002.

The fishes were placed into four different guilds based on feeding strategies taken from Jenkins and Burkhead's (1993) seminal work on Virginia freshwater fishes, plus an anadromous life cycle group (Table 1).

Bulk isotope tissue analysis, elemental analyzer, and isotope ratio mass spectrometry 

Samples of dorsal muscle tissue were dried at 60[degrees]C for three days and homogenized in preparation for analysis. The tissues were then lipid extracted by refluxing them in dichloromethane for 35 minutes (Knoff et al., 2002), except for those samples selected for compound specific analysis, which were soxlet extracted (see below; gas chromatography-mass spectrometry (GC-MS) and compound specific stable isotope analysis (CSIA)). One milligram (mg) of dried, lipid-extracted muscle was used for [delta][sup.13]C and [delta][sup.15]N analysis. Six mg was used for [delta][sup.34]S analysis. A Carlo Erba elemental analyzer (EA) (Fisons/VG/Micromass, Manchester, UK) coupled to a Micromass Optima isotope ratio mass spectrometer (IRMS) (Fisons/VG/Micromass, Manchester, UK) was used to obtain [delta][sup.13]C, [delta][sup.15]N and [delta][sup.34]S values. The [delta][sup.13]C and [delta][sup.15]N were obtained concurrently, and [delta][sup.34]S was determined during separate analytical runs.

[FIGURE 1 OMITTED]

The isotope compositions are reported in relation to standard material and follow the same procedure for all stable isotopic measurements, as follows:

[delta].sup.x]E = [([sup.x]E/[sup.y]E)sample/([sup.x]E/[sup.y]E)standard] - 1) x 1000, (1)

where E = the element analyzed (C, N, or S);

x = the molecular weight of the heavier isotope; and

y = lighter isotope (x=13, 15, 34, and y=12, 14, 32 for C, N, and S, respectively).

The standard materials to which the samples are compared are Pee Dee Belemnite for carbon, air [N.sub.2] for nitrogen, and Canyon Diablo Triolite for sulfur. Reproducibility of all measurements was typically 0.2[per thousand] or better. Between every 12 samples, a laboratory standard was analyzed. In a typical run of 60 samples (+5 standards, 65 measurements total) the standard deviations for [delta][sup.15]N and [delta][sup.13]C were <0.2[per thousand]. For [delta][sup.34]S, standard deviations were <0.3[per thousand].

Gas chromatograph-mass spectrometer (GC-MS)

Once dried, muscle samples selected for compound specific isotope analysis (CSIA) were lipid extracted (Soxhlet method from Ballentine et al., 1996) and the fatty acids had a methyl group added to the carboxyl end (derivitized) so they could be characterized by gas chromatography (GC). This was done by heating with B[F.sub.3]C[H.sub.3]OH for eight minutes (Ballentine et al., 1996). The fatty acid methyl esters (FAME) were analyzed by GC-MS using a Hewlett Packard 5890 Series II gas chromatograph (Palo Alto, CA) interfaced to a Hewlett Packard 5971A mass sensitive detector (Palo Alto, CA), with helium gas as the carrier. A 60-meter J&W DB-5 column (J&W Scientific, Folsom, CA) was used for FAME separation. The GC oven temperature program used was as follows: 100[degrees]C for 2 minutes, ramp at 3[degrees]C/min. to 210[degrees]C, hold for 20 min, ramp 1[degrees]C/min, to 220[degrees]C, hold for 10 min.

Compound specific stable isotope analysis (CSIA)

The FAMEs were analyzed for their stable carbon isotope compositions using a Hewlett Packard 5890 Series II gas chromatograph interfaced through a combustion furnace with a VG Isoprime IRMS (Fisons/VG/Micromass, Manchester, UK). The GC was equipped with the same column that was used for the GC-MS analysis and helium was the carrier gas. The GC oven temperature program was identical to that used for the GC-MS FAME identification. Time elution was used to identify peaks. The C[O.sub.2] combustion products of the fatty acids eluting from the column were introduced into the mass spectrometer after passing through a water trap.

All FAME [delta][sup.13]C values were corrected for the addition of the methyl group to the original fatty acid. The derivatization of the fatty acids to their methyl esters results in a predictable and reproducible isotope effect (Ballentine et al., 1996; Uhle et al., 1997). Adding a methyl group to the fatty acid alters its isotope signature. However, if the isotopic ratio of the methanol (in this case [delta][sup.15]C = -46[per thousand], measured by injecting the methanol into the mass spectrometer through the GC) and the fatty acid methyl ester are known, then the isotopic signature of the original fatty acid can be determined using a mass balance Equation 2.

[delta][[sup.13][C.sub.FAME] = [f.sub.FA]-[delta][[sup.13]C.sub.FA] + [f.sub.Methanol] [delta][sup.13]C Methanol (2)

where ([delta][[sup.13]C.sub.FAME], ([delta][[sup.13]C.sub.FA], and and [delta][[sup.13]C.sub.Methanol] =

the carbon isotope signatures of the FAME, the underivatized fatty acid, and the methanol, respectively; and

[f.sub.FA] and [f.sub.Methanol] =

the fractions of carbon in the FAME due to the underivatized fatty acid and methanol, respectively (Ballentine et al., 1996; Uhle et al., 1997).

Each sample was injected four to eight times (depending on the reproducibility of the analysis). Only [delta][sup.13]C values that were within 1.5[per thousand] of each other were considered to reflect the [delta][sup.13]C of the FAME (MacAvoy et al., 2002). Therefore, the [delta][sup.13]C reported for each FAME identified is represented by an average value and a standard deviation. Every sixth sample injected was an internal, laboratory standard (naphthalene-d, [delta][sup.13]C-25.7[per thousand]) to insure consistent performance of the GC, oxidation furnace, and mass spectrometer.

Statistical analysis

Kruskal-Wallis nonparametric procedures were used to test for differences in isotopic values among anadromous fish and the different guilds (predators, carnivores, generalists, and planktivores, ([alpha]=0.05)). The Dunn procedure was used to examine differences between groups (Rosner, 1990). Statview SE + Graphics (Abacus Concepts, Inc., Cary, NC), JMP In (SAS, Cary, NC) and Microsoft Excel version 5.0 (Microsoft, Inc., Redmond, WA) were used for statistical tests. The Dunn procedure reduces the risk of type-1 error inherent in multiple comparison techniques. It does so by increasing the Z-score needed to reject the null hypothesis as the number of individual groups being compared increases. In the present study, a Z-score of [+ or -] 3.02 (0.9975 confidence) was needed for a difference to be significant.

Results

The first objective of this study was to establish that the spawning anadromous fish retained the marine isotope signal more than 40 km upstream from saline waters. This was the case for all three isotopes examined. The second objective was to test whether the different guilds of fish showed the incorporation of the marine isotope signal brought to the tidal freshwater by the anadromous fishes. This was observed, but largely limited to the predator guild.

Of the groups examined, the anadromous fish were the most [sup.13]C-enriched, with mean values of approximately -19[per thousand], followed by predators and planktivores (means -21.8[per thousand] and -22.0[per thousand], respectively), which were not significantly different from each other. This suggests that, of the remaining two guilds, carnivores were significantly [sup.13]C-depleted relative to generalists (mean -24.1%, and -23.5[per thousand], respectively; Table 2). There was approximately a 10[per thousand] range in [delta][sup.13]C among the exclusively freshwater guilds (Table 2, Fig. 2).

Anadromous fish have elevated [delta][sup.15]N values relative to freshwater fish with similar feeding strategies. However, the trophic enrichment and diet-tissue discrimination associated with [delta][sup.15]N signatures make using nitrogen a less effective tracer for source than carbon or sulfur. In this study there was less variability within the guilds [delta][sup.15]N signatures, relative to [delta][sup.15]C, although the range ([per thousand]) of [delta][sup.15]N values among all fishes was similar to that observed for [delta][sup.13]C (10[per thousand]). The anadromous fish had the lowest [delta][sup.15]N values and generally grouped between 12[per thousand] and 13[per thousand]; however, their values were not lower than generalists or carnivores. The predators were the most [sup.15]N-enriched of any group (Table 2). There were no significant differences among the [delta][sup.15]N values for carnivores, generalists, and planktivores (Table 2).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Sulfur isotopes were hypothesized to be the most useful for tracing marine protein into freshwater, owing to extreme differences between the [delta][sup.34]S of marine plankton and various sulfur sources in freshwater. Predator fishes and anadromous Alosa spp. showed elevated [sup.34]S signals relative to other resident freshwater fishes, indicating that the predators incorporated Alosa spp. sulfur (protein). The range of [delta][sup.34]S values among all the fish captured was from approximately 0[per thousand] to 20[per thousand], a considerably larger range than observed for the other two isotopes (Table 2, Fig. 3). Significant differences were observed in [delta][sup.34]S among several of the separate groups. Anadromous species were highly [sup.34]S-enriched relative to all resident freshwater fish (Table 2, Fig. 2), although the striped bass (40 cm total length (TL)) had values between 0.3[per thousand] and 6.4[per thousand], the lowest of the anadromous [delta][sup.34]S values. Predators were the most [sup.34]S-enriched of the resident fish, followed by planktivores (a trend also observed for [delta][sup.34]C). Carnivores and generalists were the most [sup.34]S-depleted of the guilds and were not significantly different from each other (Table 2). Sulfur was the only stable isotope that completely separated the anadromous Alosa spp. from the full time freshwater residents. All of the Alosa spp. individual values were [sup.34]S-enriched and outside the ranges observed in the other groups (Table 2).

Fatty acid analysis

Fatty acid (FA) isotope values show that some predators derive fats from anadromous fish and that there is a large variation among FA isotope values. FA [delta][sup.13]C values were determined for one alewife (anadromous), one gizzard shad (Dorosoma cepedianum, a native freshwater planktivore), and two blue catfish (Ictalurus furcatus, an introduced piscivorous predator). For the blue catfish bulk [delta][sup.13]C and [delta][sup.34]S values from muscle tissue showed that one individual (A in Table 3) was significantly [delta][sup.13]C and [delta][sup.34]S-depleted relative to the other. This was also the case for the respective [delta][sup.13]C values of their individual FAs. The anadromous alewife and the more [delta][sup.13]C-enriched blue catfish (B) had [delta][sup.13]C FA values that, for the most part, overlapped with each other. Their 16 and 18 carbon length FAs were generally [sup.13]C-enriched relative to the gizzard shad and the second blue catfish (A) (Table 3). For all fish, except gizzard shad, the saturated 12:0, 16:0, and 18:0 FAs were more enriched (2[per thousand] to 6%[per thousand]) than the 14:0, 16:1 and 18:1 FAs. 14:0 FAs are not elongated to 16 or 18 carbons in animals, which is why they are [sup.13]C-depleted relative to saturated 16:0 and 18:0 (see Discussion). For the gizzard shad, the 12:0 FAs were 2%0 depleted relative to the 14:0 FAs. The blue catfish (B) with low 5[sup.13]C and 534S bulk values, generally had more [sup.13]C-depleted FAs than other fishes. There was up to a 10[per thousand] range among the FAs within an individual fish, with unsaturated FAs [sup.13]C-depleted relative to saturated, and longer saturated chains being generally [sup.13]C-depleted relative to shorter chain FAs (Table 3).

Discussion

The fact that the anadromous Alosa spp. were the most [sup.13]C-enriched of the groups examined was expected because they retain the [sup.13]C-enriched (relative to freshwater) signal of marine carbon fixation (Garman and Macko, 1998, MacAvoy et al., 2000, Hoffman et al., 2007). High [delta][sup.13]C in freshwater systems with anadromous fish does not necessarily indicate trophic status (Garman and Macko, 1998; MacAvoy et al., 2000; Gregory-Eaves et al., 2007). The [delta][sup.13]C-enriched predators (mostly piscivorous catfish) show a wide range in 5[sup.13]C, from -16 to -27[per thousand] (white perch also show elevated [delta][sup.13]C relative to most resident freshwater fish, but they also are [sup.34]S-depleted, indicating that their carbon signature reflects their status as a secondary carnivore, not marine carbon). The most [sup.13]C-enriched of the predators reflect the consumption of marine material, probably spawning adult Alosa spp., which had the most [sup.13]C-enriched values of any prey item found. A number of predators, however, clearly derive very little carbon from marine migrants; they are strictly freshwater feeders, as shown by their [sup.13]C-depleted carbon isotope values. Among the remaining three guilds, the planktivores (within which the anadromous Alosa spp., mainly filter feeders, were not included) were the most [sup.13]C-enriched, driven largely by the migratory and filter-feeing gizzard shad (Jenkins and Burkhead, 1993). Gizzard shad [sup.13]C enrichment probably reflects consumption of autochthonous production and not marine derived nutrients, because the gizzard shad [delta][sup.34]S are too low to reflect substantial marine material (Table 2 and see below). The ([delta][sup.13]C range among the resident freshwater fishes suggest, not surprisingly, that both autochthonous and allochthonous production contribute to carbon fixation in this tidal freshwater stream. Indeed, in the York River estuary, a few kilometers south of the Rappahannock River, Raymond and Bauer (2001) estimate that between 38% and 56% of dissolved organic carbon was derived from internal (autochthonous) sources.

Only a small percent of the residents show an exclusive allochthonous signal in the region of the Rappahannock River examined, and most of the resident freshwater fish show an autochthonous [delta][sup.13]C signature, which is characteristic of small tributaries close to the main stem of a large piedmont river. The [delta][sup.13]C range of allochthonous productivity in Virginia tidal freshwater streams is between -25[per thousand] and -28[per thousand] (Garman and Macko, 1998; Hoffman et al., 2007). Because C[O.sub.2] solubility is limited in water, systems dominated by autochthonous production tend to be [sup.13]C-enriched relative to C3 plants that appear in small streams dominated by C3 allochthonous production (Michener and Schell, 1994). Garman and Neilson (1992) note that the presence of gizzard shad and detritivores in Virginia tidal freshwater suggest that autochthonous production is important in these systems relative to non-tidal areas upstream, where fishes primarily consume terrestrial arthropods (Garman, 1991). Most of the guilds examined in this study reflected the predominance of autochthonous production and have [delta][sup.13]C values that are lower than would be expected for a C3 dominated system. The anadromous Alosa spp. were also [sup.13]C-enriched relative to other guilds. All of their [delta][sup.13]C values cluster between -22[per thousand] and -16[per thousand], whereas all other guilds range to approximately-28[per thousand] range (the most [sup.13]C-depleted values reflecting allochthonous production). This [sup.13]C enrichment in Alosa spp. is not due to incorporating autochthonous freshwater production. The [sup.13]C-enrichment is a signal from the marine environment from which the Alosa spp. biomass was derived. This interpretation is supported by the markedly [sup.34]S-enriched values of the Alosa spp., which are in most cases 7[per thousand] greater than any other fish in this study ([delta][sup.34]S value of sulfur fixed from marine S[O.sub.4] in the ocean at present is highly enriched relative to freshwater [Kaplan et al., 1963]). Therefore, the [sup.13]C enrichment of the Alosa spp. biomass (and other anadromous fishes) is due to a marine influence, not an autochthonous influence.

Of the guilds examined, predators show the highest [delta][sup.34]S value after the Alosa spp., but are not significantly enriched in [sup.13]C relative to other guilds. The elevated [sup.34]S in predators (many of whom are piscivores) shows that more marine sulfur is incorporated by this guild relative to others. The predator's elevated/PSN values place them at the top of the fish food web, although some smaller individuals (blue catfish), feed at lower trophic levels while young (Jenkins and Burkhead, 1993).

The link between anadromous Alosa spp. and the predators is also supported by the fatty acid carbon isotope signatures. Alosa spp. 16 and 18 carbon FAs were generally the most [sup.13]C-enriched of the fish examined (Table 3). The two large (53cm TL) blue catfish show two very different FA isotope profiles. One blue catfish (B in Table 3) had a series of highly [sup.13]C-enriched FAs (bulk muscle tissue [delta][sup.13]C and [delta][sup.34]S are also enriched in this individual) and the other had FAs with isotope signatures similar to allochthonus primary production (also consistent with bulk muscle tissue [delta][sup.13]C and [delta][sup.34]S). Shorter chain (12 carbon) and more saturated FAs reveal the original [delta][sup.13]C of the fats in the diet. Longer chain and unsaturated FAs can be subject to de novo transformations, which result in well established fractionations as chain length is systematically increased or as a double bond between carbons is created (making a point of unsaturation in a saturated FA). Generally, there is a 2[per thousand], depletion in [delta][sup.13]C arising from each unsaturation and another 2[per thousand]. depletion for each two carbon acetyl group addition (Deniro and Epstein, 1977). The most conservative tracer of dietary FAs, are the enriched precursors to long chain and unsaturated FAs. Among the FAs analyzed, the 12:0, 16:0, and 18:0 yield the best [delta][sup.13]C estimate for dietary FAs, which clearly show distinct isotope signals depending on the carbon sources listed below: 1) [sup.13]C-enriched marine isotope signals (represented by alewife and blue catfish B), 2) allochthonus production (represented by blue catfish A), or 3) a mix of autochthonous and allochthonus production, with the possibility of marine influences (represented by gizzard shad, although their [delta][sup.34]S values do not reflect the typical marine signal).

The [delta][sup.13]C and [delta][sup.34]S distribution and range among the freshwater fishes suggest, not surprisingly, that both autochthonous and allochthonous nutrient sources, with the allochthonous sources being terrestrial C3 vegetation and marine primary production inwelling to this tidal freshwater stream, more than 40 km from the Chesapeake Bay. Unlike streams on the West Coast of the United States, where marine derived nitrogen and carbon can be an important nutrient source to inland ecosystems (Kline et al., 1993; Bilby et al., 2003; Chaloner et al., 2002), for all fish guilds in the study reported here, except the predators, there was not significant marine nutrient uptake. Several West Coast studies have shown that marine derived nitrogen, and some marine derived carbon, contributed to invertebrates (Francis et al., 2006; Hicks et al., 2005), primary producers, and juvenile fish within or near the sites receiving the spawning anadromous fish (Bilby et al., 2003; Koyama et al., 2005). For example, Bilby et al. (1996) found that 17% and 30% of the nitrogen in collector-gathers and juvenile coho salmon (Oncorhynchus kisutch) in Washington, were derived from spawning salmon. Ben-David et al. (1998) found that salmon carcasses may have contributed to the nitrogen incorporated by some terrestrial plants, as well as deer mice, squirrels, and voles; and Wipfli et al. (2003) found that salmon carcasses fueled increased growth rates among young salmonids. However, those studies show that only some material from decaying salmon makes its way into invertebrates and riparian vegetation (Bilby et al., 1996, 1998; Francis et al., 2006). There is strong evidence however, that the nutrients deposited as a result of the postspawning death of anadromous adults did significantly sustain fry the following year (Bilby et al., 1996, 1998).

In the East Coast stream examined here, carnivores and generalists, which consume benthic invertebrates as part of their diet, did not show a marine signal. Compared with anadromous salmonids on the West Coast, East Coast herring have a lower postspawning mortality and their runs have less biomass. Both of these facts indicate that a limited amount of marine protein and nitrogen maybe be delivered to spawning streams unless it is consumed directly by predatory fish. This is consistent with findings suggesting benthic insects in Alosa spp. spawning streams do not accumulate large amounts of marine derived material, even if they are living closely with post-spawning anadromous fish carcasses (Francis et al., 2006; Garman, 1992). It should be noted that in West Coast streams associated with spawning salmon, invertebrate uptake can be substantial (Hicks et al., 2005; Chaloner et al., 2002). Unlike most West Coast streams however, some tidal streams in Virginia have large piscivorous fish (introduced from Texas, Louisiana, or Mississippi in the 1970s) and these fish clearly incorporate marine material. So, while salmon (and presumably herring) on the West Coast import nutrients to the base of the food web (terrestrial autotrophs, young-of-the-year fish, and some invertebrates), in the steams examined here the marine material enters the top of the aquatic food web where spawning adult anadromous fish are consumed by piscivorous fish. In order to fully understand the importance of a migratory or transitory nutrient source to consumers, the time required for that nutrient to be incorporated must be understood, thereby allowing a temporal evaluation of ecosystem structure. While the results of this study suggest that marine material does not form a substantial nutrient source to most of the fish community, more work needs to be done to investigate marine inputs derived from spawning anadromous fish, to other, lower order components of East Coast United States tidal freshwater systems.

Acknowledgments

We thank Mark King, Steve McIninch, and William Shuart at Virginia Commonwealth University, Department of Environmental Studies. This work was partially supported by the NOAA-Chesapeake Bay Office, a Moore Award from The University of Virginia, and a Mellon Award from American University.

Manuscript submitted 25 June 2008.

Manuscript accepted 20 October 2008. Fish. Bull. 107:165-174 (2009).

The views and opinions expressed or implied in this article are those of the author and do net necessarily reflect the position of the National Marine Fisheries Service, NOAA.

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Stephen E. MacAvoy (contact author) (1)

Greg C. Garman (2)

Stephen A. Macko (3, 4)

Email address for contact author: [e-mail]

(1) Department of Environmental Science American University 4400 Massachusetts Ave. NW Washington, DC 20016

(2) Center for Environmental Studies Virginia Commonwealth University 1000 W. Cary St., Suite 111 Richmond, Virginia 23284

(3) Environmental Science Department University of Virginia 291 McCormick Rd. Charlottesville, Virginia 22903

(4) Program in Geobiology and Low Temperature Geochemistry U.S. National Science Foundation 4201 Wilson Boulevard Arlington, Virginia 22230

Table 1
Fish species examined by guild (including an anadromous group) from
the Rappahannock River to assess the role of marine fish as nutrient
vectors. Guild assignments are based on diet as reported in Jenkins
and Burkhead (1993).

Guild         Species name               Common name

Predator      Ictalurus furcatus         blue catfish
              Lepisosteus osseus         longnose gar

Carnivore     Micropterus salmoides      largemouth bass
              Lepomis gibbosus           pumpkinseed
              Hybogreathus regius        eastern silvery minnow
              Notemigonus crysoleucas   golden shiner
              Lepomis macrochirus        bluegill
              Perca flavescens           yellow perch

Generalist    Anguilla rostrata          American eel
              Ameiurus catus             white catfish
              Ameiurus nebulosus         brown bullhead
              Ictalurus punctatus        channel catfish

Planktivore   Menidia beryllina          inland silverside
              Dorosoma cepedianum        gizzard shad
              Erimyzon oblongus          creek chubsucker

Anadromous    Alosa aestiualis           blueback herring
              Alosa pseudoharengus       alewife
              Alosa sapidissima          American shad
              Morone sasatilis           striped bass
              Morone americana           white perch

Table 2
Isotope values for all fish used in this study separated
by Family. "A" indicates anadromous, * indicates
euryhaline range. Guild assingments are based on diet
as reported in Jenkins and Burkhead (1993). "C" indicates
a group with some isotope data derived from MacAvoy et
al. (2000). White perch (Morone americana) shows elevated
[delta][sup.13]C content is probably not marine protein given
the low [delta][sup.34]S ratio; M. americana is a secondary
carnivore and the high Sr3C reflect this. Standard deviation
is given after the [+ or -] and N is in parentheses.

Family and Species         Common name

Anguillidae
  Anguilla rostrata        American eel

Atherinidae
  Menidia beryllina        inland sliverside
Catostomidae
  Erimyzon oblongus [C]    creek chubsucker

Centrarchidae
  Micropterus salmoides    smallmouth bass
  Lepomisgihhosus          pumpkinseed
  Lepomis macrochirus      bluegill
Clupeidea
  Alosa                    alewife spawning
    pseudoharegus (A,C)

  Alosa aestiualis (A,C)   blueback herring
                           spawning
  Alosa sapidissima (A,C)  juvenile American
                           shadspawning
  Dorosoma cepedianurn     gizzard shad

Cyprinidae
  Hybognathus regius       eastern silvery

  Notemigonus crysoleucas  golden shiner

Ictaluridae
  Ictalurus furcatus (C)   blue catfish
  Ictalurus punctatus      channel catfish
  Ameirus nebulosas        brown bullhead
  Ameirus catus            white catfish
Lepisosteidae
  Lepisosteus osseus       longnose gar
Moronidae
  Morone saratili. (A)     striped bass
  Morone americana (A) *   white perch

Percidae
  Perca flavescens (C)     yellow perch

Family and Species         Guild: food types

Anguillidae
  Anguilla rostrata        generalist: insects,
                           snails, fish, clams
Atherinidae
  Menidia beryllina        planktivore
Catostomidae
  Erimyzon oblongus [c]    planktivore:
                           planktonic crustaceans
Centrarchidae
  Micropterus salmoides    carnivore
  Lepomisgihhosus          carnivore: insects, worms
  Lepomis macrochirus      carnivore: insects, worms
Clupeidea
  Alosa                    anadromous: copepods,
    pseudoharegus (A,C)    diatoms, ostracods,
                           shrimp, fish
  Alosa aestiualis (A,C)   anadromous:
                           copepods, cladocerans
  Alosa sapidissima (A,C)  anadromous: copepods,
                           small invertebrates
  Dorosoma cepedianurn     planktivore: filter feeder

Cyprinidae
  Hybognathus regius       minnowcarnivore: diatoms,
                           algae, ooze detritus
  Notemigonus crysoleucas  carnivore: microcrustaceans
                           insects
Ictaluridae
  Ictalurus furcatus (c)   carnivore/piscivore
  Ictalurus punctatus      opportunistic generalist
  Ameirus nebulosas        generalist/omnivorous
  Ameirus catus            generalist/omnivorous
Lepisosteidae
  Lepisosteus osseus       predator, piscivore
Moronidae
  Morone saratili. (A)     generalist, piscivorous
  Morone americana (A) *   carnivorous: worms,
                           shrimp, fish
Percidae
  Perca flavescens (C)     carnivore: insects small fish

Family and Species                [delta][sup.13]C

Anguillidae
  Anguilla rostrata            -24.7 [+ or -] 0.7 (3)

Atherinidae
  Menidia beryllina            -23.8 [+ or -] 0.9 (3)
Catostomidae
  Erirnyzon oblongus [c]             -28.1 (1)

Centrarchidae
  Micropterus salmoides        -23.0 [+ or -] 1.9 (5)
  Lepomisgihhosus              -25.4 [+ or -] 1.1 (8)
  Lepomis macrochirus          -23.7 [+ or -] 2.2 (5)
Clupeidea
  Alosa                        -17.4 [+ or -] 1.1 (7)
    pseudoharegus (A,C)

  Alosa aestiualis (A,C)       -19.0 [+ or -] 0.6 (7)

  Alosa sapidissima (A,C)
                               -20.2 [+ or -] 0.6 (4)
  Dorosoma cepedianurn         -20.2 [+ or -] 2.1 (7)

Cyprinidae
  Hybognathus regius           -23.0 [+ or -] 2.1 (6)

  Notemigonus crysoleucas      -24.8 [+ or -] 1.1 (5)

Ictaluridae
  Ictalurus furcatus (c)       -21.6 [+ or -] 1.9 (43)
  Ictalurus punctatus          -20.5 [+ or -] 2.0 (3)
  Ameirus nebulosas            -24.0 [+ or -] 0.8 (3)
  Ameirus catus                -21.2 [+ or -] 2.7 (10)
Lepisosteidae
  Lepisosteus osseus                   -23.1
Moronidae
  Morone saratili. (A)         -25.0 [+ or -] 2.3 (2)
  Morone americana (A) *
                               -20.7 [+ or -] 1.2 (5)
Percidae
  Perca flavescens (C)         -25.1 [+ or -] 2.1 (6)

Family and Species                [delta][sup.15]N

Anguillidae
  Anguilla rostrata            11.2 [+ or -] 0.8 (3)

Atherinidae
  Menidia beryllina            15.5 [+ or -] 0.2 (3)
Catostomidae
  Erirnyzon oblongus [c]              10.9 (1)

Centrarchidae
  Micropterus salmoides        14.5 [+ or -] 1.3 (5)
  Lepomisgihhosus              13.1 [+ or -] 1.3 (8)
  Lepomis macrochirus          14.7 [+ or -] 1.8 (5)
Clupeidea
  Alosa                        12.8 [+ or -] 0.8 (7)
    pseudoharegus (A,C)

  Alosa aestiualis (A,C)       13.2 [+ or -] 0.3 (7)

  Alosa sapidissima (A,C)
                               12.6 [+ or -] 0.4 (14)
  Dorosoma cepedianurn         14.0 [+ or -] 0.9 (7)

Cyprinidae
  Hybognathus regius           12.4 [+ or -] 3.4 (6)

  Notemigonus crysoleucas      13.1 [+ or -] 1.6 (5)

Ictaluridae
  Ictalurus furcatus (c)       15.4 [+ or -] 2.0 (43)
  Ictalurus punctatus          13.4 [+ or -] 1.2 (3)
  Ameirus nebulosas            13.2 [+ or -] 0.5 (5)
  Ameirus catus                15.8 [+ or -] 2.3 (10)
Lepisosteidae
  Lepisosteus osseus                    16.8
Moronidae
  Morone saratili. (A)         13.3 [+ or -] 2.4 (2)
  Morone americana (A) *
                               16.7 [+ or -] 1.4 (5)
Percidae
  Perca flavescens (C)         14.3 [+ or -] 2.2 (6)

Family and Species                [delta][sup.34]S

Anguillidae
  Anguilla rostrata             0.9 [+ or -] 2.4 (3)

Atherinidae
  Menidia beryllina            10.0 [+ or -] 0.9 (3)
Catostomidae
  Erirnyzon oblongus [c]              5.1 (1)

Centrarchidae
  Micropterus salmoides         7.6 [+ or -] 3.2 (5)
  Lepomisgihhosus               6.5 [+ or -] 2.3 (9)
  Lepomis macrochirus           4.7 [+ or -] 2.0 (5)
Clupeidea
  Alosa                        17.9 [+ or -] 0.8 (6)
    pseudoharegus (A,C)

  Alosa aestiualis (A,C)       17.5 [+ or -] 0.4 (7)

  Alosa sapidissima (A,C)
                                8.0 [+ or -] 2.2 (4)
  Dorosoma cepedianurn          7.8 [+ or -] 2.5 (7)

Cyprinidae
  Hybognathus regius            6.5 [+ or -] 2.5 (6)

  Notemigonus crysoleucas       2.5 [+ or -] 1.7 (5)

Ictaluridae
  Ictalurus furcatus (c)       9.2 [+ or -] 3.0 (43)
  Ictalurus punctatus           8.5 [+ or -] 3.2 (3)
  Ameirus nebulosas             5.3 [+ or -] 1.6 (5)
  Ameirus catus                8.7 [+ or -] 4.7 (10)
Lepisosteidae
  Lepisosteus osseus                    8.34
Moronidae
  Morone saratili. (A)          3.4 [+ or -] 4.3 (2)
  Morone americana (A) *
                                7.5 [+ or -] 3.9 (5)
Percidae
  Perca flavescens (C)          6.9 [+ or -] 1.6 (6)

Table 3
Fatty acid (FA) [delta][sup.13]C values for Rappahannock
River fish. Means [+ or -] 1 Standard Deviation. (n=3).
Values are corrected for CH40H derevitization. FAs show
that carbon from anadromous fish has been incorporated
by Ictalarus furcatus but not by other resident fishes.
Bulk isotope values show trends similar to the FAs
and are as follows: alewife A. p.seudoharengus,
[delta][sup.13]C -19.3 [per thousand],
[delta][sup.15]N 11.9 [per thousand],
[delta][sup.34]S 17.1 [per thousand];
blue catfish Ictalarus furcatus (A)
[delta][sup.13]C -26.0 [per thousand],
[delta][sup.15]N 13.3 [per thousand];
[delta][sup.34]S 6.1 [per thousand]; I. Furcatus (B)
[delta][sup.13]C -19.3 [per thousand]
[delta][sup.15]N -16.6 [per thousand]
[delta][sup.34]S -10.8 [per thousand]
gizzard shad Dorosoma cepedianum
[delta][sup.13]C -21.5 [per thousand]
[delta][sup.15]N -14.5 [per thousand]
[delta][sup.345]N -10.2 [per thousand].

             Aloca            Ictalurus
         pseudoharengus        furcatus
Fatty       alewife          blue catfish
acid    ([per thousand])

12:0      -22.4 (0.4)        -28.5 (0.5)
14:0      -27.4 (1.8)        -33.6 (0.9)
16:1      -26.8 (0.8)        -35.4 (0.6)
16:0      -22.1 (0.1)        -30.3 (0.2)
18:1      -23.3 (0.6)        -30.5 (0.6)
18:0      -19.9 (1.8)        -28.8 (0.7)

           Ictalurus          B Dorosoma
            furcatus          cepedianum
Fatty     blue catfish       gizzard shad
acid    ([per thousand])   ([per thousand])

12:0      -22.5 (0.9)        -27.4 (1.0)
14:0      -26.9 (0.6)        -25.5 (1.4)
16:1      -25.6 (0.7)        -27.4 (0.6)
16:0      -23.3 (0.3)        -25.7 (0.6)
18:1      -24.5 (0.7)        -28.7 (0.4)
18:0      -20.4 (1.1)           -23.5