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Carbon and nitrogen stable isotopes of meiobenthos and their food resources
Estuarine, Coastal and Shelf Science (1989) 28,433-441
Carbon and Nitrogen Stable Isotopes of Meiobenthos and their Food Resources’
Carol
A. Couchb
Department of Biology and Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, U.S.A. Received 1 August I988 and in revised form 10 November 1988
meiobenthos; isotope ratios; assimilation; carbon; nitrogen; detritus; Spartina; micro-algae
Keywords:
Carbon and nitrogen stable isotope ratios were used to measure the in situ assimilation of Spartina alterni’ora and benthic micro-algae by harpacticoid copepods and nematodes. These meiofauna are important food resources for higher trophic levelsin estuarine food webs, but little has been known about their natural diets. Meiofauna, detrital Spartina, benthic micro-algae and sediment exhibited no differences between mean seasonalcarbon isotope ratios. The PC of populations of harpacticoids and nematodes are similar and indicate the use of similar foods throughout the year. The 615Nof detrital Spartina and micro-algae were not distinct and did not allow the identification of the source of nitrogen assimilated by the meiofauna. Populations of meiofauna assimilate a mixture of food resources, however, detrital Spartina may be the predominant source of carbon with micro-algae contributing some portion of the assimilated carbon and nitrogen.
Introduction Meiofauna, particularly harpacticoid copepods and nematodes are important food resources for higher trophic levels in estuarine food webs (Coull & Bell, 1979; Smith & Coull, 1987) but little is known about the extent to which Spartina alterniflora detritus and micro-algae contribute to the diet of estuarine meiofauna. Of total annual net primary production in the North Inlet Estuary of South Carolina, Spartinu contributes 66% and micro-algae 16% (Dame et al., 1986). Although micro-algal production is about onefourth of Spartinu production, micro-algal carbon may be more constantly available for consumption by meiofauna than Spurtinu carbon, which becomes available only after the senescence and microbial decomposition of the vascular plant (Gallagher & Daiber, 1974). In the present study, ratios of naturally occurring stable isotopes of carbon (‘%/12C) and nitrogen (15N/14N) of benthic micro-algae, and living and detrital Spartinu were used to examine the extent to which these resources are assimilated by meiofauna. Contribution Number 732 from the Belle W. Baruch Coastal Research, University of South Carolina. Tresent address: Institute of Ecology, University of U.S.A. 0272-7714/89/040433
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Harpacticoids and nematodes ingest micro-algae1 detritus and associated microbes (Hicks & Coull, 1983; Jensen, 1987) and species of both taxa can be selective in their ingestion of bacteria and micro-algae (Tietjen & Lee, 1973; Romeyn & Bouwman, 1983; Rieper, 1982; Carman & Thistle, 1985). The evidence regarding whether meiofaunal populations ingest more bacterial or algal carbon is conflicting. Meiofauna may ingest more bacterial than micro-algal carbon (Brown & Sibert, 1977; Montagna & Bauer, 1988), but diatoms were found to be preferentially ingested in one study of meiofaunal grazing (Montagna, 1984). In the only study attempting to resolve ingestion and assimilation, diatoms were ingested by a harpacticoid, however, only the epiphytic bacteria associated with the diatoms were assimilated while the diatoms were passed intact in the feces (Decho & Castenholz, 1986). Stable isotope studies including meiofauna are few (Spies & Des Marais, 1983; Gearing et al., 1984; Schwinghamer et al., 1983; Simenstad & Wissmar, 1985), and none have reported both stable carbon and nitrogen ratios. McConnaughey & McRoy (1979), in a stable isotope study of a Bering Sea food web, accounted for the length of the detritus based food chain by implicating meiofauna as intermediaries in the flow of carbon from detritus to benthic macrofauna. However, no isotopic date for meiofauna were presented. The use of stable isotopes in determining assimilation involves the comparison of stable isotope ratios between consumers and food resources. In comparing the isotopic ratios of consumers with suspected food resources, correction factors can be applied to compensate for isotopic fractionation occurring in consumer metabolism (Fry & Sherr, 1984). Carbon isotopes alone may be of limited value in interpreting the assimilation of salt marsh and estuarine food resources whose carbon ratios overlap (Fry & Sherr, 1984). Spartina and benthic micro-algae are reported to have distinct (Haines, 1976) as well as’overlapping (Schwinghamer et al., 1983) carbon isotope ratios. In this study, both carbon and nitrogen isotope ratios are used in attempting to resolve the assimilation of these two food resources by meiofauna.
Procedures Sample preparation
All samples were collected near Oyster Landing in the North Inlet Estuary, Georgetown, SC, USA (Lat. 33”19’N, Long. 79” 11.6’W). This site is located 4.4 km from the mouth of the inlet near the terminal end of meandering tidal creeks (Figure 1). To assessseasonal differences in stable isotope ratios, samples of meiofauna were collected in May 1986, October 1986, and March 1987. Samples of harpacticoids, nematodes, and benthic microalgae were extracted in the laboratory from intertidal mudflat sediments collected during low tide. Samples of live and standing dead Spartina were obtained from Spartina stands adjacent to the flats. Sediment samples were collected by scraping the upper 2 cm of the exposed mudflat surface into plastic buckets. This method collected the majority of meiofauna because in muddy sediment 94% of all meiofauna are located in the upper 1 cm (Coull & Bell, 1979). Benthic micro-algae were extracted from sediment which was collected by gently scraping the upper 1 mm in areas where dense, brown micro-algal mats were apparent. Care was taken to assure that samples of meiofauna and micro-algae were extracted free of contaminating debris or organisms. The extraction of harpacticoids relied on their movement from concentrated sediments into overlying seawater. The concentration of
Carbon
and nitrogen
Figure
1. Site of Oyster
stable
isotopes
Landing,
435
of meiobenthos
North
Inlet
Estuary,
South
Carolina.
the sediment was achieved by sieving as described in Couch (1988). Concentrated sediment was placed in clear plastic pans to a depth of 2 cm and covered with 4 cm of filtered seawater. The sediment was allowed to settle, and a fiber optic light was focused on the surface in a corner. Within minutes, harpacticoids were attracted to the light and swam into the water column, gathering in dense swarms. Harpacticoids were removed from the seawater with a pipette over a period of 8 h. The harpacticoids were finally sorted under a dissecting microscope and isolated to remove contaminating turbellarians which were also attracted to the light. To clear gut contents and minimize reingestion of feces, the harpacticoids were held for at least 12 h in filtered seawater and rinsed twice through 63 urn mesh nylon screen to remove fecal pellets. They were then filtered onto glass fiber filters, quickly rinsed with distilled water to remove salts, and frozen. The binderless glass fiber filters had been previously cornbusted for 24 h at 480°C to remove any contaminating organic carbon. Nematodes were extracted from sediment using the technique described in Couch (1988). Briefly, the extraction was accomplished by the downward migration of living nematodes from concentrated sediment contained in a funnel through combusted dune sand and into clean, filtered seawater. Once extracted, the nematodes were treated similarly to the harpacticoids. Prior to isotopic analysis all filters with adhering harpacticoids or nematodes were lyophilized and inspected under a dissecting microscope to remove contaminants.
436
C. A. Couch
Within 1 h of collection, sediment collected for extraction of micro-algae was spread to a depth of 1 cm in plastic pans. A nylon screen of 63 urn mesh was placed upon the sediment surface and covered with a 2 mm layer of combusted dune sand. The sand surface was thoroughly wetted with filtered seawater and covered with a second 63 urn mesh nylon screen. The pans were held under fluorescent lights in the laboratory, and their surfaces kept wet with periodic sprays of filtered seawater. The motile micro-algae migrated onto the top nylon screen through the sand layer which acted to retain debris adhering to mucus produced by the micro-algae. The top nylon screen was periodically removed and rinsed with filtered seawater to collect the motile micro-algae which were then filtered onto glass fiber filters, rinsed with distilled water and frozen. Prior to isotopic analysis, the samples were lyophilized and picked clean of any contaminants. Samples for analysis of sediment isotopic ratios were collected from the upper 2 cm of the same mudlIats from which meiofauna were collected. These samples were placed in sterile plastic bags and frozen. Frozen sediment samples were thawed overnight in a refrigerator. Portions of the thawed sediment were sieved to provide subsamples of sediment in size fractions of 63 urn to 125 urn, and 125 m to 500 urn. Spar&a detritus retained on a 500 urn sieve was removed with acid washed forceps under a dissecting microscope. Sediment and detritus samples were lyophilized, homogenized by grinding with a mortar and pestle, and stored in a dessicator until isotopic analysis. Isotopic
analysis
The purification of CO, gas and mass spectrometry was performed at the Stable Isotope Laboratory, Department of Geological Sciences, University of South Carolina. Prior to combustion, samples of detritus, Spartina, and sediments were fumed overnight with concentrated HCl to remove contaminating inorganic carbon. The conversion of organic carbon to CO, was accomplished following the method of Buchanan and Corcoran (1959) with modifications by Mucciarone (1983). The resulting CO, gas was cryogenically separated from the combustion products and the 13C/12C ratio was measured using a VG Isogas SIRA 24 isotope ratio mass-spectrometer. The purification of N, gas and measurement of 15N/14N ratios was performed by Geochron Laboratories, Cambridge, Mass., on a VG Isogas 903 isotope ratio mass-spectrometer. Stable isotope ratios are reported in conventional delta (6) notation in units ml-’ (%,): 6X={%@&
-ljx
1000
where X= 13C or 15N, and R = i3C/“C or 15N/14N. All carbon samples were compared directly to a laboratory CO, reference gas (SCB-2), and the data are reported relative to the PDB standard following the procedure of Craig (1957). All nitrogen samples were compared to standard atmospheric N,. The reproducibility of the measurements are estimated to be f 0.22% for 613C and + 0.20%0 for 615N. Results and discussion Results of 613Cand 615N analyses are presented in Table 1. 615N of sediment includes both organic and inorganic forms of sedimentary nitrogen. The 6r3C of sediment is of organic carbon only. Total sedimentary organic matter (SOM) had a seasonal mean i3i3C of - 19.54%. There was no significant difference in 613C of whole sediment (one-way ANOVA, P=O.O61) or of the 125 urn to 500 urn size fractionated subsample (one-way ANOVA, P=O.629). The seasonal mean 613C of detrital Spartina was - 16.31960.
Carbon and nitrogen stableisotopesof meiobenthos
437
Samples of harpacticoids, nematodes, and micro-algae were composed of multispecies mixtures. No attempt was made to identify species of nematodes or micro-algae. Spring and summer samples of harpacticoids were dominated by Nannopus palustris, and by Microarthridion littorale, respectively, while fall samples were composed of a broad mixture of species. The seasonal mean 613C of micro-algae was - 12*76%0, with no significant difference between seasons (one-way ANOVA, P= 0.107). Harpacticoids and nematodes had similar seasonal mean 6i3C values of - 14.39%0 and - 1489%0, respectively. Although nematodes had greater seasonal variability than harpacticoids, neither taxon exhibited significant seasonal differences in 613C (one-way ANOVA, harpacticoids P = 0.861, nematodes P = 0.275). The species composition of harpacticoid samples differed between seasons; however, the 613C of these samples did not. With the exception of live Spartina, all 613C values determined in this study are close to those found in previous stable isotope studies of salt marsh sediment, detritus and primary producers (Haines, 1976; Hackney & Haines, 1980; Hughes & Sherr, 1983; Schwinghamer et al., 1983; Ember et al., 1987). There are few published measurements of 615N for salt marsh organisms. Mariotti et al. (1983) reported 6i5N values of 6.2 f 0.7%0 and 3.9%0 for live Spartina and for benthic algae, respectively. During decomposition, the food quality of detrital Spartina improves due to its transformation by microbes and to a decrease in the C:N ratio resulting from nitrogen accumulating in microbial biomass or as adsorbed nitrogenous compounds (Rice & Tenore, 1982; Tenore et al., 1984). The flow of nutrients from detritus to meiofauna is mediated by meiofaunal assimilation of detritus-associated microbial biomass (Hicks & Coull, 1983). Because carbon stable isotope ratios of heterotrophic microbes resemble their detrital substrates (Fry & Sherr, 1984), the #‘C reported here for detrital Spartina is assumed to represent the #‘C of the associated microbes upon which the meiofauna may feed. The negative shift in 6i3C between live and detrital Spartina is attributed to the conservation of refractory components such as lignin ( - 16.34%0) and the rapid loss of more positive labile components, rather than to the proliferation of microbes whose carbon contributes to the measured ratios (Schwinghamer et al., 1983; Ember et al., 1987). The 615N measured for detrital Spartina probably results largely from nitrogen sequestered by microbes from the environment and from adsorbed nitrogenous compounds, rather than from the macrophyte substrate itself. As a dietary source of nitrogen, detrital Spartina improves with age, however, this nitrogen originates largely from sources other than the plant. To interpret the results of this study, the fractionation of isotopic ratios during consumer metabolism must be considered. During metabolism, 13C/12Cand 15N/14N ratios of assimilated food are fractionated by the selective retention of the heavier isotope and the respiration or excretion of the lighter isotope. When fed a single food of constant isotopic composition, the mean ( f 1 SD) difference between the isotopic ratios of consumers and their diet was found to be O.S& 1.1% for F13C and by 3*0f2*6%0 for 615N (DeNiro & Epstein, 1978, 1981). In food web studies, meiofauna have been found to be up to 2.2%0 more positive, or enriched in 13C, relative to than their suspected food resources (Spies & Des Marais, 1983; Gearing et al., 1984). These enrichments are consistent with the general pattern of 13C enrichment found between trophic levels in food chains (Fry & Sherr, 1984). The following discussion assumes that meiofauna fractionate carbon and nitrogen isotopes by 0.8 f 1.1% and 3.0 + 2+6%0,respectively, as determined by DeNiro and Epstein (1978, 1981). This assumption is reasonable and conservative because these ranges encompass most documented values of metabolic fractionation (Macko et al., 1982;
438
C. A. Couch
TABLE 1. PC and PN data for samples of meiofauna, resources collected at Oyster Landing. n = 2 for sample with asterisk
sediment and potential food means, except n = 3 for means
6°C
Sample
May 1986 (XkSD)
October 1986 (X+ SD)
March (X+
- 14.49 (0.75) - 14.97 (1.67) -
- 14.24 (0.34) - 15.83 (0.19) - 12.11 (0.68)
- 14.42 (0.21) -13.87(0,11) - 13.46 (0.07)
59,5.7” 5.3b 3.8”
-
- 15.94 (0.02)
- 14.27 (0.21) - 12.71 (0.01) - 16.68 (0.09)
4.3’ 29 2.8‘
-
- 18.89 (0,75)* -19.74 (1,43)*
-20.53 (0.05) -20.17 (0.41) - 19.82 (0.34)
4.3 -
type
Harpacticoids Nematodes Benthic micro-algae
1986 SD)
PN
Spartina: Live Standing dead Detritus Sediment: Total SOM 125 urn-500 urn 63 pm-125 urn “October 1986. bMixed samples ‘March 1987.
% E z
from
Micro-algae
-
Love Sportma
-
Copepods
-
Nematodes
-
Sportma Detritus
_
Sediment
-
Plankton-
October
1986 and March
1987.
n
xl-D-
l-l --U--I
-23
I
-21
L
I
-19
I
I
-17
I
I
-15
I
I,
-13
-I I
a13Carban
Figure 2. Mean 6’C ( & 1 SD) of meiofauna and potential food resources. All data except for plankton (data from Peterson et al., 1985) were measured in the present study.
Fry & Sherr, 1984). Using this assumption and the meiofaunal ratios reported in Table 1, if harpacticoids assimilated a single food resource, that resource would have isotopic values of - 15.19& 1’1 (S13C) and 2.8k2.6 (S15N). Likewise, a single food resource assimilated by nematodes would have isotopic values of - 15.69 f 1.1 (S13C)and 2.3 f 2.6 (CP’~N). Carbon isotope data alone (Figure 2) provide little resolution of the degree to which potential food resources are assimilated. The intermediate values of meiofauna could
439
Carbon and nitrogen stable isotopes of meiobenthos
6.0
H
F
0.0 c
-2.01
1
-22
1
1
-20
I
1
1
1
-I8
1
1 -16
1
’
1 -14
1
’ 2
a?arbon
Figure 3. PC and ?PN of potential food resources and meiofauna. P = plankton, SOM = sedimentary organic matter, M = micro-algae, H = harpacticoids, N = nematodes. X = 6% and PN of harpacticoids minus 0.8 and 3.0, respectively, to correct for metabolic fractionation. Boxed area () denotes the standard deviations of the metabolic fractionations, + 1.1 for 6% and k2.6 for PN. The same adjustments have been applied to derive Y from N. Boxed area (------) denotesstandard deviationsof metabolic fractionations of PC and P5N by nematodes. X and Y are isotopic values for hypothetical food resources which could solely constitute the diet of the meiofauna.
result either from the assimilation of a mixture of phytoplankton, benthic micro-algae, and Spar&a detritus, or from the assimilation of only Spartina detritus. In the North Inlet, phytoplankton production contributes less than 376 of total annual net primary production (Dame et al., 1986). Consequently, the contribution of phytoplankton to a mixed diet may be very limited. Due to its heterogenous composition, SOM cannot be considered as a single food resource, and it is not possible to determine its specific contribution to a mixed diet. Figure 3 exhibits both carbon and nitrogen isotope data. In Figure 3, arrows connect the measured isotopic values of meiofauna to hypothetical points (X,Y) produced by subtracting 0.8 + 1.1 and by 3.0 + 2.6 for 613C and 615N, respectively, to correct for metabolic fractionation. The areas surrounding points X and Y enclose the values which may be assumed by a single food resource if it alone constituted the diet of meiofauna. From these results, if meiofauna assimilate both carbon and nitrogen from a single food resource, only detrital Spurtinu could be considered as that resource. However, detritus-associated microbes may not be the source of both carbon and nitrogen in detritivore nutrition (Findlay & Tenore, 1982). The meiofaunal isotopic values could result from the assimilation of detrital Spurtinu carbon and nitrogen from detritus or micro-algae. The measured nitrogen isotopic values are not sufficiently distinct to allow for a unique identification of the source of dietary nitrogen. As suggested by Fry and Sherr (1984), the usefulness of 615N as a food web tracer in estuaries is limited by the small differences in 615N found among potential food resources, However, carbon isotope data do preclude micro-algae as a sole meiofaunal food resource. During no season does the 613C of meiofauna assume a value that indicates the predominant assimilation of micro-algal carbon.
440
C. A. Couch
Detrital Spartina is not likely to be the only food resource assimilated by meiofaunal populations. Some harpacticoids thrive in culture on micro-algae exclusively (Sellner, 1976), and some nematodes are morphologically adapted for feeding on diatoms (Jensen, 1987). However, given the close correspondence between meiofaunal and detrital Spartina values, and the low production of phytoplankton in the North Inlet, the bulk of carbon assimilated by meiofaunal populations may be derived from Spurtinu detritus. This conclusion is supported by studies showing the preferential ingestion or assimilation of bacterial over algal carbon by meiofauna (Brown & Sibert, 1977; Decho & Castenholz, 1986; Montagna & Bauer, 1988). Perhaps the assimilation of micro-algal carbon and nitrogen is important during autumn and winter when fresh Spurtinu detritus may be low in associated microbial biomass. Acknowledgements The author acknowledges the contributions of a number of individuals whose assistance was essential to the completion of this project: Andy Barnard, Lauren Billheimer, Maria Ellis, Bob Ferguson, Brian Hentschel, Amy Nelson and Debbie Salamy. I am grateful for the support and advice of Bruce Coull, Bettye Dudley, Leon Ember, Bob Feller, and Doug Williams. This research was supported by NSF Grant OCE8521345 to B. C. Coull and R. J. Feller, and by funds from the Stable Isotope Laboratory, Department of Geological Sciences, University of South Carolina. References Brown, T. J. & Sibert, J. R. 1977 Food of some harpacticoid copepods. Journal of the Fisheries Research Board of Canada 34,1028-1031. Buchanan, D. & Corcoran, B. 1959 Sealed tube combustions for the determination of 6°C and total carbon. Analytical Chemistry 31, 16351638. Carman, K. R. &Thistle, D. 1985 Microbial food partitioning by three species of benthic copepods. Marine Biology 88,143-148. Coull, B. C. &Bell, S. S. 1979 Perspectives of marine meiofaunal ecology. In: Ecological Processes in Coasral and Marine Systems (Livingston, R. J., ed.). New York: Plenum Press, pp. 189-216. Couch, C. A. 1988 A procedure for extracting large numbers of debris-free, living nematodes from muddy marine sediments. Transactions of the American Microscopical Society 107,96-100. Craig, H. 1957 Isotopic standards for carbon and oxygen correction factors for mass-spectrometric analysis of carbon dioxide. Geochimica et Cosmochimica Acta 12,133-149. Dame, R., Chrxanowski, T., Bildstein, K., Kjerfve, B., McKellar, H., Nelson, D., Spurrier, J., Stancyk, S., Stevenson, H.,Vemberg, J. & Zingmark, R. 1986 The outwelling hypothesis and North Inlet, South Carolina. Marine Ecology Progress Series 33,217-229. Decho, A. W. & Castenholz, R. W. 1986 Spatial patterns and feeding of meiobenthic harpacticoid copepods in relation to microbial flora. Hydrobiologia 131,217-229. DeNiro, M. J. & Epstein, S. 1978 Influence of diet on the distribution of carbon isotopes in animals. Geochimica et Cosmochimica Acta 42,495-506. DeNiro, M. J. & Epstein, S. 1981 Inlluence of diet on the distribution of nitrogen isotopes in animals. Geochimica et Cosmochimica Acta 45,341-351. Ember, L. M., Williams, D. F. &Morris, J. T. 1987 Processes that influence carbon isotope variations in salt marsh sediments. Marine Ecology Progress Series 36,33-42. Findlay, S. & Tenore, K. 1982 Nitrogen source for a detritivore: detritus substrate versus associated microbes. Science 218,371-372. Fry, B. & Sherr, E. B. 1984 6i3C measurements as indicators of carbon flow in marine and freshwater ecosystems. Contributions in Marine Science 27, 13-47. Gallagher, J. L. & Daiber, F. C. 1974 Primary production of edaphic algal communities in a Delaware salt marsh. Limnology and Oceanography 19,390-395. Gearing, J. N., Gearing, P. J., Rudnick, D. T., Requejo, A. G. & Hutchins, M. J. 1984 Isotopic variability of organic carbon in a phytoplankton-based temperate estuary. Geochimica et Cosmochimica Acta 48, 1089-1098.
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and nitrogen
stable
isotopes
of meiobenthos
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Hackney, C. T. & Haines, E. B. 1980 Stable carbon isotope composition of fauna and organic matter collected in a Mississippi estuary. Estuarine and Coastal Marine Science 10,703-708. Haines, E. B. 1976 Stable carbon isotope ratios in biota, soils, and tidal water of a Georgia salt marsh. Estuarine and Coastal Marine Science 4,609-616. Hicks, G. R. F. & Coull, B. C. 1983 The ecology of marine meiobenthic harpacticoid copepods. Oceanography and Marine Biology Annual Review 21,67-175. Hughes, E. H. & Sherr, E. B. 1983 Subtidal food webs in a Georgia estuary: 6°C analysis. rournal of Experimental Marine Biology and Ecology 67,227-242. Jensen, P. 1987 Feeding ecology of free-living aquatic nematodes. Marine EcoZogy Progress Series 35, 187-196. Macko, S. A., Lee, W. Y. & Parker, P. L. 1982 Nitrogen and carbon isotope fractionation by two species of marine amphipods: laboratory and field studies. Journal of Experimental Marine Biology and Ecology 63, 145-149. Mariotti, A., Letolle, R. & Sherr, E. 1983 Distribution of stable isotopes in a salt marsh estuary. Esruaries 6, 304-305. McConnaughy, T. & McRoy, C. P. 1979 Food web structure and the fractionation of carbon isotopes in the Bering Sea. Marine Biology 53,257-262. Montagna, P. A. 1984 In situ measurement of meiobenthic grazing rates on sediment bacteria and edaphic diatoms. Marine Ecology Progress Series l&119-130. Montagna, P. A. & Bauer, J. E. 1988 Partitioning radiolabeled thymidine uptake by bacteria and meiofauna using metabolic blocks and poisons in benthic feeding studies. Marine Biology 98, 101-l 10. Mucciarone, D. 1983 Stable Isotope Laboratory Operations Manual. University of South Carolina, Columbia. Peterson, B. J., Howarth, R. W. & Garitt, R. H. 1985 Multiple stable isotopes used to trace the flow of organic matter in estuarine food webs. Science 227,1361-1363. Rice, D. L. & Tenore, K. R. 1981 Dynamics of carbon and nitrogen during decomposition of detritus derived from estuarine macrophytes. Estuarine Coastal and Shelf Science 13,681-690. Rieper, M. 1982 Feeding preferences of marine harpacticoid copepods for various species of bacteria. Marine Ecology Progress Series 7,303-307. Romeyn, K. & Bouwman, L. A. 1983 Food selection and consumption by estuarine nematodes. Hydrobiological Bulletin 17, 103-109. Schwinghamer, P. F., Tan, F. C. & Gordan, D. C., Jr. 1983 Stable carbon isotope studies in Pecks Cove mudflat ecosystem in the Cumberland Basin, Bay of Fundy. CanadianJournal of Fisheries and Aquatic Science 40 (Supplement 1), 262-272. Sellner, B. W. 1976 Survival and metabolism of the harpacticoid copepod Thomsonula hyaenae fed on different diatoms. Hydrobiologica 50,223-238. Simenstad, C. A. & Wissmar, R. C. 1985 6°C evidence of origins and fates of organic carbon in estuarine and nearshore food webs. Marine Ecology Progress Series22,141-152. Smith, L. D. & Coull, B. C. 1987 Juvenile spot (Pisces) and grass shrimp predation on meiobenthos in muddy and sandy substrata. Journal of Experimental Marine Biology and Ecology 105, 123-136. Spies, R. B. & Des Marais, D. J. 1983 Natural isotope study of trophic enrichment of marine benthic communities by petroleum seepage. Marine Biology 73,67-71. Tenore, K. R., Hanson, R. B., McClain, J., Maccubbin, A. E. & Hodsen, R. E. 1984 Changes in composition and nutritional value to a benthic deposit feeder of decomposing detritus pools. Bulletin of Marine Science 35,299-311. Tietjen, J. H. & Lee, J. J. 1973 Life history and feeding habits of the marine nematode Chromadora macrolaimoides Steiner. Oecologia (Berlin) 12,303-314.
Carbon and Nitrogen Stable Isotopes of Meiobenthos and their Food Resources’
Carol
A. Couchb
Department of Biology and Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, U.S.A. Received 1 August I988 and in revised form 10 November 1988
meiobenthos; isotope ratios; assimilation; carbon; nitrogen; detritus; Spartina; micro-algae
Keywords:
Carbon and nitrogen stable isotope ratios were used to measure the in situ assimilation of Spartina alterni’ora and benthic micro-algae by harpacticoid copepods and nematodes. These meiofauna are important food resources for higher trophic levelsin estuarine food webs, but little has been known about their natural diets. Meiofauna, detrital Spartina, benthic micro-algae and sediment exhibited no differences between mean seasonalcarbon isotope ratios. The PC of populations of harpacticoids and nematodes are similar and indicate the use of similar foods throughout the year. The 615Nof detrital Spartina and micro-algae were not distinct and did not allow the identification of the source of nitrogen assimilated by the meiofauna. Populations of meiofauna assimilate a mixture of food resources, however, detrital Spartina may be the predominant source of carbon with micro-algae contributing some portion of the assimilated carbon and nitrogen.
Introduction Meiofauna, particularly harpacticoid copepods and nematodes are important food resources for higher trophic levels in estuarine food webs (Coull & Bell, 1979; Smith & Coull, 1987) but little is known about the extent to which Spartina alterniflora detritus and micro-algae contribute to the diet of estuarine meiofauna. Of total annual net primary production in the North Inlet Estuary of South Carolina, Spartinu contributes 66% and micro-algae 16% (Dame et al., 1986). Although micro-algal production is about onefourth of Spartinu production, micro-algal carbon may be more constantly available for consumption by meiofauna than Spurtinu carbon, which becomes available only after the senescence and microbial decomposition of the vascular plant (Gallagher & Daiber, 1974). In the present study, ratios of naturally occurring stable isotopes of carbon (‘%/12C) and nitrogen (15N/14N) of benthic micro-algae, and living and detrital Spartinu were used to examine the extent to which these resources are assimilated by meiofauna. Contribution Number 732 from the Belle W. Baruch Coastal Research, University of South Carolina. Tresent address: Institute of Ecology, University of U.S.A. 0272-7714/89/040433
+09
$03.00/O
Institute
for Marine
Georgia,Athens, @ 1989 Academic
Biology
Georgia,
and 30602,
Press Limited
434
c. A. Couch
Harpacticoids and nematodes ingest micro-algae1 detritus and associated microbes (Hicks & Coull, 1983; Jensen, 1987) and species of both taxa can be selective in their ingestion of bacteria and micro-algae (Tietjen & Lee, 1973; Romeyn & Bouwman, 1983; Rieper, 1982; Carman & Thistle, 1985). The evidence regarding whether meiofaunal populations ingest more bacterial or algal carbon is conflicting. Meiofauna may ingest more bacterial than micro-algal carbon (Brown & Sibert, 1977; Montagna & Bauer, 1988), but diatoms were found to be preferentially ingested in one study of meiofaunal grazing (Montagna, 1984). In the only study attempting to resolve ingestion and assimilation, diatoms were ingested by a harpacticoid, however, only the epiphytic bacteria associated with the diatoms were assimilated while the diatoms were passed intact in the feces (Decho & Castenholz, 1986). Stable isotope studies including meiofauna are few (Spies & Des Marais, 1983; Gearing et al., 1984; Schwinghamer et al., 1983; Simenstad & Wissmar, 1985), and none have reported both stable carbon and nitrogen ratios. McConnaughey & McRoy (1979), in a stable isotope study of a Bering Sea food web, accounted for the length of the detritus based food chain by implicating meiofauna as intermediaries in the flow of carbon from detritus to benthic macrofauna. However, no isotopic date for meiofauna were presented. The use of stable isotopes in determining assimilation involves the comparison of stable isotope ratios between consumers and food resources. In comparing the isotopic ratios of consumers with suspected food resources, correction factors can be applied to compensate for isotopic fractionation occurring in consumer metabolism (Fry & Sherr, 1984). Carbon isotopes alone may be of limited value in interpreting the assimilation of salt marsh and estuarine food resources whose carbon ratios overlap (Fry & Sherr, 1984). Spartina and benthic micro-algae are reported to have distinct (Haines, 1976) as well as’overlapping (Schwinghamer et al., 1983) carbon isotope ratios. In this study, both carbon and nitrogen isotope ratios are used in attempting to resolve the assimilation of these two food resources by meiofauna.
Procedures Sample preparation
All samples were collected near Oyster Landing in the North Inlet Estuary, Georgetown, SC, USA (Lat. 33”19’N, Long. 79” 11.6’W). This site is located 4.4 km from the mouth of the inlet near the terminal end of meandering tidal creeks (Figure 1). To assessseasonal differences in stable isotope ratios, samples of meiofauna were collected in May 1986, October 1986, and March 1987. Samples of harpacticoids, nematodes, and benthic microalgae were extracted in the laboratory from intertidal mudflat sediments collected during low tide. Samples of live and standing dead Spartina were obtained from Spartina stands adjacent to the flats. Sediment samples were collected by scraping the upper 2 cm of the exposed mudflat surface into plastic buckets. This method collected the majority of meiofauna because in muddy sediment 94% of all meiofauna are located in the upper 1 cm (Coull & Bell, 1979). Benthic micro-algae were extracted from sediment which was collected by gently scraping the upper 1 mm in areas where dense, brown micro-algal mats were apparent. Care was taken to assure that samples of meiofauna and micro-algae were extracted free of contaminating debris or organisms. The extraction of harpacticoids relied on their movement from concentrated sediments into overlying seawater. The concentration of
Carbon
and nitrogen
Figure
1. Site of Oyster
stable
isotopes
Landing,
435
of meiobenthos
North
Inlet
Estuary,
South
Carolina.
the sediment was achieved by sieving as described in Couch (1988). Concentrated sediment was placed in clear plastic pans to a depth of 2 cm and covered with 4 cm of filtered seawater. The sediment was allowed to settle, and a fiber optic light was focused on the surface in a corner. Within minutes, harpacticoids were attracted to the light and swam into the water column, gathering in dense swarms. Harpacticoids were removed from the seawater with a pipette over a period of 8 h. The harpacticoids were finally sorted under a dissecting microscope and isolated to remove contaminating turbellarians which were also attracted to the light. To clear gut contents and minimize reingestion of feces, the harpacticoids were held for at least 12 h in filtered seawater and rinsed twice through 63 urn mesh nylon screen to remove fecal pellets. They were then filtered onto glass fiber filters, quickly rinsed with distilled water to remove salts, and frozen. The binderless glass fiber filters had been previously cornbusted for 24 h at 480°C to remove any contaminating organic carbon. Nematodes were extracted from sediment using the technique described in Couch (1988). Briefly, the extraction was accomplished by the downward migration of living nematodes from concentrated sediment contained in a funnel through combusted dune sand and into clean, filtered seawater. Once extracted, the nematodes were treated similarly to the harpacticoids. Prior to isotopic analysis all filters with adhering harpacticoids or nematodes were lyophilized and inspected under a dissecting microscope to remove contaminants.
436
C. A. Couch
Within 1 h of collection, sediment collected for extraction of micro-algae was spread to a depth of 1 cm in plastic pans. A nylon screen of 63 urn mesh was placed upon the sediment surface and covered with a 2 mm layer of combusted dune sand. The sand surface was thoroughly wetted with filtered seawater and covered with a second 63 urn mesh nylon screen. The pans were held under fluorescent lights in the laboratory, and their surfaces kept wet with periodic sprays of filtered seawater. The motile micro-algae migrated onto the top nylon screen through the sand layer which acted to retain debris adhering to mucus produced by the micro-algae. The top nylon screen was periodically removed and rinsed with filtered seawater to collect the motile micro-algae which were then filtered onto glass fiber filters, rinsed with distilled water and frozen. Prior to isotopic analysis, the samples were lyophilized and picked clean of any contaminants. Samples for analysis of sediment isotopic ratios were collected from the upper 2 cm of the same mudlIats from which meiofauna were collected. These samples were placed in sterile plastic bags and frozen. Frozen sediment samples were thawed overnight in a refrigerator. Portions of the thawed sediment were sieved to provide subsamples of sediment in size fractions of 63 urn to 125 urn, and 125 m to 500 urn. Spar&a detritus retained on a 500 urn sieve was removed with acid washed forceps under a dissecting microscope. Sediment and detritus samples were lyophilized, homogenized by grinding with a mortar and pestle, and stored in a dessicator until isotopic analysis. Isotopic
analysis
The purification of CO, gas and mass spectrometry was performed at the Stable Isotope Laboratory, Department of Geological Sciences, University of South Carolina. Prior to combustion, samples of detritus, Spartina, and sediments were fumed overnight with concentrated HCl to remove contaminating inorganic carbon. The conversion of organic carbon to CO, was accomplished following the method of Buchanan and Corcoran (1959) with modifications by Mucciarone (1983). The resulting CO, gas was cryogenically separated from the combustion products and the 13C/12C ratio was measured using a VG Isogas SIRA 24 isotope ratio mass-spectrometer. The purification of N, gas and measurement of 15N/14N ratios was performed by Geochron Laboratories, Cambridge, Mass., on a VG Isogas 903 isotope ratio mass-spectrometer. Stable isotope ratios are reported in conventional delta (6) notation in units ml-’ (%,): 6X={%@&
-ljx
1000
where X= 13C or 15N, and R = i3C/“C or 15N/14N. All carbon samples were compared directly to a laboratory CO, reference gas (SCB-2), and the data are reported relative to the PDB standard following the procedure of Craig (1957). All nitrogen samples were compared to standard atmospheric N,. The reproducibility of the measurements are estimated to be f 0.22% for 613C and + 0.20%0 for 615N. Results and discussion Results of 613Cand 615N analyses are presented in Table 1. 615N of sediment includes both organic and inorganic forms of sedimentary nitrogen. The 6r3C of sediment is of organic carbon only. Total sedimentary organic matter (SOM) had a seasonal mean i3i3C of - 19.54%. There was no significant difference in 613C of whole sediment (one-way ANOVA, P=O.O61) or of the 125 urn to 500 urn size fractionated subsample (one-way ANOVA, P=O.629). The seasonal mean 613C of detrital Spartina was - 16.31960.
Carbon and nitrogen stableisotopesof meiobenthos
437
Samples of harpacticoids, nematodes, and micro-algae were composed of multispecies mixtures. No attempt was made to identify species of nematodes or micro-algae. Spring and summer samples of harpacticoids were dominated by Nannopus palustris, and by Microarthridion littorale, respectively, while fall samples were composed of a broad mixture of species. The seasonal mean 613C of micro-algae was - 12*76%0, with no significant difference between seasons (one-way ANOVA, P= 0.107). Harpacticoids and nematodes had similar seasonal mean 6i3C values of - 14.39%0 and - 1489%0, respectively. Although nematodes had greater seasonal variability than harpacticoids, neither taxon exhibited significant seasonal differences in 613C (one-way ANOVA, harpacticoids P = 0.861, nematodes P = 0.275). The species composition of harpacticoid samples differed between seasons; however, the 613C of these samples did not. With the exception of live Spartina, all 613C values determined in this study are close to those found in previous stable isotope studies of salt marsh sediment, detritus and primary producers (Haines, 1976; Hackney & Haines, 1980; Hughes & Sherr, 1983; Schwinghamer et al., 1983; Ember et al., 1987). There are few published measurements of 615N for salt marsh organisms. Mariotti et al. (1983) reported 6i5N values of 6.2 f 0.7%0 and 3.9%0 for live Spartina and for benthic algae, respectively. During decomposition, the food quality of detrital Spartina improves due to its transformation by microbes and to a decrease in the C:N ratio resulting from nitrogen accumulating in microbial biomass or as adsorbed nitrogenous compounds (Rice & Tenore, 1982; Tenore et al., 1984). The flow of nutrients from detritus to meiofauna is mediated by meiofaunal assimilation of detritus-associated microbial biomass (Hicks & Coull, 1983). Because carbon stable isotope ratios of heterotrophic microbes resemble their detrital substrates (Fry & Sherr, 1984), the #‘C reported here for detrital Spartina is assumed to represent the #‘C of the associated microbes upon which the meiofauna may feed. The negative shift in 6i3C between live and detrital Spartina is attributed to the conservation of refractory components such as lignin ( - 16.34%0) and the rapid loss of more positive labile components, rather than to the proliferation of microbes whose carbon contributes to the measured ratios (Schwinghamer et al., 1983; Ember et al., 1987). The 615N measured for detrital Spartina probably results largely from nitrogen sequestered by microbes from the environment and from adsorbed nitrogenous compounds, rather than from the macrophyte substrate itself. As a dietary source of nitrogen, detrital Spartina improves with age, however, this nitrogen originates largely from sources other than the plant. To interpret the results of this study, the fractionation of isotopic ratios during consumer metabolism must be considered. During metabolism, 13C/12Cand 15N/14N ratios of assimilated food are fractionated by the selective retention of the heavier isotope and the respiration or excretion of the lighter isotope. When fed a single food of constant isotopic composition, the mean ( f 1 SD) difference between the isotopic ratios of consumers and their diet was found to be O.S& 1.1% for F13C and by 3*0f2*6%0 for 615N (DeNiro & Epstein, 1978, 1981). In food web studies, meiofauna have been found to be up to 2.2%0 more positive, or enriched in 13C, relative to than their suspected food resources (Spies & Des Marais, 1983; Gearing et al., 1984). These enrichments are consistent with the general pattern of 13C enrichment found between trophic levels in food chains (Fry & Sherr, 1984). The following discussion assumes that meiofauna fractionate carbon and nitrogen isotopes by 0.8 f 1.1% and 3.0 + 2+6%0,respectively, as determined by DeNiro and Epstein (1978, 1981). This assumption is reasonable and conservative because these ranges encompass most documented values of metabolic fractionation (Macko et al., 1982;
438
C. A. Couch
TABLE 1. PC and PN data for samples of meiofauna, resources collected at Oyster Landing. n = 2 for sample with asterisk
sediment and potential food means, except n = 3 for means
6°C
Sample
May 1986 (XkSD)
October 1986 (X+ SD)
March (X+
- 14.49 (0.75) - 14.97 (1.67) -
- 14.24 (0.34) - 15.83 (0.19) - 12.11 (0.68)
- 14.42 (0.21) -13.87(0,11) - 13.46 (0.07)
59,5.7” 5.3b 3.8”
-
- 15.94 (0.02)
- 14.27 (0.21) - 12.71 (0.01) - 16.68 (0.09)
4.3’ 29 2.8‘
-
- 18.89 (0,75)* -19.74 (1,43)*
-20.53 (0.05) -20.17 (0.41) - 19.82 (0.34)
4.3 -
type
Harpacticoids Nematodes Benthic micro-algae
1986 SD)
PN
Spartina: Live Standing dead Detritus Sediment: Total SOM 125 urn-500 urn 63 pm-125 urn “October 1986. bMixed samples ‘March 1987.
% E z
from
Micro-algae
-
Love Sportma
-
Copepods
-
Nematodes
-
Sportma Detritus
_
Sediment
-
Plankton-
October
1986 and March
1987.
n
xl-D-
l-l --U--I
-23
I
-21
L
I
-19
I
I
-17
I
I
-15
I
I,
-13
-I I
a13Carban
Figure 2. Mean 6’C ( & 1 SD) of meiofauna and potential food resources. All data except for plankton (data from Peterson et al., 1985) were measured in the present study.
Fry & Sherr, 1984). Using this assumption and the meiofaunal ratios reported in Table 1, if harpacticoids assimilated a single food resource, that resource would have isotopic values of - 15.19& 1’1 (S13C) and 2.8k2.6 (S15N). Likewise, a single food resource assimilated by nematodes would have isotopic values of - 15.69 f 1.1 (S13C)and 2.3 f 2.6 (CP’~N). Carbon isotope data alone (Figure 2) provide little resolution of the degree to which potential food resources are assimilated. The intermediate values of meiofauna could
439
Carbon and nitrogen stable isotopes of meiobenthos
6.0
H
F
0.0 c
-2.01
1
-22
1
1
-20
I
1
1
1
-I8
1
1 -16
1
’
1 -14
1
’ 2
a?arbon
Figure 3. PC and ?PN of potential food resources and meiofauna. P = plankton, SOM = sedimentary organic matter, M = micro-algae, H = harpacticoids, N = nematodes. X = 6% and PN of harpacticoids minus 0.8 and 3.0, respectively, to correct for metabolic fractionation. Boxed area () denotes the standard deviations of the metabolic fractionations, + 1.1 for 6% and k2.6 for PN. The same adjustments have been applied to derive Y from N. Boxed area (------) denotesstandard deviationsof metabolic fractionations of PC and P5N by nematodes. X and Y are isotopic values for hypothetical food resources which could solely constitute the diet of the meiofauna.
result either from the assimilation of a mixture of phytoplankton, benthic micro-algae, and Spar&a detritus, or from the assimilation of only Spartina detritus. In the North Inlet, phytoplankton production contributes less than 376 of total annual net primary production (Dame et al., 1986). Consequently, the contribution of phytoplankton to a mixed diet may be very limited. Due to its heterogenous composition, SOM cannot be considered as a single food resource, and it is not possible to determine its specific contribution to a mixed diet. Figure 3 exhibits both carbon and nitrogen isotope data. In Figure 3, arrows connect the measured isotopic values of meiofauna to hypothetical points (X,Y) produced by subtracting 0.8 + 1.1 and by 3.0 + 2.6 for 613C and 615N, respectively, to correct for metabolic fractionation. The areas surrounding points X and Y enclose the values which may be assumed by a single food resource if it alone constituted the diet of meiofauna. From these results, if meiofauna assimilate both carbon and nitrogen from a single food resource, only detrital Spurtinu could be considered as that resource. However, detritus-associated microbes may not be the source of both carbon and nitrogen in detritivore nutrition (Findlay & Tenore, 1982). The meiofaunal isotopic values could result from the assimilation of detrital Spurtinu carbon and nitrogen from detritus or micro-algae. The measured nitrogen isotopic values are not sufficiently distinct to allow for a unique identification of the source of dietary nitrogen. As suggested by Fry and Sherr (1984), the usefulness of 615N as a food web tracer in estuaries is limited by the small differences in 615N found among potential food resources, However, carbon isotope data do preclude micro-algae as a sole meiofaunal food resource. During no season does the 613C of meiofauna assume a value that indicates the predominant assimilation of micro-algal carbon.
440
C. A. Couch
Detrital Spartina is not likely to be the only food resource assimilated by meiofaunal populations. Some harpacticoids thrive in culture on micro-algae exclusively (Sellner, 1976), and some nematodes are morphologically adapted for feeding on diatoms (Jensen, 1987). However, given the close correspondence between meiofaunal and detrital Spartina values, and the low production of phytoplankton in the North Inlet, the bulk of carbon assimilated by meiofaunal populations may be derived from Spurtinu detritus. This conclusion is supported by studies showing the preferential ingestion or assimilation of bacterial over algal carbon by meiofauna (Brown & Sibert, 1977; Decho & Castenholz, 1986; Montagna & Bauer, 1988). Perhaps the assimilation of micro-algal carbon and nitrogen is important during autumn and winter when fresh Spurtinu detritus may be low in associated microbial biomass. Acknowledgements The author acknowledges the contributions of a number of individuals whose assistance was essential to the completion of this project: Andy Barnard, Lauren Billheimer, Maria Ellis, Bob Ferguson, Brian Hentschel, Amy Nelson and Debbie Salamy. I am grateful for the support and advice of Bruce Coull, Bettye Dudley, Leon Ember, Bob Feller, and Doug Williams. This research was supported by NSF Grant OCE8521345 to B. C. Coull and R. J. Feller, and by funds from the Stable Isotope Laboratory, Department of Geological Sciences, University of South Carolina. References Brown, T. J. & Sibert, J. R. 1977 Food of some harpacticoid copepods. Journal of the Fisheries Research Board of Canada 34,1028-1031. Buchanan, D. & Corcoran, B. 1959 Sealed tube combustions for the determination of 6°C and total carbon. Analytical Chemistry 31, 16351638. Carman, K. R. &Thistle, D. 1985 Microbial food partitioning by three species of benthic copepods. Marine Biology 88,143-148. Coull, B. C. &Bell, S. S. 1979 Perspectives of marine meiofaunal ecology. In: Ecological Processes in Coasral and Marine Systems (Livingston, R. J., ed.). New York: Plenum Press, pp. 189-216. Couch, C. A. 1988 A procedure for extracting large numbers of debris-free, living nematodes from muddy marine sediments. Transactions of the American Microscopical Society 107,96-100. Craig, H. 1957 Isotopic standards for carbon and oxygen correction factors for mass-spectrometric analysis of carbon dioxide. Geochimica et Cosmochimica Acta 12,133-149. Dame, R., Chrxanowski, T., Bildstein, K., Kjerfve, B., McKellar, H., Nelson, D., Spurrier, J., Stancyk, S., Stevenson, H.,Vemberg, J. & Zingmark, R. 1986 The outwelling hypothesis and North Inlet, South Carolina. Marine Ecology Progress Series 33,217-229. Decho, A. W. & Castenholz, R. W. 1986 Spatial patterns and feeding of meiobenthic harpacticoid copepods in relation to microbial flora. Hydrobiologia 131,217-229. DeNiro, M. J. & Epstein, S. 1978 Influence of diet on the distribution of carbon isotopes in animals. Geochimica et Cosmochimica Acta 42,495-506. DeNiro, M. J. & Epstein, S. 1981 Inlluence of diet on the distribution of nitrogen isotopes in animals. Geochimica et Cosmochimica Acta 45,341-351. Ember, L. M., Williams, D. F. &Morris, J. T. 1987 Processes that influence carbon isotope variations in salt marsh sediments. Marine Ecology Progress Series 36,33-42. Findlay, S. & Tenore, K. 1982 Nitrogen source for a detritivore: detritus substrate versus associated microbes. Science 218,371-372. Fry, B. & Sherr, E. B. 1984 6i3C measurements as indicators of carbon flow in marine and freshwater ecosystems. Contributions in Marine Science 27, 13-47. Gallagher, J. L. & Daiber, F. C. 1974 Primary production of edaphic algal communities in a Delaware salt marsh. Limnology and Oceanography 19,390-395. Gearing, J. N., Gearing, P. J., Rudnick, D. T., Requejo, A. G. & Hutchins, M. J. 1984 Isotopic variability of organic carbon in a phytoplankton-based temperate estuary. Geochimica et Cosmochimica Acta 48, 1089-1098.
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and nitrogen
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of meiobenthos
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Hackney, C. T. & Haines, E. B. 1980 Stable carbon isotope composition of fauna and organic matter collected in a Mississippi estuary. Estuarine and Coastal Marine Science 10,703-708. Haines, E. B. 1976 Stable carbon isotope ratios in biota, soils, and tidal water of a Georgia salt marsh. Estuarine and Coastal Marine Science 4,609-616. Hicks, G. R. F. & Coull, B. C. 1983 The ecology of marine meiobenthic harpacticoid copepods. Oceanography and Marine Biology Annual Review 21,67-175. Hughes, E. H. & Sherr, E. B. 1983 Subtidal food webs in a Georgia estuary: 6°C analysis. rournal of Experimental Marine Biology and Ecology 67,227-242. Jensen, P. 1987 Feeding ecology of free-living aquatic nematodes. Marine EcoZogy Progress Series 35, 187-196. Macko, S. A., Lee, W. Y. & Parker, P. L. 1982 Nitrogen and carbon isotope fractionation by two species of marine amphipods: laboratory and field studies. Journal of Experimental Marine Biology and Ecology 63, 145-149. Mariotti, A., Letolle, R. & Sherr, E. 1983 Distribution of stable isotopes in a salt marsh estuary. Esruaries 6, 304-305. McConnaughy, T. & McRoy, C. P. 1979 Food web structure and the fractionation of carbon isotopes in the Bering Sea. Marine Biology 53,257-262. Montagna, P. A. 1984 In situ measurement of meiobenthic grazing rates on sediment bacteria and edaphic diatoms. Marine Ecology Progress Series l&119-130. Montagna, P. A. & Bauer, J. E. 1988 Partitioning radiolabeled thymidine uptake by bacteria and meiofauna using metabolic blocks and poisons in benthic feeding studies. Marine Biology 98, 101-l 10. Mucciarone, D. 1983 Stable Isotope Laboratory Operations Manual. University of South Carolina, Columbia. Peterson, B. J., Howarth, R. W. & Garitt, R. H. 1985 Multiple stable isotopes used to trace the flow of organic matter in estuarine food webs. Science 227,1361-1363. Rice, D. L. & Tenore, K. R. 1981 Dynamics of carbon and nitrogen during decomposition of detritus derived from estuarine macrophytes. Estuarine Coastal and Shelf Science 13,681-690. Rieper, M. 1982 Feeding preferences of marine harpacticoid copepods for various species of bacteria. Marine Ecology Progress Series 7,303-307. Romeyn, K. & Bouwman, L. A. 1983 Food selection and consumption by estuarine nematodes. Hydrobiological Bulletin 17, 103-109. Schwinghamer, P. F., Tan, F. C. & Gordan, D. C., Jr. 1983 Stable carbon isotope studies in Pecks Cove mudflat ecosystem in the Cumberland Basin, Bay of Fundy. CanadianJournal of Fisheries and Aquatic Science 40 (Supplement 1), 262-272. Sellner, B. W. 1976 Survival and metabolism of the harpacticoid copepod Thomsonula hyaenae fed on different diatoms. Hydrobiologica 50,223-238. Simenstad, C. A. & Wissmar, R. C. 1985 6°C evidence of origins and fates of organic carbon in estuarine and nearshore food webs. Marine Ecology Progress Series22,141-152. Smith, L. D. & Coull, B. C. 1987 Juvenile spot (Pisces) and grass shrimp predation on meiobenthos in muddy and sandy substrata. Journal of Experimental Marine Biology and Ecology 105, 123-136. Spies, R. B. & Des Marais, D. J. 1983 Natural isotope study of trophic enrichment of marine benthic communities by petroleum seepage. Marine Biology 73,67-71. Tenore, K. R., Hanson, R. B., McClain, J., Maccubbin, A. E. & Hodsen, R. E. 1984 Changes in composition and nutritional value to a benthic deposit feeder of decomposing detritus pools. Bulletin of Marine Science 35,299-311. Tietjen, J. H. & Lee, J. J. 1973 Life history and feeding habits of the marine nematode Chromadora macrolaimoides Steiner. Oecologia (Berlin) 12,303-314.