112 
Fishery Bulletin 111(2) 
Intrapopulation variability in SI compositions can 
be particularly difficult to interpret in species and eco- 
systems with multiple overlapping trophic pathways. 
Omnivorous fish species, such as Eurasian Perch {Per- 
ea fluviatilis ) and Spotted Seatrout ( Cynoscion nebulo- 
sus), hold important positions in aquatic food webs as 
trophic mediators of primary production, but they have 
complex stable isotope signatures that require a close 
examination of assumptions regarding intrapopulation 
variability (Quan et ah, 2007; Quevedo et ah, 2009). 
The estuarine habitat of Spotted Seatrout, in particu- 
lar, is highly complex; it contains both potential aquat- 
ic sources (e.g., phytoplankton, microphytobenthos, 
submerged aquatic vegetation, marsh macrophytes) 
and terrestrial sources of primary production that can 
vary in importance for estuarine food webs, both tem- 
porally and spatially. One of the key questions with 
the food-web approach to EFM that can be addressed 
with stable isotope data is an estimation of the relative 
importance of different sources of organic matter for 
the production of estuarine consumers. Yet, individual 
fishes exposed to the same suite of prey resources may 
have very different isotopic values, and that variation 
may overemphasize the importance of omnivory and 
confound detection of any population-level differences 
in isotope ratios (Zanden et ah, 2010). The utility of 
stable isotope data for analysis of estuarine ecosystems 
will be greatly enhanced when sources of uncertainty 
have been well-quantified for important populations. 
The goal of this analysis was to measure intrapopu- 
lation variability in carbon (S 13 C) and nitrogen (5 15 N) 
SI values in a representative estuarine omnivore (Spot- 
ted Seatrout) to improve understanding and interpre- 
tation of SI data in an estuarine ecosystem in the 
northern Gulf of Mexico. The study objectives were 1) 
to quantify individual variation in carbon and nitrogen 
isotopic values of Spotted Seatrout in the laboratory 
and in wild populations; 2) to examine spatial and sea- 
sonal changes in the isotopic value of various tissue 
types with different turnover rates; and 3) to quantify 
intrapopulation variability for carbon and nitrogen iso- 
topes in the laboratory and compare these estimates 
with field-based and literature reported values for iso- 
tope variability. A characterization of these sources of 
variability will allow for a clearer interpretation of how 
opportunistic changes in fish diet may be reflected in 
seasonal or spatial variances in isotope values. 
Methods 
Spotted Seatrout were collected from regular surveys 
conducted monthly by the University of Southern Mis- 
sissippi Center for Fisheries Research and Develop- 
ment at 8 sites in coastal Mississippi (Fig. 1). Addition- 
al fish were also captured at 2 additional sites as part 
of a second survey of composition of fish communities 
associated with oyster reefs in the western Mississippi 
Sound (Fig. 1). Both surveys were conducted from 2007 
to 2009. All fish were captured during 1-h sets of an 
experimental gillnet with 5 panels (30.48x1.83 m with 
mesh sizes of 50.8, 63.5, 76.2, 88.9, and 101.6 mm). 
All Spotted Seatrout collected were identified based 
on taxonomic descriptions of Hoese and Moore (1977), 
measured to the nearest 1 mm (total length [TL] ) and 
weighed to the nearest 1 g (wet weight), returned to 
the laboratory on ice, and immediately frozen at -20° C. 
In the laboratory, fish were thawed and tissue samples 
were collected: 2 samples of white muscle were collect- 
ed from the left and right side of the fish dorsal to the 
midline, and 2 samples were collected of liver tissue. 
Both muscle and liver samples were freeze-dried to a 
constant weight, ground to a fine power, homogenized, 
and stored in a desiccator for stable isotope analysis. 
Stable isotope ratios for 8 13 C and § 15 N were em- 
ployed to delineate spatial and temporal changes in 
the SI values of individual fish within and between lo- 
cal populations. The high lipid content of liver tissue 
necessitated the need for a correction factor because 
lipids are typically depleted in 13 C relative to carbohy- 
drates and protein (Deniro and Epstein, 1977). Ten liv- 
er samples were split into 2 parts; lipid was extracted 
from half the sample and the other half was unaltered. 
Each lipid subset of liver tissue samples was extract- 
ed sequentially with 2:1 chloroform: methanol and 1:2 
chloroforrmmethanol mixtures, dried, and homogenized 
before analysis (Bligh and Dyer, 1959; Ruiz et ah, 
2007). The 8 13 C results of the lipid-extracted samples 
were plotted against the results of the nonextracted 
samples, and the resultant linear equation was used to 
correct the liver 8 13 C values for lipid content: 
5 ,3 C „ r „ cted = (0.9975 x 5«C + 2.0578,(1) 
where the coefficient of determination [r 2 ] = 0.93. 
Samples were analyzed with a DELTA V Advantage 1 
stable isotope ratio mass spectrometer (Thermo Scien- 
tific, Waltham, MA) coupled to an ECS 4010 elemental 
combustion system (Costech Analytical Technologies, 
Inc., Valencia, CA). All samples were analyzed in du- 
plicate and referenced to known isotopic standards and 
are reported in per mil notation {%o). Nitrogen is ref- 
erenced to atmospheric nitrogen and carbon to the Pee 
Dee Belemnite (PDB) standard. 
The 8 15 N value was used to calculate mean trophic 
level by site and month based on a general formula 
reported by Rooker et al. (2006): 
Trophic level = 1.0 + (8 15 N — 6.0)/3.2, (2) 
where 3.2 is the reported mean fractionation factor for 
nitrogen in fish (Peterson and Fry, 1987; Post, 2002) 
and 6.0 is the mean 8 15 N value of primary producers 
in Gulf of Mexico coastal estuaries on the basis of both 
analysis of samples collected as part of separate proj- 
ects in Mississippi Sound (senior author, unpubl. data) 
1 Mention of trade names or commercial companies is for 
identification purposes only and does not imply endorsement 
by the National Marine Fisheries Service, NOAA. 
