2008 NORTHEASTERN NATURALIST 15(1):35–56 Assessing Habitat Quality of Mount Hope Bay and Narragansett Bay Using Growth, RNA:DNA, and Feeding Habits of Caged Juvenile Winter Flounder (Pseudopleuronectes americanus Walbaum) Lesa Meng1,2, David L. Taylor3,*, Jonathan Serbst1, and J. Christopher Powell4 Abstract - Somatic growth rates, RNA:DNA, and feeding habits of juvenile Pseudopleuronectes americanus (Winter Flounder) were used to asses small-scale spatio-temporal variations in the habitat quality of Mount Hope Bay and Narragansett Bay, RI. Three successive caging experiments (14–16 d each) were conducted with flounder (initial size = 25–35 mm total length) in June and July 2003 in shallow water habitats (<1 m) of Spar Island, Common Fence Point, and Hog Island; the first two sites were located in Mount Hope Bay, and the latter in Narragansett Bay. The average growth rate of flounder ranged between 0.51 and 0.95 mm d-1 and was inversely related with increased incidences of hypoxic conditions (i.e., amount of time dissolved oxygen was ≤4.0 mg L-1). RNA:DNA, a surrogate measure of growth and feeding condition, corroborated somatic growth trends, and therefore exhibited similar spatio-temporal variability. In contrast to somatic growth, however, water temperature was the most important factor affecting flounder condition, such that RNA:DNA was inversely related to the amount of time water temperature was >20 ºC. Benthic core samples indicated that food availability was greatest at Spar Island and was attributable to the numerical dominance of Crepidula fornicata Linnaeus (slipper limpet) during the early summer. Moreover, stomach contents of flounder reflected differences in prey species composition, whereby individuals from Spar Island consumed a higher percentage of molluscs relative to the other sites, where the preferred prey items were harpacticoid copepods and small decapods (primarily brachyuran crabs). Despite the observed discrepancies in feeding habits across sites, the extent of stomach fullness for flounder did not vary spatially (mean fullness = 44–49% across sites). It is concluded that the somatic growth, RNA:DNA, and feeding behavior of juvenile flounder in Mount Hope Bay and Narragansett Bay varies significantly across small spatio-temporal scales in response to changes in dissolved oxygen and thermal conditions. Introduction The functional significance of estuaries as nursery habitat for youngof- the-year (YOY) fish is defined by the survival of resident species. 1US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Laboratory, Atlantic Ecology Division, 27 Tarzwell Drive, Narragansett, RI 02882. 2Deceased. 3Roger Williams University, Department of Biology and Marine Biology, One Old Ferry Road, Bristol, RI 02809. 4Division of Fish and Wildlife-Marine Fisheries, Fort Wetherill Marine Laboratory, 3 Fort Wetherill Drive, Jamestown, RI 02835. *Corresponding author - dtaylor@rwu.edu. 36 Northeastern Naturalist Vol. 15, No. 1 Moreover, the number of YOY fish surviving to subsequent life-history stages is affected by individual growth rates (Houde 1987, Pepin 1990, Rice et al. 1993). For example, rapid growth during ontogeny can increase the survival of early-stage fish by reducing size-dependent predation (Anderson 1988, Parker 1971, Post and Evans 1989). Fast growth also confers a survival advantage because fish that attain larger body sizes at the end of the summer growing season have lower over-wintering mortality (Hurst and Conover 1998, Schultz et al. 1998, Sogard 1997). To this end, fisheries scientists frequently evaluate the quality of nursery habitats by measuring the growth of YOY fish. High-quality nurseries are those in which the growth of YOY fish is enhanced because these habitats presumably offer adequate prey resources and optimal environmental growth conditions. The biological and physical factors that regulate habitat-specific growth of fish, however, often vary over small spatial and temporal scales (Manderson et al. 2002). Thus, determining habitat quality on the basis of growth performance of fish is difficult because associations between individuals and their habitat are complex. Nevertheless, evaluating the functional significance of nurseries is necessary to properly identify and manage areas that are important for fish year-class formation and recruitment. Pseudopleuronectes americanus Walbaum (Winter Flounder) is a pleuronectid flatfish that has traditionally supported valuable commercial and recreational fisheries. This species is distributed along the northwestern Atlantic coast extending as far north as Labrador and southward to North Carolina and Georgia (Pereira et al. 1999). The primary concentration of Winter Flounder occurs in inshore regions, and early life-history stages are estuarine-dependent (Able and Fahay 1998). Specifically, Winter Flounder spawning occurs in estuaries during the winter and early spring (January to April; Collette and Klein-MacPhee 2002). After hatching, larval Winter Flounder are pelagic for ≈60 d (Chambers and Leggett 1987), after which metamorphosis occurs, and the resulting juveniles settle to the benthos during the spring and early summer (April to June; Collette and Klein-MacPhee 2002). The small size of Winter Flounder at metamorphosis (8 to 9 mm total length [TL]; Chambers and Leggett 1987) exposes the juveniles to intense predator-induced mortality during settlement and several months thereafter (Manderson et al. 1999, 2000; Taylor 2003, 2005). Variation in the growth rates of juvenile flounder is therefore likely to influence post-settlement survival by regulating the amount of time fish are susceptible to different predators (Taylor 2003, Taylor and Collie 2003). The growth of post-settlement Winter Flounder is responsive to several habitat-specific characteristics, including prey availability, temperature, and dissolved oxygen (Manderson et al. 2002, Meise et al. 2003, Phelan et al. 2000). Previous studies on the growth response of Winter Flounder to these environmental variables have focused primarily on 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 37 main effects over broad spatial scales (Manderson et al. 2002). The habitats of temperate estuaries of the northeastern United States, however, are generally heterogeneous at fine spatial scales (<5 km), and seasonal environmental conditions vary simultaneously over small temporal scales (weeks). In this study, caging experiments were used to measure juvenile Winter Flounder somatic growth rates and to assess small-scale spatio-temporal variability in the habitat quality of Mount Hope Bay and Narragansett Bay, RI. RNA:DNA and feeding habits of juvenile flounder were also used as alternative measures of fish condition within specific habitats. Winter Flounder growth and condition were subsequently examined relative to habitat-specific environmental variables to ascertain inter-estuarine dynamics in habitat quality. Methods Study area and experimental sites The Narragansett Bay Estuary (≈260 km2 area) is contiguous with Block Island Sound at its mouth and extends northward into Rhode Island and Massachusetts (Fig. 1). Mount Hope Bay is a semi-enclosed estuary (≈35 km2) that adjoins Narragansett Bay at the East Passage and Sakonnet River (Fig. 1). Both estuaries are relatively shallow (mean depth = 7.8 and 5.7 m for Narragansett Bay and Mount Hope Bay, respectively), and are characterized by a small salinity range of 24–30 ppt, a large annual Figure 1. A= the collection site (▲) of Winter Flounder used in the study (Wickford Habor). B= Experimental caging sites (●) in Mount Hope Bay (Spar Island and Common Fence Point) and Narragansett Bay (Hog Island). 38 Northeastern Naturalist Vol. 15, No. 1 temperature range from -0.5 to 27 ºC, and weak seasonal stratification (Oviatt and Nixon 1973). Three sites were chosen for the caging experiments: (1) Spar Island in central Mount Hope Bay, (2) Common Fence Point along the southeastern edge of Mount Hope Bay, and (3) Hog Island in the East Passage of Narragansett Bay (Fig. 1). To ensure access to experimental cages, sites had water depths <1 m at mean low tide and were located in areas distant from boating and other human disturbances. Moreover, sites were chosen based on consistent substrates, bathymetry, and wind and wave exposure. Sites were positioned along the northern edge of landmasses to maintain consistency in physical forcing and substrates of all sites were primarily sand, cobble, and shell hash. Growth experiments Juvenile Winter Flounder somatic growth was monitored in 1-m2 cages composed of wooden and welded metal frames. Cages were 70 cm tall and covered with 3-mm stiff plastic netting on the sides and top. Galvanized steel edges (5 cm deep) around the bottom of the frame allowed for burial of cages into the sediment. Cages were further secured into the sediment by driving 70-cm stakes through fixtures at the lower corners of the cages. To allow access to the inside of cages, removable tops were constructed and cages were placed in water that was approximately 60 cm deep at mean low tide. Four cages were used per experimental site, with the exception of experiment 2 at Spar Island where one cage was lost. Three successive experiments were conducted between 10 June and 22 July 2003, each lasting 14 to 16 d. Juvenile Winter Flounder were collected one day prior to the start of a caging experiment. Fish were collected from one site outside of Wickford Harbor, Narragansett Bay (Fig. 1), with seine hauls (61- x 3.05-m beach-seine set with 0.64-cm mesh size and 0.48-cm bunt). Winter Flounder from 25 to 35 mm TL were measured to the nearest mm TL and individually marked with visible implant fluorescent elastomer. To assess handling mortality, fish were held overnight in the field before placing them in cages. Dead Winter Flounder (<1% of total) were replaced with healthy marked fish. Prior to each experiment, cages were cleared of resident fish and decapods using bar seines and dip nets. Four randomly chosen fish were placed into each cage, a density comparable to juvenile Winter Flounder numbers measured in several temperate estuaries of the northwest Atlantic (Curran and Able 2002, Sogard et al. 2001). Cages were serviced every 3 d to make repairs and brush the plastic netting to minimize fouling. At the end of an experiment, flounder were retrieved from cages using bar seines and dip nets (89% retrieval rate). Retrieved fish were immediately placed on ice, after which TL (± 0.5 mm) was measured in the laboratory. 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 39 At each caging site, several variables related to habitat quality were measured. Hydrolabs were deployed at each station that measured water temperature (°C), dissolved oxygen (mg L-1), and salinity (ppt) at 45-min intervals. A handheld YSI Model 85 (YSI Incorporated, Yellow Springs, OH) was used to take similar measurements every 3 d to verify data collected by the Hydrolabs. Three 1-L samples of water were also collected at each site at the end of experiments, and subsequently analyzed for chlorophyll-a concentration using high-performance liquid chromatography. A stepwise multiple linear regression model was used to examine relationships between flounder somatic growth, RNA:DNA (see below), and the following abiotic and biotic variables: average water temperature, maximum water temperature, proportion of time water temperature was >25 °C, proportion of time water temperature was >20 °C, average salinity, average dissolved oxygen, minimum dissolved oxygen, proportion of time dissolved oxygen was ≤2.0 mg L-1, proportion of time dissolved oxygen was ≤4.0 mg L-1, chlorophyll-a concentration, and initial fish size. Nine observations were used for the analyses (3 sites x 3 experiments), and data represented as proportions were arc-sin square-root transformed to meet assumptions of normality and homogeneity of variance. The effect of caging site (Spar Island, Common Fence Point, and Hog Island) and experiment (experiments 1–3) on flounder growth (mm d-1) and RNA:DNA were also assessed with independent, two-way analysis of variance (ANOVA) models. Mean rates of growth and RNA:DNA across three levels of sites and experiments were contrasted with a Ryan–Einot–Gabriel–Welsch (Ryan’s Q) multiple comparison test (Day and Quinn 1989). RNA:DNA analysis Juvenile Winter Flounder RNA:DNA, a surrogate measure of short-term (1–3 d) instantaneous growth and feeding condition (Kuropat et al. 2002), was measured using techniques described by Caldarone et al. (2001). Briefly, this method used N-lauroylsarcosine to dissociate proteins from the nucleic acids, and the fluorophore ethidium bromide (EB: 3,8-diamino-6-phenyl-5- ethylphenanthridinium bromide) to measure total nucleic acids. Fluorescence was detected using a 96-well fluorescence microplate reader (BioTek FL500, BioTek Instruments, Inc., Winooski, VT). RNase was added to differentiate RNA from DNA, and when residual fluorescence was significant (>7%), DNase was also added to determine the true DNA content. Standard curves were constructed from genomic ultrapure calf thymus DNA, and molecular grade 18S- and 28S-rRNA. Prey availability and stomach content analysis Prey availability at each caging site was measured by four core samples (6.7 cm diameter by 5 cm deep) taken inside and outside of the cages at the end of the first and third experiments (16 cores total for each site). Samples were sieved on 0.25-mm screens, and all prey was identified to the lowest 40 Northeastern Naturalist Vol. 15, No. 1 practical taxonomic level. A three-way ANOVA model was used to compare mean differences in prey availability between sites, inside and outside of cages, and at the end of the first and third experiments. Variables used in the analysis were number of molluscs, polychaetes, copepods, amphipods, “other” organisms (98% of which were nematodes), total number of organisms, and number of taxa. The stomach contents of 118 flounder used in caging experiments were analyzed and averaged by cage (n = 35). Stomach fullness was estimated visually by comparing relative amounts of food in the stomachs. Specific prey items recovered from flounder stomachs were identified to the lowest taxon possible, and further categorized as molluscs, polychaetes, copepods, amphipods, and “other” organisms (82% of which were decapods). Moreover, the amount of each food category was recorded as the percent volume for each stomach. Independent, two-way ANOVA models were used to examine differences in stomach fullness and percent volume of each food category across sites and experiments. Data represented as percentages (proportions) were arc-sin square-root transformed to meet assumptions of normality and homogeneity of variance, and mean values of stomach fullness and prey contents were contrasted with a Ryan’s Q multiple comparison test across three levels of sites and experiments. Results Environmental conditions Water temperatures generally increased throughout the duration of the study and ranged from 14.0 to 26.9 °C, with the lowest temperature at Spar Island and the highest at Hog Island (Table 1, Fig. 2). Average temperatures for Common Fence Point, Hog Island, and Spar Island were 20.1, 20.8, and 20.6 °C, respectively. Average salinities were 27.2, 27.2, and 25.8 ppt for Common Fence Point, Hog Island, and Spar Island, respectively (Table 1). Common Fence Point had the highest average dissolved oxygen content at 6.01 mg L-1, followed by Spar Island (5.34 mg L-1) and Hog Island (4.59 mg L-1) (Table 1, Fig. 2). By the third experiment, dissolved oxygen levels were ≤4.0 mg L-1 60% of the time at Hog Island, followed by 51% at Spar Island and 32% at Common Fence Point. The highest chlorophyll-a concentrations were at Common Fence Point during the first experiment (19.8 μg L-1), followed by Spar Island during the third experiment with 14.8 μg L-1 (Table 1). All remaining chlorophyll-a concentrations were <10 μg L-1. Growth experiments Winter Flounder somatic growth rates differed significantly across experiments and caging sites (ANOVA: experiment p < 0.0001, site p < 0.05) (Table 1, Fig. 3a). Specifically, average growth across sites decreased with each experiment, with the fastest growth occurring during experiment 1 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 41 Table 1. Environmental conditions (means and ranges) of the three experimental sites in Mount Hope Bay and Narragansett Bay, and the initial lengths, growth rates, and RNA:DNA of Winter Flounder used in three caging experiments (experiment 1 = June 10–25; experiment 2 = June 25–July 9, and experiment 3 = July 9–22, 2003). Chlorophyll-a, initial fish length, growth, and RNA:DNA are means per experiment ± 1 SE. Values represent the average of four replicates per site, with the exception of experiment 2 at Spar Island where one cage was lost. Common Fence Point Hog Island Spar Island Envronmental Conditions 1 2 3 1 2 3 1 2 3 Temperature (ºC) 16.8 21.8 21.8 17.2 23.1 22.1 16.9 22.4 22.5 Range 14.3–19.2 18.2–26.0 18.7–25.1 14.4–21.6 20.0–26.9 19.3–25.6 14.0–19.7 18.0–26.0 19.7–24.7 Salinity (ppt) 28.2 25.8 27.6 27.8 25.5 28.3 26.3 24.1 27 Range 23.6–30.5 19.4–28.8 24.8–29.7 23.1–30.1 22.2–27.9 26.4–29.5 23.2–29.6 18.5–27.8 23.9–29.3 Dissolved oxygen (mg L-l) 6.4 6.9 4.8 5.3 4.6 3.9 5.2 6.8 4.1 Range 2.8–11.3 1.2–15.1 1.7–9.1 1.7–9.3 0.1–13.2 0.5–10.1 1.6–9.2 1.9–12.5 0.3–9.1 Chlorophyll-a (μg L-l) 19.8 ± 0.5 3.3 ± 0.1 5.0 ± 0.5 8.9 ± 0.2 2.7 ± 0.04 4.1 ± 0.2 9.3 ± 0.5 8.3 ± 0.03 14.8 ± 0.7 Initial fish length (mm) 29.1 ± 0.4 33.8 ± 0.4 32.3 ± 0.5 27.9 ± 0.7 33.8 ± 0.9 37.4 ± 1.5 29.3 ± 0.8 34.7 ± 1.0 34.7 ± 0.2 Growth (mm d-1) 0.71 ± 0.01 0.86 ± 0.06 0.59 ± 0.05 0.70 ± 0.02 0.61 ± 0.01 0.67 ± 0.04 0.95 ± 0.03 0.79 ± 0.06 0.51 ± 0.05 RNA:DNA 8.0 ± 0.3 7.4 ± 0.3 5.8 ± 0.4 7.8 ± 0.1 6.7 ± 0.1 6.0 ± 0.4 9.1 ± 0.3 6.9 ± 0.4 6.5 ± 0.1 42 Northeastern Naturalist Vol. 15, No. 1 Figure 2. Maximum temperature (ºC) and minimum dissolved oxygen (mg L-1) measured by Hydrolabs deployed at Common Fence Point, Hog Island, and Spar Island during the three experiments (experiment 1 = June 10–25; experiment 2 = June 25–July 9, and experiment 3 = July 9–22, 2003). 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 43 (0.79 mm d-1), followed by 0.76 and 0.59 mm d-1 for experiments 2 and 3, respectively. Moreover, average growth across experiments was highest at Spar Island (0.75 mm d-1), followed by Common Fence Point (0.72 mm d-1) and Hog Island (0.66 mm d-1). Figure 3. Juvenile Winter Flounder growth rates (a) and RNA:DNA (b) for three experiments at Common Fence Point, Hog Island, and Spar Island. Values represent the average of four replicates (cages) per site (+ 1 SE), with the exception of experiment 2 at Spar Island where one cage was lost. 44 Northeastern Naturalist Vol. 15, No. 1 The interaction effect between experiment and caging site on flounder growth was significant (ANOVA: experiment x site p < 0.0001), thereby precluding contrasts across the main effects (Table 1, Fig. 3a). The interaction effect was attributed to significantly faster growth at Common Fence Point during experiment 2, relative to experiments 1 and 3. Conversely, flounder growth did not vary by experiment at Hog Island, and at Spar Island growth was 54–86% slower during experiment 3 relative to the previous experiments. Also during experiment 1, growth rates of flounder at Spar Island were 10–36% faster relative to the other two locations, and conversely during experiment 2, flounder at Hog Island experienced 30–41% slower growth when compared to flounder at the alternative sites. The amount of time dissolved oxygen was ≤4.0 mg L-1 was the only environmental factor that significantly affected juvenile Winter Flounder growth (regression: p = 0.05, R2 = 0.432). The estimated coefficient for the dissolved oxygen variable was negative (-0.436), suggesting an inverse relationship between hypoxic events and flounder growth. RNA:DNA analysis There was a strong positive correlation between Winter Flounder RNA:DNA and daily somatic growth rates (regression: p < 0.0001, R2 = 0.471). Correspondingly, trends in Winter Flounder RNA:DNA generally corroborated observed spatial and temporal patterns of flounder growth, only differing in two instances (experiments 2 and 3 at Common Fence Point and Spar Island, respectively) (Table 1, Fig. 3b). RNA:DNA was significantly different among experiments and caging sites (ANOVA: experiment p < 0.0001, site p < 0.05), and the experiment-site interaction effect was not significant (ANOVA: experiment x site p = 0.086). Flounder RNA:DNA declined significantly at each site from the first to third experiment, and when averaged across experiments, fish at Spar Island had significantly higher RNA:DNA relative to conspecifics at Hog Island. Moreover, the RNA:DNA of Winter Flounder was negatively correlated with the amount of time water temperature exceeded 20 °C (regression: p < 0.005), and elevated temperatures accounted for 72% of the variation observed in flounder condition. Prey availability and stomach content analysis The number of benthic organisms in core samples indicated that food availability varied among experimental sites and inside versus outside of the cages (i.e., across the cage boundary) (ANOVA: site p < 0.0001, cage boundary p < 0.05; Fig. 4a). Moreover, the interaction between sites and the cage boundary was not significant (ANOVA: site x cage boundary p = 0.26). Relative to the prey measured at Hog Island and Common Fence Point, Spar Island had significantly more total food items (mean = 1433 individuals m-2 versus 366 and 84 individuals m-2 for Hog Island and Common Fence Point, respectively) and taxa (mean = 19 taxa m-2 versus 12 and 10 taxa m-2 for Hog 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 45 Island and Common Fence Point, respectively) (Table 2, Fig. 4a). All of the food groups varied significantly among sites (except amphipods; Table 2), and the greatest discrepancy occurred for molluscs with Crepidula fornicata Figure 4. Mean number of benthic organisms m-2 collected (a) inside (“In”) and outside (“Out”) of experimental cages, and (b) at the end of experiment 1 (“Initial”) and experiment 3 (“Final”). Benthic organisms were collected using a 6.7-cm-diameter core sampler (5 cm deep) at three locations: Common Fence Point, Hog Island, and Spar Island. Four core samples were taken inside and outside of the cages at the end of the first and third experiments (16 cores total for each site; n = 8). Error bars represent + 1 SE. 46 Northeastern Naturalist Vol. 15, No. 1 Table 2. Stomach fullness and contents of Winter Flounder used in three caging experiments (118 flounder were examined and averaged by cage; n = 35), and benthic organisms found in 6.7-cm-diameter core samples. Core samples were taken inside and outside of cages after experiment 1 and 3 (n = 48). Values represent the average (± 1 SE) percent volume of food item eaten per stomach (“Eaten”) and the average number of prey items found m-2 (“Cage”). Common Fence Point Hog Island Spar Island Experiment 1 2 3 1 2 3 1 2 3 % Stomach fullness 43.1 ± 3.4 53.0 ± 4.7 40.3 ± 3.3 44.2 ± 6.9 34.4 ± 4.0 55.7 ± 3.8 63.3 ± 4.3 41.7 ± 5.3 36.8 ± 5.1 Molluscs Eaten 39.0 ± 5.1 20.7 ± 5.8 0.6 ± 0.4 4.7 ± 3.9 0.8 ± 0.4 0.3 ± 0.3 62.1 ± 5.6 7.0 ± 3.6 0.7 ± 0.4 Cage 5.5 ± 0.9 13.9 ± 6.8 3.6 ± 1.2 2.0 ± 1.2 2170.0 ± 750 208.0 ± 146 Polychaetes Eaten 1.8 ± 1.3 2.0 ± 0.9 2.1 ± 1.1 13.8 ± 7.6 22.9 ± 7.3 4.6 ± 2.5 1.0 ± 0.7 3.2 ± 1.8 Cage 7.6 ± 1.5 20.3 ± 5.0 65.8 ± 20.5 52.1 ± 8.8 182.0 ± 35.3 150.0 ± 26.4 Copepods Eaten 14.9 ± 3.2 8.7 ± 2.0 30.9 ± 7.3 15.8 ± 6.6 4.3 ± 1.2 5.4 ± 2.4 3.9 ± 0.8 5.4±1.2 14.8 ± 0.7 Cage 1.4 ± 0.4 2.1 ± 1.1 6.3 ± 2.8 2.0 ± 0.9 25.8 ± 6.1 1.1 ± 0.9 Amphipods Eaten 8.1 ± 3.5 13.3 ± 3.7 4.1 ± 3.1 44.1 ± 9.0 30.4 ± 7.8 51.4 ± 2.4 11.7 ± 4.3 70.2±9.6 58.9 ± 7.7 Cage 0.1 ± 0.1 0.5 ± 0.3 3.6 ± 2.0 24.8 ± 21.2 52.8 ± 19.9 6.5 ± 3.0 Other Eaten 40.3 ± 5.4 57.6 ± 5.9 61.6 ± 8.8 24.5 ± 7.9 45.9 ± 7.6 39.9 ± 7.3 24.3 ± 4.0 9.6±5.5 39.6 ± 7.3 Cage 96.0 ± 15.8 4.3 ± 1.7 187.0 ± 38.6 316.0 ± 79.0 44.3 ± 15.9 17.9 ± 7.5 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 47 Linnaeus (slipper limpet) being the numerically dominant species (mean number of molluscs = 1189, 10, and 3 individuals m-2 at Spar Island, Common Fence Point, and Hog Island, respectively). Copepods and the number of taxa were significantly greater outside the cages, but all other prey categories were not significantly different across the cage boundary. Food availability differed significantly as a function of time and caging site (ANOVA: time p < 0.0001, site p < 0.05), but the interaction effect was also significant (ANOVA: time x site p < 0.001), precluding contrasts across the main effects (Fig. 4b). The interaction effect was due to a ten-fold decrease in molluscs at Spar Island at the end of the third experiment (Table 2). Otherwise, there was no difference in food availability inside and outside of the cages when initial and final cores were compared. The stomach contents of Winter Flounder differed significantly by experiment, caging site, and the experiment-site interaction effect (ANOVA: experiment p < 0.0001, site p < 0.0001, experiment x site p < 0.0001; Fig. 5). Conversely, stomach fullness was similar among sites (mean = 44–49%) and experiments (mean = 43–52%) (ANOVA: experiment p > 0.05, site p > 0.05). The interaction effect between experiment and site on stomach fullness was significant (ANOVA: experiment x site p < 0.001), however, and reflects a decline in the mean stomach fullness of flounder at Spar Island from 63% in the first experiment to 37% in the third (Table 2). Stomach contents of caged Winter Flounder reflected spatial differences in prey composition, and therefore, patterns of feeding habits differed considerably across sites (Table 2, Fig. 5). For example, Winter Flounder at Spar Island consumed significantly more molluscs than flounder at Common Fence Point and Hog Island (ANOVA: p < 0.0001). This result indicates opportunistic foraging by flounder on the abundant slipper limpet, as this mollusc accounted for >83% of the total number of available prey at Spar Island. Conversely, harpacticoid copepods were the preferred food resource of flounder at Common Fence Point and Hog Island, accounting for 52–78% of the total number of prey identified in stomach contents. The consumption of copepods at Common Fence Point and Hog Island suggests a selective foraging strategy, considering the relatively low abundance of this prey item at the respective sites (1–2% of the total number of available prey). Notwithstanding the numerical dominance of copepods in the diet of juvenile Winter Flounder (particularly at Common Fence Point and Hog Island), this prey category accounted for a relatively low percent volume of the total stomach contents (8.5–18.2%). In contrast, amphipods were not regarded as a prominent food resource based on availability or numerical identification in stomach contents, yet this prey category routinely ranked among the top items by percent volume of the total diet (8.5–47%). The availability of polychaetes was consistent across sites (range = 12–18% of total prey), but this prey item was only consumed by flounder at appreciable 48 Northeastern Naturalist Vol. 15, No. 1 Figure 5. Food items available compared to the prey consumed by juvenile Winter Flounder in experimental cages at three sites: Common Fence Point, Hog Island, and Spar Island. For each prey category, values represent the percent of the total number of food items in core samples (“Available”), percent of total number of prey items eaten (“Number eaten”), and percent volume of prey items eaten (“Volume eaten”). 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 49 levels at Hog Island (ANOVA: p < 0.005). Within the “other” prey category, nematodes were relatively abundant in the field (accounting for 98% of the organisms within the category), but this prey item was not consumed by flounder in high quantities. Conversely, the majority of “other” prey consumed (based on percent volume of stomach contents) were small decapods (>82%), and this food resource was eaten in significantly greater quantities by flounder at Common Fence Point relative to the alternative sites (ANOVA: p < 0.0001). Discussion Patterns of somatic growth, RNA:DNA, and feeding habits of juvenile Winter Flounder varied on small spatio-temporal scales in response to changes in dissolved oxygen, thermal conditions, and prey composition, respectively. Specifically, the average growth rate of caged Winter Flounder decreased throughout the duration of this study and was negatively correlated with the occurrence of hypoxic events (dissolved oxygen ≤4.0 mg L-1). The RNA:DNA of Winter Flounder corroborated somatic growth trends, which was also reported in similar investigations that compared the growth and condition of caged juvenile Winter Flounder (Kuropat et al. 2002). In contrast to the growth patterns observed in this study, however, RNA:DNA were inversely related to elevated water temperatures (>20 ºC). Diet analysis of Winter Flounder further revealed that differences in feeding habits across experimental sites reflected habitat-specific variability in prey composition. The mollusc C. fornicata, for example, was opportunistically consumed at Spar Island when this species was numerically dominant, whereas harpacticoid copepods and small decapods (i.e., brachyuran crabs) were preferentially selected as a food resource at alternative sites (Common Fence Point and Hog Island). Cumulatively, these results underscore how biotic and abiotic factors influence the function of habitats at small spatial and temporal scales, and consequently, the importance of evaluating biological responses at the appropriate scale. The somatic growth of post-settlement flatfish, including Winter Flounder, is responsive to a multitude of biotic and abiotic factors. Temperature, for example, is one the most significant factors underlying variations in the growth of early-stage flatfish (Malloy and Targett 1991, Manderson et al. 2002, Meng et al. 2000, Rose et al. 1996). For juvenile Winter Flounder, growth as a function of temperature exhibits a unimodal response with optimal growth occurring at ≈15 ºC (Rose et al. 1996). In contrast to similarly designed experiments (Meng et al. 2000, 2001; Phelan et al. 2000; Sogard 1992), water temperature during this study did not account for a significant level of variation in observed flounder growth. There was, however, an inverse relationship between flounder RNA:DNA and the amount of time water temperature was >20 ºC. 50 Northeastern Naturalist Vol. 15, No. 1 RNA concentration reflects active protein synthesis, and therefore represents an indirect measurement of short-term (1–3 d) instantaneous growth (Kuropat et al. 2002). Moreover, RNA content normalized with DNA concentration (i.e., RNA:DNA) is frequently used as a tool to assess variability in fish growth and recent feeding history (Buckley et al. 1999). For example, Malloy and Targett (1991) noted increased RNA: DNA in juvenile Paralichthys dentatus Linnaeus (Summer Flounder) when temperature and foraging activity increased simultaneously. The opposite trend was observed in this study, whereby Winter Flounder RNA: DNA was inversely correlated with periods of elevated water temperature (>20 ºC). Water temperatures ranging between 20 and 29 °C may inhibit the feeding of juvenile Winter Flounder (Casterlin and Reynolds 1982). Results from this investigation, however, do not support this supposition because flounder stomach fullness remained relatively constant (mean fullness across experiment = 43–52%) despite changing temperature conditions. More likely, warmer temperatures depress protein synthesis in juvenile Winter Flounder because a disproportionate amount of energy is devoted to increased metabolism, which in turn, leaves less energy for somatic development (Buckley et al. 1999). This physiological response also partially explains the general decrease in Winter Flounder somatic growth rates during the investigation (mean growth = 0.79 and 0.59 mm d-1 during experiments 1 and 3, respectively). Concentrations of dissolved oxygen decrease as water temperature increases, which in turn negatively affects fish growth. In this study, levels of dissolved oxygen ≤4.0 mg L-1 explained 43% of the variability in Winter Flounder growth. This is consistent with Bejda et al. (1992), who found lower growth in Winter Flounder under conditions of fluctuating and low dissolved oxygen. In a Narragansett Bay caging study, Winter Flounder growth decreased dramatically as dissolved oxygen fell with increasing temperatures (Meng et al. 2001). Caging studies in New Jersey showed growth and survival were depressed in tidal marsh creeks and vegetated macroalgae habitats where dissolved oxygen was often less than 2 mg L-1, due to high temperatures and the breakdown of macroalgae (Phelan et al. 2000). In Long Island Sound, areas with low dissolved oxygen had fewer and smaller Winter Flounder (Howell and Simpson 1994). Thus, prolonged periods of low dissolved oxygen can have a distinctly negative affect on Winter Flounder growth. Chlorophyll-a concentrations may also have indirectly negatively affected growth during this study by contributing to low dissolved oxygen concentrations. Chlorophyll-a is a surrogate measurement for nutrient enrichment that is known to degrade fish habitat by lowering dissolved oxygen, increasing turbidity, and changing sediment characteristics (Valiela et al. 1992). Increased nutrients may also benefit fish by producing more food (Meng et al. 2002, Tsai et al. 1991), but it is unclear how nutrients interact 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 51 with other factors, such as temperature. In this study, there was no clear trend in chlorophyll-a concentrations. Adequate food resources, which are often associated with substrate characteristics (Manderson et al. 2002, Phelan et al. 2001), are also necessary to sustain maximal growth rates of post-settlement flatfish (Gibson 1994 and references therein). Variations in the growth of newly settled plaice, for example, have been partially attributed to food quality and quantity (Poxton 1983, Van der Veer and Witte 1993). Similarly, Meise et al. (2003) observed a positive relationship between juvenile Winter Flounder growth and benthic food abundance in the Navesink River/Sandy Hook Bay estuary in New Jersey. In this study, somatic growth of flounder was fastest at Spar Island during experiment 1 (0.95 mm d-1) and may be related to the availability and subsequent consumption of the abundant slipper limpets. This mollusc was also abundant at Common Fence Point and Mount Hope Bay, albeit at a lesser extent, and was eaten in proportions indicative of their overall availability. The steady decline in flounder growth at Spar Island (experiment 3 growth rate = 0.51 mm d-1) may have been related to lower prey availability and, specifically, the lower abundance of slipper limpets. Stomach fullness decreased markedly over time at Spar Island (63% and 37% fullness after experiments 1 and 3, respectively), coinciding with a ten-fold decrease in prey density during the same time period. As previously discussed, water temperatures between 20 and 29 °C might cause feeding inhibition in juvenile flounder (Casterlin and Reynolds 1982), and in this study, may have interacted with lower prey densities to decrease stomach fullness at Spar Island. In contrast to Winter Flounder opportunistically foraging on slipper limpets, the frequency of harpacticoid copepods, amphipods (primarily Microdeutopus gryllotalpa Costa), and small decapods (brachyuran crabs) in fish stomach contents was disproportional to the preys’ overall habitatspecific abundance. Harpacticoid copepods were also a major constituent in the diet of post-settlement Winter Flounder (≈12–36 mm TL) in the Mystic River, CT (Pearcy 1962) and the Pettaquamscutt River, RI (Mulkana 1966), but not from the Navesink River/Sandy Hook Bay estuary (Stehlik and Meise 2000). In another study in Narragansett Bay, Polydora cornuta Bosc (mud polychaete) was a favored food item (Meng et al. 2001). In this study, however, despite the relatively high abundance of polychaetes such as P. cornuta, this prey category was eaten less frequently than expected. Winter Flounder are generally considered opportunistic feeders, foraging on the most abundant and available prey resource (Carlson et al. 1997, Stehlik and Meize 2000). Results from this study partially verify this supposition, most notably through the presence of slipper limpets in the stomach contents of flounder from Spar Island. However, flounder from alternative sites apparently exhibit a selective foraging strategy, whereby fish preferentially consume prey items that were in relatively low abundance. 52 Northeastern Naturalist Vol. 15, No. 1 Somatic growth rates measured in this study are slightly higher than other northeastern estuaries, but comparable to other studies on Winter Flounder in this size range. Winter Flounder growth averaged from 0.51 to 0.95 mm d-1 in this study, compared to 0.22 to 0.60 mm d-1 in the West Passage of Narragansett Bay (Meng et al. 2001) and 0.29 to 0.44 mm d-1 in Rhode Island’s coastal lagoons (Meng et al. 2000). DeLong et al. (2001) developed a length-based model for Winter Flounder growth based on data from Narragansett Bay. Using their model for 30-mm fish, similar in size to those used in our experiments, growth was estimated at 0.32–0.37 mm d-1. In the Mystic River estuary, growth averaged from 0.28 to 0.35 mm d-1 (Pearcy 1962). Phelan et al. (2000) recorded growth of -0.3 to 0.69 mm d-1 when they compared Connecticut and New Jersey estuaries, with higher rates in New Jersey. Growth of caged fish in New Jersey estuaries ranged from 0 to 1.3 mm d-1 (Sogard 1992), and that of free-ranging fish was calculated from otoliths at 0.3 to 1.7 mm d-1 (Sogard and Able 1992). Another caging study in the Navesink River/Sandy Hook Bay estuary recorded rates of 0 to 0.9 mm d-1 and noted that growth was most rapid at cool temperatures (<21 °C) (Manderson et al. 2002). The gradient of growth rates from north to south suggests that warmer temperatures may be beneficial up to a point, but when temperatures exceed 25 °C, as they did in the Rhode Island coastal lagoon study, Winter Flounder growth is depressed (Meng et al. 2000). As previously stated, modeling studies have indicated that the optimum temperature for juvenile Winter Flounder growth is ≈15 °C (DeLong et al. 2001, Rose et al. 1996), but it is likely that the optimum temperature varies among estuaries and through the growing season, and is dependent on the variation of other factors, such as salinity (Manderson et al. 2002). This study demonstrates that the quality of estuarine nurseries is dynamic because of variations in habitat-specific environmental factors that regulate fish growth. Specifically, juvenile Winter Flounder growth and condition (RNA:DNA and feeding habits) varied considerably over relatively small spatial scales (<5 km) and temporal scales (weeks) in response to seasonal changes in dissolved oxygen concentrations, thermal conditions, and prey species composition. These results underscore the importance of measuring the biological responses of early-stage fish to dominant controlling factors at the appropriate scale. Acknowledgments We thank Phil Colarusso of the US Environmental Protection Agency, Region 1 in Boston, MA, for suggesting this study and guiding us through the process of acquiring the funds through the Regional Acquired Research Effort program to conduct the work. Many people helped with the field effort, including Nora Sturgeon, Adam Frimodig, Steve Raciti, Lee von Kraus, Adam Memon, Sarah Pierce, Julie St. Andre, and Nicole Calabrese. We also thank Sheldon Pratt for 2008 L. Meng, D.L. Taylor, J. Serbst, and J.C. Powell 53 identifying and enumerating the benthic prey, and Jean St. Onge-Burns and Melissa Wagner for analyzing RNA:DNA and fish stomach contents. We are grateful to Saro Jayaraman for analyzing the chlorophyll samples. Jim Heltshe’s suggestions on the statistics were invaluable. We thank the many people who reviewed this manuscript, including Marty Chinata, Beth Hinchey, and Walter Berry. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. This paper is contribution number AED-05-095 of the US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Laboratory’s Atlantic Ecology Division. Although the research described in this article has been funded by the US Environmental Protection Agency, it has not been subjected to Agency-level review. Therefore, it does not necessarily reflect the views of the Agency. Literature Cited Able, K.W., and M.P. Fahay. 1998. 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The food and growth of 0-group flatfish on nursery grounds in the Clyde Sea Area. Estuarine, Coastal and Shelf Science 17:319–337. Rice, J.A., J.A. Miller, K.A. Rose, L.B. Crowder, E.A. Marshall, A.S. Trebitz, and D.L. DeAngelis. 1993. Growth-rate variation and larval survival: Inferences from an individual-based size-dependent predation model. Canadian Journal of Fisheries and Aquatic Sciences 50:133–142. Rose, K.A., J.A. Tyler, R.C. Chambers, G. Klein-MacPhee, and D.J. Danila. 1996. Simulating Winter Flounder population dynamics using coupled individualbased young-of-the-year and age-structured adult models. Canadian Journal of Fisheries and Aquatic Sciences 53:1071–1091. Schultz, E.T., D.O. Conover, and A. Ehtisham. 1998. The dead of winter: Size-dependent variation and genetic differences in seasonal mortality among Atlantic Silversides (Atherinidae: Menidia menidia) from different latitudes. Canadian Journal of Fisheries and Aquatic Sciences 55:1149–1157. Sogard, S.M. 1992. Variability in growth rates of juvenile fishes in different estuarine habitats. Marine Ecology Progress Series 85:35–53. Sogard, S.M. 1997. Size-selected mortality in the juvenile stages of teleost fishes: A review. Bulletin of Marine Science 60:1129–1157. 56 Northeastern Naturalist Vol. 15, No. 1 Sogard, S.M., and K.W. Able. 1992. Growth variation of newly settled Winter Flounder (Pseudopleuronectes americanus) in New Jersey estuaries as determined by otolith microstructure. Netherlands Journal of Sea Research 29:163–172. Sogard, S.M., K.W. Able, and S.M. Hagan. 2001. Long-term assessment of settlement and growth of juvenile Winter Flounder (Pseudopleuronectes americanus) in New Jersey estuaries. Journal of Sea Research 45:189–204. Stehlik, L.L., and C.J. Meise. 2000. Diet of Winter Flounder in a New Jersey estuary: Ontogenetic change and spatial variation. Estuaries 23:381–391. Taylor, D.L. 2003. Size-dependent predation on post-settlement Winter Flounder Pseudopleuronectes americanus by sand shrimp Crangon septemspinosa. Marine Ecology Progress Series 263:197–215. Taylor, D.L. 2005. Predatory impact of the green crab (Carcinus maenas Linnaeus) on post-settlement Winter Flounder (Pseudopleuronectes americanus Walbaum) as revealed by immunological dietary analysis. Journal of Experimental Marine Biology and Ecology 324:112–126. Taylor, D.L., and J.S. Collie. 2003. A temperature- and size-dependent model of sand shrimp (Crangon septemspinosa) predation on juvenile Winter Flounder (Pseudopleuronectes americanus). Canadian Journal of Fisheries and Aquatic Sciences 60:1133–1148. Tsai, C., M. Wiley, and A. Chai. 1991. Rise and fall of Potomac River Striped Bass stock: A hypothesis of the role of sewage. Transactions of the American Fisheries Society 120:1–22. Valiela, I., K. Foreman, M. LaMontagne, D. Hersh, J. Costa, P. Peckol, B. DeMeo- Anderson, C. D’Avanzo, M. Babione, C. Sham, J. Brawley, and K. Lajtha. 1992. Couplings of watersheds and coastal waters: Sources and consequences of nutrient enrichment in Waquoit Bay, Massachusetts. Estuaries 15:443–457. Van der Veer, H.W., and J.I.J. Witte. 1993. The “maximum growth/optimal food condition” hypothesis: A test for 0-group Plaice Pleuronectes platessa in the Dutch Wadden Sea. Marine Ecology Progress Series 101:81–90. Further research on Mount Hope Bay is available in Special Issue #4 of the Northeastern Naturalist: Natural and Anthropogenic Influences on the Mount Hope Bay Ecosystem. The papers in this special issue were presented as part of a day-long symposium to determine the state of knowledge of the Mount Hope Bay ecosystem and to examine how natural and anthropogenic factors affect estuarine systems. The symposium was convened as part of a joint meeting of the New England Estuarine Research Society and the Southern New England Chapter of the American Fisheries Society. 204 pp. To order a copy, please contact Dan MacDonald at: School for Marine Science and Technology, University of Massachusetts Dartmouth, New Bedford, MA 02744; dmacdonald@umass.edu.