Pierre-Yves Pascal
email: pierreyves.pascal@gmail.com

John W Fleeger

Henricus T S Boschker

Hanan M Mitwally

David S Johnson

RESPONSE OF THE BENTHIC FOOD WEB TO SHORT-AND LONG-TERM NUTRIENT ENRICHMENT IN SALTMARSH MUDFLATS

We examined the responses of biota at or near the base of the benthic food web to nutrient enrichment in saltmarsh mudflats in Plum Island Estuary (Massachusetts, USA). To simulate eutrophication, nitrate and phosphate loading rates were increased 10-15x in creeks fertilized for 2 months (i.e., short-term enrichment) and 6 years (chronic enrichment). We found that benthic invertebrate community structure was not altered by nutrient enrichment, although the abundance of epifaunal, but not infaunal, grazers increased. Short-term enrichment had no effect on the food web but significant changes were detected with chronic enrichment. Grazing experiments with 15 N-enriched bacteria and 13 C-enriched benthic algae revealed higher per capita ingestion rates of benthic microalgae by nematodes, copepods and hydrobiid snails in the creek with chronic nutrient enrichment where isotope composition also indicated that algae increased in dietary importance. The fraction of bacterial biomass grazed was not affected by nutrient enrichment; however, the fraction of benthic algal biomass grazed increased by 235% with chronic enrichment. This higher grazing pressure was partly the result of dietary changes (increases in per capita feeding rate or a change in selection) but was mostly due to an increased abundance of the grazing consumer with the highest biomass, the snail Nassarius obsoletus. This increased top-down control partially masked the bottomup effects of nutrient enrichment on algal biomass and helps explain the slow and inconsistent response of microalgal biomass to chronic nutrient enrichment previously observed in this estuary. Our research shows that eutrophication may subtlety affect benthic food webs before large, sustained increases in algal biomass are observed.

Introduction

Anthropogenic activities have contributed to a doubling of the rate of nitrogen entering the global biogeochemical cycle [START_REF] Galloway | Transformation of the nitrogen cycle: recent trends, questions and potential solutions[END_REF][START_REF] Gruber | An earth-system perspective of the global nitrogen cycle[END_REF][START_REF] Canfield | The evolution of Earth's nitrogen cycle[END_REF]. Through river, groundwater and atmospheric transport, a large fraction of this excess nutrient load passes through coastal ecosystems [START_REF] Howarth | Regional nitrogen budgets and riverine N and P fluxes for the drainage to the North Atlantic Ocean: natural and human influences[END_REF][START_REF] Nixon | The fate of nitrogen and phosphorus at the land-sea margin of the North Atlantic Ocean[END_REF] causing harmful algal blooms, hypoxia and fisheries losses [START_REF] Conley | Controlling eutrophication: nitrogen and phosphorus[END_REF].

Nutrient loading and subsequent eutrophication has been documented worldwide in coastal environments (Diaz & Rosenberg 2008) becoming major issues for both scientists and managers. Among coastal environments, salt marshes appear particularly sensitive experiencing accelerated degradation and habitat loss [START_REF] Silliman | Human impacts on salt marshes: a global perspective[END_REF][START_REF] Deegan | Coastal eutrophication as a driver of saltmarsh loss. Nature Diaz RJ, Rosenberg R[END_REF].

The effects of eutrophication on saltmarsh function are difficult to interpret and predict.

One reason is that benthic food webs are complex, and in general, the nature and strength of interactions are poorly understood. Chronic nutrient enrichment is expected to increase the biomass and/or productivity of the two dominant primary producers in salt marshes: cordgrass, Spartina spp., primarily as aboveground production, and benthic algae [START_REF] Mccomb | Eutrophic shallow estuaries and lagoons[END_REF][START_REF] Bertness | Anthropogenic modification of New England salt marsh landscapes[END_REF][START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF][START_REF] Ferreira | Overview of eutrophication indicators to assess environmental status within the European Marine Framework Strategy Framework Directive[END_REF]. This bottom-up stimulation extends to benthic bacteria because bacteria utilize labile organic matter released by benthic microalgae, which increases with nutrient enrichment (Bowen et al. 2009a).

Bottom-up stimulation may also extend to infaunal and epifaunal invertebrates that exploit benthic bacteria and algae as food resources [START_REF] Posey | Interactive effects of nutrient additions and predation on infaunal communities[END_REF], Johnson & Fleeger 2009[START_REF] Wimp | Increased primary production shifts the structure and composition of a terrestrial arthropod community[END_REF]). An increased per capita consumption by benthic grazers associated with an increased food supply may, over time, stimulate an increase in invertebrate biomass [START_REF] Bertness | Eutrophication and consumer control of New England salt marsh primary productivity[END_REF][START_REF] Johnson | High-marsh invertebrates are susceptible to eutrophication[END_REF]. Increases in per capita grazing rates and/or grazer biomass both have the effect of increasing grazing pressure on bacteria and algae, possibly enhancing top-down control which, in turn, may limit the increase in biomass of benthic algae caused by eutrophication [START_REF] Sardá | Decadal shifts in a salt marsh macroinfaunal community in response to sustained long-term experimental nutrient enrichment[END_REF][START_REF] Posey | Top-down versus bottom-up limitation in benthic infaunal communities: direct and indirect effects[END_REF]. Because nutrient enrichment tends to increase primary producer biomass and grazing tends to decrease it, primary producer responses to nutrient enrichment depend on the relative strength of bottom-up and top-down forces [START_REF] Hillebrand | Marine microbenthic community structure regulated by nitrogen loading and grazing pressure[END_REF][START_REF] Hillebrand | Control of microbenthic communities by grazing and nutrient supply[END_REF][START_REF] Haglund | The effect of grazing and nutrient supply on periphyton associated bacteria[END_REF]. Studies comparing the relative importance of these forces as they affect organismal abundance are becoming common (e.g. [START_REF] Posey | Top-down versus bottom-up limitation in benthic infaunal communities: direct and indirect effects[END_REF][START_REF] Hillebrand | Consumer versus resource control of producer diversity depends on ecosystem type and producer community structure[END_REF], Johnson et al. 2009). Our study however differs from previous work because we also examined the rates of consumption of benthic algae by both infaunal and epifaunal consumers to better elucidate the mechanisms by which benthic food webs change with nutrient enrichment.

We took advantage of a whole-ecosystem manipulation (The 'TIDE' project ; [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF][START_REF] Deegan | Coastal eutrophication as a driver of saltmarsh loss. Nature Diaz RJ, Rosenberg R[END_REF]) designed to investigate the effects of nutrient enrichment on salt marshes in the Plum Island Estuary, Massachusetts, USA. TIDE mimics the nutrient loadings and the mechanism of nutrient delivery typically associated with anthropogenic eutrophication in marsh creeks. Dissolved N and P have been added to creeks with the flooding tide throughout whole growing seasons since 2004. Excess fertilizer and phytoplankton production are removed on the falling tide due to the local 3-m tidal range and nutrient are diluted in the surrounding larger waterways [START_REF] Drake | Salt marsh ecosystem biogeochemical responses to nutrient enrichment: a paired 15 N tracer study[END_REF]). The large spatial (60,000 m 2 ) and temporal (> 6 years of seasonal nutrient enrichment) scales over which TIDE is conducted make this experimental perturbation more similar to human-induced eutrophication than the typical application of dry fertilizer to small plots. These features should contribute to a better understanding of the rate and mechanisms of change associated with chronic nutrient enrichment.

We examined variation in natural abundance isotope concentrations and conducted grazing experiments, with simultaneous dual labeling of bacteria and benthic algae (Pascal et al. 2008a), to clarify our understanding of the effects of nutrient enrichment history on mudflat food webs. The results allowed us to estimate per capita and, based on density estimates, population-level grazing rates across the diversity of benthic grazers that occur with a high biomass or a high abundance. Our labeling and isotope studies allowed an evaluation of the effects of nutrient enrichment on the ability of each grazer to select food, quantifying changes in diet. These methods also allowed an appraisal of the influence of nutrient enrichment on top-down control by summing the quantity of bacteria and benthic algae consumed by the total community of benthic grazers. The goal of the present study is therefore to evaluate food-web changes induced by nutrient enrichment in the low intertidal marsh environment, the mudflat, in TIDE creeks.

Materials and methods

TIDE Project

TIDE stands for "Trophic Cascades and Interacting Control Processes in a Detritus-Based Aquatic Ecosystem" and is an ecosystem-level nutrient enrichment experiment in the salt marshes of Plum Island Estuary (PIE), Massachusetts, (42°44'N, 70°52'W), USA (Figure 1). PIE is a 25-km long mesotidal estuary with a mean tidal range of 3 m. Spartina alterniflora and S. patens are the dominant macrophytes on the creek edge and marsh platform in the seawater-dominated intertidal zone where the salinity ranges from 8 to 28‰.

Plant zonation in PIE is considered typical of New England estuaries [START_REF] Niering | Vegetation patterns and processes in New England salt marshes[END_REF], and PIE has historically been unaffected by anthropogenic nutrient loading (background nutrients: <5 µM NO 3 -; 1 µM PO 4

3-

). Located in the lower intertidal area, mudflats consist of gently sloping creek habitat of poorly consolidated sediments without macrophytes (Figure 1). Nutrient-enrichment experiments were conducted in three creeks (West, Clubhead, Sweeney) having similar water chemistry, plant and infaunal communities (details in [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF], Johnson et al. 2007). Physical dimensions were also similar with the watershed area (including the marsh platform) ranging between 8.4-12.4 ha and the mean tidal exchange volume ranging from 10,560-21,170 m 3 . Furthermore, all creeks drain completely at low tide before being replenished with unenriched water from surrounding waterways on the rising tide [START_REF] Drake | Salt marsh ecosystem biogeochemical responses to nutrient enrichment: a paired 15 N tracer study[END_REF]. Sweeney (hereafter referred to as the Chronically Fertilized Creek or CFC) was enriched for 6 field seasons (2004)(2005)(2006)(2007)(2008)(2009) , resulting in a loading rate of ~ 40 gN m -2 y -1 . For SFC, nutrient enrichment was identical to CFC but began about 2 months before our experiments were conducted. Nutrient concentrations were chosen because they designate an estuary to be "moderately to severely eutrophied" (US EPA 2002). Northeastern US estuaries have experienced a large range in anthropogenically derived nutrient enrichment mostly due to variation in sewage input and differences in the regional frequency and types of agriculture practiced. However, the total inorganic nitrogen concentrations achieved by fertilization in CFC and SFC are similar to levels in found in eutrophic Jamaica Bay, New York, USA, although TIDE nitrogen loading rates were about an order of magnitude lower than most plotlevel nutrient enrichment studies that add dry fertilizer to saltmarsh sediments [START_REF] Deegan | Coastal eutrophication as a driver of saltmarsh loss. Nature Diaz RJ, Rosenberg R[END_REF]. Detailed background information of experimental design and biogeochemistry of the tidal creeks can be found in [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF][START_REF] Deegan | Coastal eutrophication as a driver of saltmarsh loss. Nature Diaz RJ, Rosenberg R[END_REF] and [START_REF] Drake | Salt marsh ecosystem biogeochemical responses to nutrient enrichment: a paired 15 N tracer study[END_REF].

Natural Abundance Isotope Analysis

All biological samples (i.e., for isotope analysis, density estimates and grazing experiments) were collected in the mudflats of each creek, at similar tidal elevation in areas with notable physical similarities. Sample sites in nutrient-enriched creeks were located 100 m upstream from the point at which fertilizer was added. An analogous location was selected for collections in the reference creek. All samples were collected during the second week of July, 2009.

For natural abundance stable isotope analysis of meiofauna, the upper 0.5-cm layer of sediment was collected from 3 replicate plots of 900 cm 2 in each creek and frozen. In the laboratory, samples were thawed, centrifuged with Ludox (de Jonge & Bouwman 1977), and  700-nematode-,  300-copepod, and  10-ostracod specimens were haphazardly removed and retained from each replicate. After centrifugation, foraminifers in the pellet fraction were stained with Rose Bengal and 300 individuals were picked from each replicate. Each specimen was cleaned of debris and rinsed. Taxa containing high concentrations of calcium carbonate (i.e., foraminifera and ostracods) were decalcified with 2.5% HCl. All specimens from each taxon were pooled from a sample and then dried for isotope analysis (n =3).

Snails were collected by hand near each of the plots used to sample meiofauna. Fifteen specimens of hydrobiid snails were removed from the sediment from each sample and snail tissue was subsequently separated from their shells using forceps and pooled (mean longer shell height  sd = 1.8 mm  0.4). Three groups of 15 small (mean longer-shell height  sd = 6.7 mm  1.3) and ten large (mean longer-shell height  sd = 16.3 mm  1.5) specimens of Nassarius obsoletus were also located and pooled by size. Snail tissues were homogenized using a tissue grinder and freeze dried before isotope analyses (n=3).

Samples were analyzed at the Isotope Facility at the University of California, Davis, using an elemental-analyzer isotope ratio mass spectrometer. Samples were reported relative to the standards, atmospheric N 2 and Vienna PeeDee Belemnite carbon. Stable isotope values are reported in  notation:

 13 C or  15 N = [ (R sample /R standard ) -1] X 10 3
where R is respectively 13 C/ 12 C or 15 N/ 14 N.

Grazing Experiments

15 N-enrichment of bacteria was carried out using the methods of Pascal et al. (2008b).

Briefly, surficial sediment was sampled to a depth of 0.5 cm during the ebbing tide in RC.

Bacteria were cultured in a liquid bacterial medium containing 1 g L -1 of 15 NH 4 Cl (> 99%

15 N-enriched). Cultured bacteria were collected from their medium by centrifugation, frozen in liquid nitrogen and stored in a freezer (-20°C) until used in grazing experiments. Enriched bacteria abundance was 6.4 10 9 bacteria mL -1 , and bacteria-produced nitrogen content was 12.28% 15 N.

In each creek mudflat, 4 randomly placed plots of 900 cm 2 were staked. NaH 13 CO 3 (> 99% 13 C-enriched) was dissolved in sea water, and applied daily over 4 days with a garden sprayer directly to sediment surface of each plot in each creek at a concentration of 1 g m -2 [START_REF] Middelburg | The fate of intertidal microphytobenthos carbon. An in situ 13 C labelling study[END_REF]. After spraying was completed, surficial sediment (0.5 cm) of each plot was collected, pooled from each creek, sieved and the sediment fraction retained on a 63µm mesh containing meiofauna was removed. Three subsamples of sediment from each creek aggregate were freeze dried, phospholipid-derived fatty acids (PLFA) were extracted and their isotopic composition was determined (n=3) using a gas-chromatograph combustion-interface isotope-ratio mass spectrometer (GC-c-IRMS) as in [START_REF] Boschker | The contribution of macrophytederived organic matter to microbial biomass in salt-marsh sediments: stable carbon isotope analys of microbial biomarkers[END_REF]. Concentrations and  13 C PLFA specific to diatoms (C20:46; C20:53; C22:53 and C22:63), green algae and cyanobacteria (C18:26c; C18:33; C18:4) and bacteria (i14:0, i15:0, ai15:0, i16:0 and cy19:0) (Cy19:0) were used to estimate the relative contribution of these groups to the total PLFA pool and their weighted-average  13 C composition (Table 1).

13 C-enriched sediment from each creek was mixed with 15 N-enriched bacteria, and 157 ml of this slurry was amended to each of 3 experimental and 3 control incubation microcosms (314 cm 2 ) established for each creek. Bacterial abundance was estimated (see below) and the ratios of total bacteria to 15 N-enriched bacteria in slurry sediments from RC, SFC and CFC were respectively 1.86, 1.80 and 1.81. Superficial sediment (upper 0.5 cm) was collected in each creek and 157 ml of this sediment was sieved on a 63-µm mesh. This fraction of sediment containing meiofauna was added to the experimental microcosms from each creek along with 10 specimens of hydrobiid snails, 10 large and 15 small specimens (size previously described) of N. obsoletus collected from the same creek. Three controls were also performed from each creek in which fauna were added to microcosms in the quantities described above but after being frozen (-20°C) for 24 h to induce mortality. All incubations were for 5 h and were conducted at the same time, in similar conditions of light and temperature, close to natural ones. To ensure a near instantaneous cessation of feeding, incubations were ended by sieving sediment on 63-µm mesh and freezing the retained sediment (-20°C). Samples were later thawed and all isotope samples were prepared and analyzed as previously described.

Isotopic analysis, including the measurement of carbon and nitrogen isotope ratios and carbon and nitrogen content, was conducted on pooled specimens of each microcosm (n=6).

Incorporation of 15 N from bacteria into grazers is defined as excess above background 15 N (control experiment) and is expressed in term of specific uptake (I). I was calculated as the product of excess 15 N (E) and biomass of N per taxon. I was converted to bacterial carbon grazed using C/N ratio of bacteria. E is the difference between the background (F background RairN 2 . The uptake of bacteria was calculated as [I x (% C enriched bacteria / % N enriched bacteria ) / (F enriched bacteria x incubation time)]. This uptake was multiplied by the ratio between the abundance of total and enriched bacteria. Incorporation of 13 C, and thus benthic microalgae (BMA), into grazers was calculated analogously, with

F = 13 C / ( 13 C + 12 C) = R / (R + 1),
RairN 2 is replaced by R VPDB and Uptake = I / (F enriched BMA X incubation time). F enriched BMA was determined using  13 C of PLFA specific to diatoms.

Taxon-specific, individual weights were derived from stable isotope samples and were used in grazing rate calculations. Weights averaged  sd were 344.1 ng  122.2 per nematode (a total of 189,000 specimens were examined isotopically from which weight was derived), 757.4 ng  276.8 per copepod (81,000 specimens), 17.1 µg  3.4 per foraminifera (81,000 specimens, after acid treatment), 64.5 µg  13.1 per ostracod (270 specimens, after acid treatment), 92.7 µg  32.4 per hydrobiid (n=405 specimens, after shell removal), 4.2 mg  2.6 per small N. obsoletus (405 specimens, after shell removal) and 41.9 mg  10.4 per large N.

obsoletus (270 specimens, after shell removal).

Population and Community Grazing Rates

In order to estimate the biomass of bacteria and benthic algae on a surface area basis at the time of grazing experiments, the upper 0.5-cm layer of sediment was collected from 3 plots of 900 cm 2 in each creek and homogenized. Three subsamples of this sediment (20 ml)

were drawn from each homogenate for bacterial and algal analysis. Bacteria were extracted from sediment particles [START_REF] Carman | Microautoradiographic detection of microbial activity[END_REF], stained using DAPI (2500 µg L -1 ), filtered onto 0.2µm Nucleopore black filters, and then counted under an epifluorescence microscope to estimate abundance (n=3 from each creek). Bacterial carbon content was estimated from bacterial abundances using conversion factors [START_REF] Epstein | Microbial food webs in marine sediments. I. Trophic interactions and grazing rates in two tidal flat communities[END_REF]. Benthic microalgal biomass was assessed using chlorophyll (chl) a concentration as a proxy (n=3 from each creek), and measured after extraction with acetone using a Turner fluorometer (APHA 1992). Values were compared to the studies of Bowen et al. (2009a) and Mitwally & Fleeger (In review) who examined the effects of nutrient enrichment on benthic algae in CFC and RC. Algal biomass in units of carbon was estimated using a carbon:chl a ratio of 40 (Jonge 1980).

Microalgal production was estimated using empirically developed relationships between benthic chl a biomass and microalgal production for estuarine mudflats [START_REF] Pinckney | Benthic community responses to nutrient enrichment and predator exclusion: influence of background nutrient concentrations and interactive effects[END_REF]):

production (mgC m -1 y -1 ) = (3.2087* biomass (mg Chl a m -2 )) -0.0903. Mean meiofaunal (n=2 in each creek) and epifaunal densities (n=10 in each creek) were multiplied by mean grazing rates (n=3 per taxon in each creek) to evaluate the total amount of bacteria and algae grazed by each taxon. Grazing was also summed across all taxa to estimate a community grazing rate. This total amount was compared to bacterial and algal biomasses (n=3 in each creek) to evaluate the amount of prey standing stock grazed per unit of time.

Data Analyses

To examine variation in nematode trophic guild composition and copepod community structure among creeks, analysis of similarity (ANOSIM) was conducted with PRIMER 5.2.9 software [START_REF] Clarke | Change in marine communities: an approach to statistical analysis and interpretation[END_REF]. One-way analysis of variance (ANOVA) was used to test for differences in abundances, individual grazing rates and total amount of prey grazed in each creek.

Results

Population Responses and Community Structure

Bacterial biomass at the time of the study was equivalent in all creeks (Table 2).

Microalgal biomass ranged from 15-45 mg chl a m 2 and was less than similar estimates derived from previous studies conducted in the same creeks: i.e., 73-88 (Bowen et

Natural Abundance Isotopic Composition

The natural isotope abundance of large N. obsoletus contained relatively more 13 C than did other grazers in RC (Figure 2). Small N. obsoletus, hydrobiid snails, foraminiferans and ostracods in RC had similar natural abundance carbon isotope abundance but copepods were more depleted in 13 C compared to the other taxa. Ostracods were depleted in 15 N compared to other taxa (Figure 2).

Confidence intervals for natural abundance isotope composition among creeks generally overlapped (Figure 3). However, two trends were observed in all creeks: all grazers from RC were more enriched in 15 N and except for small N. obsoletus all taxa from CFC were more depleted in 13 C. The most common method of fertilizer production is industrial fixation of atmospheric nitrogen, resulting in fertilizer products with  15 N values close to zero. Nutrient enrichment in CFC likely led to decreased algal  15 N ratios, and grazers consuming those algae should to be similarly depleted.

Individual Grazing Rates

Mean per capita grazing rates on bacteria measured in each creek ranged between 12-20 pgC ind -1 h -1 for nematodes, 78-118 pgC ind -1 h -1 for copepods, 15-26 pgC ind -1 h -1 for foraminiferans, 336-397 pgC ind -1 h -1 for ostracods, 31-50 ngC ind -1 h -1 for hydrobiid snails, 1.1-1.4 µgC ind -1 h -1 for small N. obsoletus and 3.3-3.4 µgC ind -1 h -1 for large N. obsoletus (Figure 4). Nematodes from CFC grazed bacteria at a significantly lower per capita rate than nematodes from the other creeks (ANOVA, p < 0.01). All other taxa ingested bacteria at a similar rate, regardless of nutrient enrichment history (ANOVA, ns).

Mean per capita grazing rates of benthic algae measured in each creek ranged between 41-395 pgC ind -1 h -1 for nematodes, 1012-2369 pgC ind -1 h -1 for copepods, 31-77 pgC ind -1 h - 1 for foraminifera, 344-6046 ngC ind -1 h -1 for ostracods, 47-167 ngC ind -1 h -1 for hydrobiid snails, 0.5-1.0 µgC ind -1 h -1 for small N. obsoletus and 1.1-2.3 µgC ind -1 h -1 for large N.

obsoletus (Figure 5). Ingestion of algae was significantly higher in CFC than in the other two creeks for copepods (ANOVA, p < 0.05) and hydrobiid snails (ANOVA, p < 0.01). Only nematodes and ostracods differed with lower per capita ingestion at SFC (ANOVA, p < 0.05 and p < 0.01 respectively). Foraminiferans and N. obsoletus (small and large) ingested benthic microalgae at similar rates in all creeks (ANOVA, ns).

Specific fatty acids of diatoms and bacteria allow evaluation of their respective biomasses. The ratio of diatoms:bacteria in RC sediment used for grazing experiments was 1.9 ± 1.2 (Table 1). Ingestion rates of bacteria and benthic microalgae (BMA) were measured simultaneously during the grazing experiment. When compared to the ratio of food sources available, the ratio of BMA:bacteria grazed indicates food selection (Pascal et al. 2008a). In RC, this ratio was 10.5 ± 1.9 for nematodes, 17.1 ± 3.1 for copepods, 6.4 ± 4.5 for foraminifera, 16.9 ± 2.4 for ostracods, 2.4 ± 0.6 for hydrobiids, 0.7 ± 0.1 for small N.

obsoletus and 0.6 ± 0.2 for large N. obsoletus. In RC, this ratio was not significantly different between small and large N. obsoletus (ANOVA, ns). This ratio was significantly higher for nematodes and hydrobiids from CFC than in the other two creeks (ANOVA, p < 0.01).

Population and Community Grazing Rates

Population-level grazing rates (per m 2 ) on bacteria were calculated as the product of the per capita grazing rate and density for each taxon (Table 3). Nematodes and large N.

obsoletus from CFC and copepods from SFC ingested significantly more bacteria than in the other creeks (ANOVA, p < 0.05 and p < 0.01 respectively). Hydrobiid snails and small N.

obsoletus from RC ingested significantly fewer bacteria than in the other creeks (ANOVA, p < 0.01 and p < 0.05 respectively). The amount of bacteria grazed by total meiofauna, total epifauna and total fauna did not differ significantly in the three treatment creeks (ANOVA, ns). Together, the entire community of benthic invertebrates ingested daily respectively 4.2, 9.3 and 14.5% of bacterial biomass in RC, SFC and CFC.

Population-level grazing rates (per m 2 ) on benthic algae were calculated as the product of the per capita grazing rate and density for each taxon (Table 4). Nematodes, hydrobiids snails and large N. obsoletus ingested significantly more algae in CFC than in the other creeks (ANOVA, p < 0.001, p < 0.01 and p < 0.01 respectively). Population-level grazing was summed across all taxa, and was significantly higher in CFC than in other creeks (ANOVA, p < 0.01). Together, the community of benthic invertebrates ingested daily 235% more benthic algae in CFC than in RC. Compared to the algal biomass of each creek, the community of grazers ingested daily 1.9, 5.1 and 4.7% of algal biomass respectively in RF, SFC and CFC.

The grazing community ingested 9.9, 26.3 and 23.9% of benthic algal production respectively in RF, SFC and CFC. Overall, hydrobiids snails consumed more benthic algae than any other taxon. However, the fraction ingested by N. obsoletus showed the largest increase in CFC, and N. obsoletus accounted for about 40% of all benthic microalgae grazed under the conditions of chronic nutrient enrichment.

Discussion

We studied the effects of nutrient enrichment on benthic foods webs in mudflats, with emphasis on the potential for top-down control over benthic microalgae and bacteria. Nutrient enrichment may alter the food web in three ways that could act singly or in combination;

(1) benthic grazers may change their dietary preference by becoming more selective in their diet and (2) per capita grazing rates of individual taxa on bacteria or algae may vary and (3) changes in the population density of grazing species may affect the total consumption of algae or bacteria. We found that the food web was altered in the creek with a long history (six years) of nutrient enrichment through all three mechanisms. Some grazers became more selective in their diet, increasing their preference for algae over bacteria, and most grazers increased per capita consumption rates of algae with chronic nutrient enrichment. In addition, the epifaunal snail N. obsoletus responded to nutrient enrichment with increased abundance [START_REF] Johnson | Chronic nutrient enrichment increase the density and biomass of the mudsnail, Nassarius obsoletus. Estuar Coast Jonge VND (1980) Fluctuations in the organic carbon to chlorophyll-a ratios in estuarine benthic diatom populations[END_REF], and contributed most to the increased grazing pressure in nutrient enriched creeks. As a result, total community grazing on benthic algae increased with chronic nutrient enrichment.

Diet composition

The diet of most benthic grazers differed in the chronically nutrient-enriched creek.

Isotope composition integrates diet over long-time intervals, and benthic invertebrates in PIE have relatively depleted natural abundance  13 C values compared to saltmarsh macrophyte detritus [START_REF] Galván | Stable isotope addition reveals dietary importance of phytoplankton and microphytobenthos to saltmarsh infauna[END_REF]present study, Galván et al. 2011), indicating an importance of benthic algae to their diet. However, five of the six taxa we examined had lower  13 C values in CFC, consistent with increased long-term consumption of benthic algae (Figure 3). We also calculated the ratio of ingested algae and bacteria from our simultaneous, dual-label experiment. This ratio is a function of the ability of the grazing species to select diatoms from the sediment-algae-bacteria aggregate. In the reference creek, ratios observed for nematodes (10.5), copepods (17.1) and ostracods (16.9) indicate that benthic diatoms were the primary carbon source, while ratios observed for hydrobiid snails (2.4) and for N. obsoletus (0.6) were smaller, indicating a limited capacity to select algae. Previous research has also found that many meiofauna selectively ingest food [START_REF] Buffan-Dubau | Feeding selectivity of the harpaticoid copepod Canuella perplexa in benthic muddy environments demonstrated by HPLC analyses of chlorin and carotenoid pigments[END_REF][START_REF] Moens | Selective attraction of marine bacterivorous nematodes to their bacterial food[END_REF][START_REF] Buffan-Dubau | Diel feeding behavior of meiofauna and their relationships with microalgal resources[END_REF][START_REF] Wyckmans | Effect of food diversity on diatom selection by harpaticoid copepods[END_REF]), often preferring diatoms, while estuarine snails have limited selectivity [START_REF] Brown | The structure and function of the digestive system of the mud snail Nassarius obsoletus (Say)[END_REF][START_REF] Lopez | Epipsammic browsing and deposit-feeding in mud snails (Hydrobiidae)[END_REF][START_REF] Levinton | The body size-prey-size hypothesis and Hydrobia[END_REF][START_REF] Haubois | Ingestion rate of the deposit-feeder Hydrobia ulvae (Gastropoda) on epipelic diatoms: effect of cell size and algal biomass[END_REF]. However, nematodes and hydrobiid snails became significantly more selective in their diet in the chronically nutrient-enriched creek, increasing their preference for benthic microalgae over bacteria. Finally, short-term grazing rate experiments revealed that nematodes, copepods and hydrobiid snails ingested more microalgae on a per capita basis under conditions of chronic eutrophication. The per capita grazing rate on bacteria was altered in only one taxon (such that nematode grazing rates were significantly lower in CFC).

Previous research has shown that opportunistic species may benefit from nutrient enrichment leading to communities dominated by generalist feeders [START_REF] Heip | Eutrophication and zoobenthos dynamics[END_REF][START_REF] Keats | The effect of nitrogen loading on a brackish estuarine faunal community: a stable isotope approach[END_REF]). However we found no evidence for a change in benthic community structure in our study creeks as the macroinfauna community (Johnson & Fleeger 2009), the relative abundance of nematode trophic groups, and the species composition of meiobenthic copepods were all unaffected by nutrient enrichment. Moreover N. obsoletus was the most abundant species of epifauna regardless of nutrient enrichment history [START_REF] Johnson | Chronic nutrient enrichment increase the density and biomass of the mudsnail, Nassarius obsoletus. Estuar Coast Jonge VND (1980) Fluctuations in the organic carbon to chlorophyll-a ratios in estuarine benthic diatom populations[END_REF]. The dietary changes we observed at the community scale were therefore due to changes in feeding behavior or population density at the species level rather than a change in community composition. Previous studies in estuaries also indicate considerable diet plasticity in benthic grazers. Diet become more varied [START_REF] Armitage | Stable isotope reveal complex changes in trophic relationships following nutrient addition in a coastal marine ecosystem[END_REF] and omnivores switched to herbivory [START_REF] Keats | The effect of nitrogen loading on a brackish estuarine faunal community: a stable isotope approach[END_REF][START_REF] Fox | Restructuring of benthic communities in eutrophic estuaries: lower abundance of preys leads to trophic shifts from omnivory to grazing[END_REF]) when the abundance of the basal resource was altered by nutrient enrichment. In a previous study of nutrient enrichment in PIE two surface feeding polychaete species switched to a microalgal resource after 10-weeks of enrichment [START_REF] Galván | The diet of saltmarsh consumers[END_REF]. Estuarine grazers appear to readily adapt to changing food resource availability in their environment under conditions of nutrient enrichment.

Total consumption of bacteria and algae

We calculated population-level grazing rates by multiplying per capita grazing rates and population density. We then employed an estimate of grazing by the benthic community as a whole by summing grazing across the populations of benthic grazers. At the population level, the non-selective epifaunal snails, hydrobiids and N. obsoletus, were responsible for the greatest grazing pressure on bacteria and microalgae (averaging from 64 to 98% of all grazing) (Fig. 4, 5 and 6). The potential to exert top-down control over benthic algae and bacteria is therefore much greater for epifauna than meiofauna as has been found in other mudflats [START_REF] Van Oevelen | The fate of bacterial carbon in sediments: modeling an in situ isotope tracer experiment[END_REF][START_REF] Pascal | Seasonal variation in consumption of benthic bacteria by meio-and macrofauna in an intertidal mudflat[END_REF].

Previous research at our study site has shown that nutrient enrichment indirectly benefits the microbial community because of increased algal exudation, although bacterial productivity and community structure were unaffected by nutrient enrichment (Bowen et al. 2009a, b, Bowen et al. 2011). A similar indirect nutrient effect was observed on bacteria associated with periphyton [START_REF] Hepinstall | Periphyton reactions to different light and nutrient levels and the response of bacteria to these manipulations[END_REF][START_REF] Hillebrand | Control of microbenthic communities by grazing and nutrient supply[END_REF]. Our research

shows that consumption of bacteria by the grazing community was not affected by nutrient enrichment even though the most important grazer, N. obsoletus, increased in density and population-level consumption of bacteria in CFC. We estimate that the total community of grazers ingested between 3 and 14% of bacterial standing stock each day. These observations suggest grazing by benthic invertebrates did not mask a bottom-up stimulation of bacterial production, and that increased grazing had little effect on the biomass of bacteria. Mudflat bacterial communities appear to be strongly resistant to change from exogenous perturbation to the food web (Bowen et al. 2009b[START_REF] Bowen | Microbial community composition in sediments resists perturbation by nutrient enrichment[END_REF], including from grazing invertebrates.

Algal biomass in mudflats was subtly if at all, rather than dramatically, altered in nutrient enriched creeks after 6 years of fertilization in PIE (Mitwally & Fleeger In review).

This absence of a persistent and easily perceivable change in algal biomass in the chronically nutrient-enriched creek mudflat is of interest because algal biomass is often used as an indicator of eutrophication [START_REF] Juanes | Macroalgae, a suitable indicator of the ecological status of coastal rocky communities in the NE Atlantic[END_REF][START_REF] Giordani | Simple tools for assessing water quality and trophic status in transitional water ecosystems[END_REF][START_REF] Ferreira | Overview of eutrophication indicators to assess environmental status within the European Marine Framework Strategy Framework Directive[END_REF]. Our research allowed us to determine what role, if any, benthic grazing had in maintaining the levels of algal biomass in mudflats experiencing nutrient enrichment.

Daily grazing by the community of meiofauna and small macroinfauna has been shown

to exceed the benthic algal standing stock [START_REF] Montagna | Effect of production and biomass of intertidal microphytobenthos on meiofaunal grazing rates[END_REF][START_REF] Carman | Response of a benthic food web to hydrocarbon contamination[END_REF]. Daily consumption by epifauna has also been shown to exceed benthic algal biomass [START_REF] Connor | The effect of feeding by mud snails, Ilyanassa obsoleta (Say), on the structure and metabolism of a laboratory benthic algal community[END_REF]. Although grazing pressure by benthic invertebrates at our study site did not reach the levels observed in the studies cited above, it was substantial. Meiofauna and epifauna together ingested 2 to 5% of algal stock daily, and the proportion of the daily algal primary production consumed increased from 10% in the reference creek to 24% in the creek with chronic nutrient enrichment. Total grazing pressure on benthic algae in the creek with chronic nutrient enrichment was increased by 235% compared to the reference creek. The difference in algal production grazed between RC and CFC (about 20% more of primary production was grazed per day at CFC) suggests that algal biomass at CFC would increase in the absence of benthic grazers and that algae would reach a higher maximum biomass [START_REF] Blanchard | Dynamic behaviour of benthic microalgal biomass in intertidal mudflats[END_REF]). This difference is relatively small in the absolute amount of benthic algal biomass, but the increased grazing pressure on benthic algae lessened potential increases in algal biomass associated with chronic eutrophication. Similar results were observed in a sandy environment [START_REF] Hillebrand | Marine microbenthic community structure regulated by nitrogen loading and grazing pressure[END_REF]. Our results indicate that at least part of the slow and inconsistent response of benthic algae to nutrient enrichment in PIE mudflats was due to top-down control, because increased grazing restrained changes in algal biomass.

Our results also show that nutrient enrichment effects on food webs may take longer than a single growing season to occur. Findings in the creek with short-term nutrient enrichment and the reference creek were very similar and contrasted sharply with those from the chronically fertilized creek. For example, natural abundance isotopes and per capita grazing rates in RFC were not increased compared to RC for any taxon. Similarly, benthic algal biomass did not increase during the first year ( 2004) of nutrient enrichment in CFC (data in [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF]), but increased by 60% in the second year of enrichment in treatments in which a nektonic grazer was reduced in abundance. Previous research has identified effects of nutrient enrichment on abundance or consumer diet that occurred within weeks to months after nutrient enrichment was begun [START_REF] Pinckney | Benthic community responses to nutrient enrichment and predator exclusion: influence of background nutrient concentrations and interactive effects[END_REF][START_REF] Mcfarlin | Impact of fertilization on a salt marsh food web in Georgia[END_REF], while other studies have found that food web changes or abundance changes take longer to develop. For example, in a tundra river fertilization experiment effects were also first noted in the second year nutrient enrichment [START_REF] Peterson | Biological response of a tundra river to fertilization[END_REF][START_REF] Sardá | Decadal shifts in a salt marsh macroinfaunal community in response to sustained long-term experimental nutrient enrichment[END_REF] found that an infauna community was affected by nutrient enrichment but over decadal time scales. Although some infaunal species at our study site responded quickly to nutrient enrichment by changes in dietary selectivity [START_REF] Galván | Stable isotope addition reveals dietary importance of phytoplankton and microphytobenthos to saltmarsh infauna[END_REF], large non-selective epifauna were the most important grazers at our mudflat site. These large species are slow growing and, changes in abundance occur over long time scales perhaps explaining the lack of a rapid change in grazing pressure on benthic algae in nutrient-enriched creeks at our study site.

Other considerations

Saltmarsh nekton can consume benthic bacteria and algae but were not considered here.

Mummichog (Fundulus heteroclitus) is a key omnivorous fish able to ingest algae, bulk detritus and the microbial community as well as animal prey (D'avanzo [START_REF] Valiela | Use of detrital foods and assimilation of nitrogen by coastal detritivores[END_REF]).

However adults likely graze filamentous algae not found on mudflats [START_REF] Lockfield | Chronic nutrient enrichment effects on mummichog, Fundulus heteroclitus in a northeastern Massachusetts salt marsh[END_REF], and juveniles can graze on mudflat microalgae only for a short time as the tide rises over the mudflat before epipelic diatoms vertically migrate. The grass shrimp (Palaemonetes pugio)

are not effective grazers on the epipelic microalgal community [START_REF] Fleeger | Consumption of microalgae by the grass shrimp Palaemonetes pugio[END_REF].

Macroinfauna also consume benthic microalgae [START_REF] Galván | Stable isotope addition reveals dietary importance of phytoplankton and microphytobenthos to saltmarsh infauna[END_REF] although the biomass of epifaunal snails was more than two orders of magnitude higher than the combined biomasses of all macroinfauna (Johnson & Fleeger 2009[START_REF] Johnson | Chronic nutrient enrichment increase the density and biomass of the mudsnail, Nassarius obsoletus. Estuar Coast Jonge VND (1980) Fluctuations in the organic carbon to chlorophyll-a ratios in estuarine benthic diatom populations[END_REF]. Our estimation of the total community grazing therefore focuses on the most abundant and highest biomass members of the infaunal (the meiofauna) and epifaunal (snail) communities.

Estimates of biomass are needed to evaluate the trophic fate of benthic microalgae and bacteria. Our algal and bacterial biomass estimates were based on 3 large (900 cm 2 ) sediment samples from each creek that were subsequently homogenized and subsampled. This procedure should reduce small-scale variation and increase confidence in the means we generated [START_REF] Moreno | Scales of variability in the sediment chlorophyll content in the shallow Palmones River Estuary, Spain[END_REF]. To allow comparisons with algal grazing rates, algal abundance must be converted from units of chl a to carbon using a conversion factor known to vary (Jonge 1980[START_REF] Frost | Ecological stoichiometry in freshwater benthic ecosystems: an introduction[END_REF]. Bacterial biomass measurements are also potentially biased because a conversion factor from volume to carbon content [START_REF] Bratbak | Bacterial biovolume and biomass estimations[END_REF] is required, and a physical separation of attached bacteria from sediment particles is necessary for accurate counts [START_REF] Carman | Microautoradiographic detection of microbial activity[END_REF]. Like all methods previously developed and applied to measure benthic microalgal and bacterial biomasses, the methods used in the present study present potential methodological shortcomings that must be kept in mind when interpreting results.

Our calculation of algal ingestion is based on the assumption that enrichment of grazer tissue is due only to the ingestion of 13 C-enriched diatoms. However other potential food resources may also have become enriched in 13 C. Fatty acid isotopic analysis revealed that green algae and cyanobacteria were, on average, 20% more enriched than diatoms. However, their contribution to grazer enrichment was limited because their biomasses were on average one order of magnitude lower than diatoms. Dominance by diatoms is common [START_REF] Vander Grinten | Dominance of diatoms over cyanobacterial species in nitrogen-limited biofilms[END_REF], particularly in muddy sediments [START_REF] Watermann | Competition between benthic cyanobacteria and diatoms as influenced by different grain sizes and temperatures[END_REF]. PLFA specific-markers for bacteria were also enriched in 13 C but bacterial dietary contributions may be limited because they were on average 5 times less enriched than diatoms. Those potential biases could lead to a small overestimation of diatom importance in diet of grazers. However, grazing rates measured using the 15 N pre-labeled bacteria method were comparable with that reported in a French intertidal mudflat (Pascal et al. 2008b[START_REF] Pascal | Seasonal variation in consumption of benthic bacteria by meio-and macrofauna in an intertidal mudflat[END_REF].

Another uncertainty in our calculations of the impact of the grazing community on microalgae and bacteria is associated with faunal density estimates. N. obsoletus was the most important contributor to changes in the food web in CFC, and its density was surveyed at the same time as the present study [START_REF] Johnson | Chronic nutrient enrichment increase the density and biomass of the mudsnail, Nassarius obsoletus. Estuar Coast Jonge VND (1980) Fluctuations in the organic carbon to chlorophyll-a ratios in estuarine benthic diatom populations[END_REF]. However, meiofauna were sampled with minimal replication at the time of our study because density was estimated simultaneously in the same creeks as part of a long-term assessment of nutrient enrichment effects (Mitwally & Fleeger In review). This long-term assessment could not detect consistent effects on meiofauna associated with nutrient enrichment in the mudflat habitat, and mean values were very similar to those used in our calculations. To determine if the conclusions of our estimates of grazing would be altered by using the long-term meiofauna density estimates, we recalculated grazing rates using meiofauna densities of Mitwally & Fleeger (In review).

The amount of algae grazed hourly by meiofauna increased by 11 and 10% whereas amount of bacteria grazed increased by 24 and 4% respectively in RC and CFC. These reduced fluctuations suggest that bias due to low replication of density measurements is limited.

Conclusions

Increased algal biomass is often used as an indicator of eutrophication [START_REF] Juanes | Macroalgae, a suitable indicator of the ecological status of coastal rocky communities in the NE Atlantic[END_REF][START_REF] Giordani | Simple tools for assessing water quality and trophic status in transitional water ecosystems[END_REF][START_REF] Ferreira | Overview of eutrophication indicators to assess environmental status within the European Marine Framework Strategy Framework Directive[END_REF]. Similar to the conclusion of Shramm (1999), our research shows that top-down control by an extant benthic invertebrate community may mask the effects of eutrophication when measured by algal biomass. Although changes in diet preference and increases in species-specific per capita grazing rates both contributed, grazing pressure on benthic algae increased largely because the abundance of the non-selective feeding snail, N. obsoletus, increased with chronic nutrient enrichment. This increased abundance accounted for about 40% of the increase in community grazing rates. Our results also indicate that nutrient-induced changes in the benthic food web were observed sometime after one field season and before 6 years of fertilization, and that large changes in benthic algal biomass were not observed during this time. Thus, we detected changes in food-web structure that occurred prior to any large, sustained increases in algal biomass, suggesting that effects of nutrient enrichment on food webs may take place before eutrophication is apparent. 4. Amount of algal carbon grazed daily (µgC m -2 d -1 ) and fraction of algal standing stock grazed daily (%) by meiofauna (nematodes, copepods, foraminiferans and ostracods) and epifauna (Hydrobiid snails, small and large N. obsoletus) in surficial sediment (0.5 cm) of three study creeks, see text for creek designations (means  SD; n=3). % corresponds to fraction of total bacteria ingested by each grazer. * indicates significant differences (p < 0.05) with two other creeks (ANOVA; Tukey test).

Tables

  , Clubhead (hereafter referred to as the Short-term Fertilized Creek or SFC) was enriched for about 2 months (2009) and West (hereafter referred to as the Reference Creek or RC) was not enriched. In order to maintain a relatively constant enrichment through each tidal cycle, fertilizer was pumped into the creek at flow rates based on a hydrologic model that estimates the volume of creek flooding water throughout each tidal cycle. In CFC for approximately 150 d during the growing season, nitrate and phosphate levels were increased on each rising tide to approximately 70 µmol L

  ) and the sample (F sample ) 15 N fraction: E = F sample -F background , with F = 15 N/( 15 N+ 14 N) = R/(R+2) and R = the nitrogen isotope ratio. For F background , we used values measured with the control (frozen) taxa. R was derived from the measured  15 N values: R = [( 15 N / 1000) + 1] x

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Natural isotopic composition of nitrogen ( 15 N) and carbon ( 13 C) of meiofauna

Figure 3 .

 3 Figure 3. Natural isotopic composition of nitrogen ( 15 N) and carbon ( 13 C) of meiofauna

Figure 4 .

 4 Figure 4. Individual grazing rates of bacterial carbon, per hour and per individual taxa of

Figure 5 .

 5 Figure 5. Individual grazing rates of algal carbon, per hour and per individual grazer of

Figure 6 .

 6 Figure 6. Fluxes between benthic bacteria and algae and their meiofaunal and epifaunal grazers (gC m -2 d -1 ) in three study creeks. * indicates significant (p < 0.01) differences in flux

Figure

  Figure

  Figure 2.

  Figure 3. 819

  Figure 4. 821

  Because this analysis did not reveal a creek effect, data from all creeks were pooled and the nematode community overall was composed of 0.5% deposit feeders, 1.1% scavengers, 41.1% epistrate feeders and 57.3% predators. Similarly, species composition of adult benthic copepods did not differ among creeks (ANOSIM, ns) and data from each creek were consequently pooled. The relative abundances of five most abundant copepod species of this community were Stenhelia sp. (20%), Pseudobradya sp. (17%), Enhydrosoma sp. (17%),

	Scottolana canadensis (15%) and Microarthridon littorale (8%).
	Mean total infauna abundances at the time of the grazing experiments were very similar
	to the long-term averages found by Mitwally & Fleeger (In review), and ranges expressed as
	10 3 ind m -2 almost completely overlapped for nematodes 287-1167 vs. 172-1783 and
	ostracods 33-98 vs. 2-112. However for copepods, means were higher at the time of the
	grazing experiments (267-562 vs. 20-130). The density of N. obsoletus was significantly (4-x)
	higher in CFC than RC (Johnson & Short 2012).

al. 2009a)

and 51-101

(Mitwally & Fleeger In review)

. Benthic algal biomass significantly differed among creeks at the time of the experiment, with the highest concentration of chl a in CFC and lowest in SFC.

We used the composition of trophic groups as a surrogate for the functional diversity of nematodes. Trophic groups did not differ among creeks based on Analysis of Similarity (ANOSIM, ns).

Table 1 .

 1 Contribution (in %) of fatty acids specific to diatoms, green algae/cyanobacteria, and bacteria to total fatty acid methyl esters (FAME) and weighted  13 C of each compound in three study creeks, see text for creek designations (means  SD; n=3).

		RC	SFC	CFC
	% FAME
	Diatom	15.9  1.6	11.3  0.7	17.0  0.9
	Green algae/cyanobacteria	1.8  0.3	1.8  0.1	1.6  0.0
	Bacteria	8.3  0.1	10.8  0.2	6.6  0.1
	Weighted  13 C
	Diatom	2973  123	2697  73	2180  38
	Green algae/cyanobacteria	3164  105	2633  194	3654  217
	Bacteria	472  7	323  6	808  8

Acknowledgements

Pierre-Yves Pascal conducted this research while being supported by a postdoctoral fellowship funded by Department of Energy Office of Biological and Environmental Research Award DE-FG02-05ER64070 and the Louisiana State University College of Science.

This material is based upon work supported by the National Science Foundation under Grant Nos. 0213767 and 9726921. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. We thank the Marshview field crew who maintained the nutrient enrichment experiment. We also would like to thank Jennifer L.

Bowen for her help in calculation of bacterial production.