Our Students and Their Research

Bacteria are central players in many important processes within aquatic ecosystems, yet little is known about the diversity, distribution and precise functions of these organisms. This is due mainly to the difficulty in culturing these organisms in the laboratory. However, the application of molecular techniques to the field of marine microbiology is beginning to unveil the mysteries of bacterial roles in marine systems. The DNA of cells can be directly examined without need to grow the bacterial cells. In this study a fosmid library was created storing the genetic information of bacteria present in surface waters of the mid Chesapeake Bay. Partial analysis of the library via ITS-LH-PCR and limited sequencing has revealed some of the diversity of bacterioplankton in the bay. 60% of the bacterial groups identified belonged to the actinobacteria class, which was traditionally associated with freshwater bacteria only. 25% of the species identified belonged to the bacteroidetes phylum, 9% to the Roseobacter group and 6% to the gamma subclass of the proteobacteria phylum. Further analysis of the library may reveal a greater diversity of species and the genetic information stored could even be used for other purposes in the future such screening for genes of particular interest or searching for the presence of antibiotic-producing organisms.

In the event of an oil spill, timely and informed response decisions must be made based on sound scientific research. In coastal areas, shallow water and rocky shoals serve as barriers to the use of recovery equipment and leave coastal life vulnerable to surface slicks. Dispersants could provide a solution to this problem; however, careful research must be conducted to determine the effects on benthic and water column life. This research studied the effects of chemically dispersed oil on the sensitive symbiotic soft coral Xenia elongata to determine toxicity levels in this common cnidarian. Three experiments were conducted testing the acute and sub-lethal toxicity of water accommodated fractions of Arabian Light crude oil (WAF), chemically enhanced water accommodated fractions (CEWAF), as well as the toxicity of the dispersant alone. A multi-tiered array of behavioral and physical endpoints were analyzed including pulse rate, pulse intensity, rigidity, as well as physical measurements of poly-aromatic hydrocarbons (PAHs), dissolved oxygen, chlorophyll content and DNA damage. The corals showed high sensitivity to both the chemically dispersed oil (CEWAF) and the dispersant alone. LC50s for dispersant alone were in the 50-60 range and concentrations at 25 ppm and higher produced strong adverse behavioral responses. In the acute experiment, 20 ppm dispersant treatments and CEWAF treatments at the 1 g oil/L with a 1:20 dispersant to oil ratio produced a complete paralysis of pulsing. All showed significant recovery in clean artificial sea water within two weeks with CEWAF treatments resulting in the slowest recovery. Chlorophyll levels and algal cell counts did not differ significantly between treatments when normalized to protein levels although there were lower levels of both protein and chlorophyll overall in the high acute CEWAF treatments. Further analysis of water and tissue samples will illuminate PAH uptake and accumulation levels as well as assess levels of DNA damage.

Methane is important to study because of its role as a greenhouse gas. In our attempts to solve environmental problems, it is important to fully understand the system we are dealing with so as to not inadvertently create a new problem, or add to a preexisting one. This study was focused on the relationship between denitrification and methanogenesis in areas dominated by agriculture. According to past lab studies, there should be a negative correlation between methanogenesis and denitrification in soils rich in nitrate. Three different farms were studied using a subset of 8 groundwater wells. Samples were taken and analyzed for dissolved methane, nitrogen gas, and oxygen. These variables were regressed to test the relationship predicted by the literature. Overall, there was found to be no significant correlation between methanogenesis and denitrification; however, there was a significant negative correlation found at one of the three farms when examined separately (r² = 0.58, p = 0.02). A highly significant inverse quadratic relationship was also found between the concentration of methane and the percent saturation of oxygen in the samples (r² = 0.33, p < 0.01). These results show that microsites in soils can produce methane despite high levels of nitrate and denitrification in the bulk soil properties.

This study examined the relationship between exposure to polychlorinated biphenyls (PCBs) and behavior and metabolic rates measured as oxygen consumption of juvenile snapping turtles, Chelydra serpentina. Two behavioral assays were done: a predator avoidance study and a foraging study. The turtles fed contaminated food were found be more active than those fed reference food in the first of three trials in the predator avoidance study. There was no significant difference found among treatments in the ability of juveniles to forage for live food. In the metabolism study the juveniles fed contaminated food were found to have higher rates of oxygen consumption than those fed reference food in two of three months. These results suggest that PCB contamination through food may affect the metabolism of the organism, but perhaps may not have a significant effect on behavior.

Aquatic microbial species have been proven to be difficult to culture and thus hard to study. The use of genetic analysis has begun to fill the knowledge regarding aquatic microbes. Analyses of ribosomal RNA genes in fosmid libraries constructed from bacterioplankton from surface waters of the Chesapeake Bay has revealed the presence of several groups of marine/estuarine microbes. Through PCR, sequencing, and phylogentic analysis, several aquatic bacterial groups have been identified. These include, but are not limited to Thiobacillus (Betaproteobacteria), Delftia (Betaproteobacteria), Verrucomicrobia, Synechococcus, Bacteroidetes, Paenibacillus (Firmicutes), and Actinobacteria.

Seagrass plays an important role in its ecosystem, but it has declined in the Chesapeake Bay to as low as 10% of historic levels. Increased seston concentrations, due to eutrophication and increased sediment loads, are believed to be the main cause of the seagrass decline because seston increases light attenuation, thereby decreasing photosynthesis. Oysters reduce seston concentrations through suspension feeding, and oyster reefs and breakwaters can also decrease seston through wave attenuation. A numerical model that included seagrass, waves, oysters, light, and seston (SWOLS model) was used to determine if oyster reefs and breakwaters could improve seagrass growth by reducing suspended sediment concentrations. Seagrass growth potential was calculated using different wave heights, the presence or lack of oysters and/or breakwaters, and two configurations of oyster reefs/breakwaters. Wave height was found to be the dominant factor influencing seagrass growth potential, with higher waves predictably causing decreases in seagrass growth potential. Breakwaters were found to improve seagrass habitat quality only in the presence of the highest wave heights simulated (0.4 m). Oysters did not affect seagrass growth in any of the simulations conducted, mostly likely due to their seston-dependent filtration rates which reduced filtration to less than 20% of full capacity in low (< 5 mg l^-1) and high (> 35 mg l^-1) suspended sediment concentrations. A dynamic phytoplankton component and wave reflection should be incorporated into the model in order to assess the influence of oyster filtration on both the organic and inorganic components of seston, and the effect of breakwaters on seagrass growth potential in high wave conditions.

Bacterial community composition was surveyed across three separate species and sites. Leaf and root samples from each bed per species were collected and bacterial community composition was characterized by denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA. Multidimensional scaling analysis revealed the dense canopy-forming Stuckenia pectinata and Potamogeton perfoliatus to have very similar leaf bacterial communities, between 86 and 90% degree similarity, whereas Zostera marina demonstrated much more variability within and across its beds. Differences in seasonality, salinity and plant structure affected bacterial community composition. Across species, bacterial community populations on Z. marina tended to be less similar on its leaves and roots than those on the roots and leaves of S. pectinata or P. perfoliatus. Bacterial cultures of 96 isolates found some overlap in community but no substantial matching, proving cultivation to be more difficult than anticipated for restoration purposes.

Microphytobenthos are important contributors to oxygen in the sediment, food sources for grazers, and stabilizers of sediment. However, little study on microphytobenthos has been performed in North America, specifically in the Chesapeake Bay. In this study, the assemblage of microphytobenthos was characterized and vertical migration of the microphytobenthos was assessed in the Choptank River, a tributary of the Chesapeake Bay. A new tool, the spectrofluorometer, was used to investigate both characterization and migration. Diatoms and cryptophytes were the dominant species in the benthos and results suggested that both migrated with each other, not independently. Overall, light had little effect on migration. The spectrofluorometer was a useful tool for quick species and migration assessment of the microphytobenthos.

Bacterioplankton play crucial roles in marine and estuarine environments. They comprise one of the largest biomass pools in these systems and are responsible in great part for important environmental processes such as nutrient cycling and organic matter decomposition. As part of a larger effort to understand these organisms, we have sought to characterize the presence and relative abundance of bacterioplankton in surface waters of Station 834 in the mid Chesapeake Bay. Specifically, we have constructed a Fosmid library that was subsequently screened for rRNA fragments. A series of PCR reactions using fluorescently labeled primers were performed in order to amplify three specific rRNA fragments. Such fragments have natural length variations the combinations of which are phylotype-specific. The results showed a very diverse bacterial community and suggested year-to-year variation in community structure. Five of the phylotypes present were putatively identified based on comparison with existing operon libraries, three of which belong to freshwater Actinobacteria in groups ACI-C, ACIV-D, and ACIV-C, one was a member of the Alphaproteobacterium from the Roseobacter group, and one a member of the Bacteroidetes phylum.

Presently, many species of marine fish are being captured for their decorative characteristics in a fish tank, rather than their nutritional qualities on the dinner table. These fish are called marine ornamentals, and they are currently on the decline due to over harvesting and loss of habitat. One of the main fish in this category is the clownfish. Raising these fish in a controlled aquaculture setting is a possible solution to the declining populations. I have tested several nutritional enrichments on rotifer/Artemia diets for clownfish larvae. The three enrichments are 1) Aquagrow Advantage 10% docosahexaenoic acid (DHA, 22:6n - 3), 10% protein, 23% fat, 9% carbohydrate, 2) Aquagrow Advantage-EN 10% DHA + 750 mg/kg astaxanthin, 24% protein, 24% fat, 24% carbohydrate, and 3) Aquagrow Advantage-BL 10% DHA, 2% arachidonic acid (ARA, 20:4n - 6) and eicosapentaenoic acid (EPA, 20:5n - 3), 13% protein, 25% fat, 12% carbohydrate. The control for this experiment is rotifers fed nanochloropsis algae (0.22% DHA) followed by newly hatched Artemia (1.1% DHA). The survival, growth, and rate of metamorphosis of these clownfish were tested for each of the four diets. I predict that the enriched diets will lead to better survival, more growth, and faster metamorphosis than the unenriched diet.

Nitrogen is considered to be the most common nutrient limiting primary production and growth in the oceans (Capone, 2001; Blackburn and Sorensen, 1988). Nitrogen fixation was studied in the rinsed roots, rhizomes, and blades with associated sediment of several bay grasses including Vallisnaria americana, Potamogeton perfoliatus, Hydrilla verticillata. Nitrogen fixation was also studied in pelagic areas containing cyanobacterial blooms, and sediment loaded with different organic treatments in mesocosms. Nitrogen fixation was determined using the Acetylene Reduction Assay and detected using a flame ionization detector, Gas Chromatograph. Organic content seemed to play the major factor influencing nitrogen fixation in sediment. The mesocosms that contained different organic treatments had higher rates of nitrogenase activity in the higher percentages of organic treatments. Bay grass roots, Myriophyllum sp. yielded higher rates of nitrogenase activity. Rates of nitrogenase acitivity in the pelagic realm was higher in the cyanobacterial bloom rather than outside of the bloom. Overall in the Chesapeake Bay nitrogen fixation was ubiquitous, variable, and relatively high in localized areas and maybe a source of "new" nitrogen.

Sediment cores were taken from the main stem of the Chesapeake Bay and the Saint Martin River and analyzed for trace metal contamination. The goal of this study was to determine the recent history of trace metal contamination in both sites. A flame atomic absorption spectrometer was used to analyze for the metals copper, nickel, iron, lead, and manganese. Due to an inability to date the cores with ²¹⁰Pb, ages were estimated assuming the lead concentration peak occurred in 1975. Past research has shown a decrease in trace metal contamination since the mid 1970s. Other than copper in all the cores and the northern Chesapeake Bay, this study upholds this trend.

The potential influence of large-scale climatic processes on freshwater flow into Chesapeake and Delaware Bays was assessed using indices of the North Atlantic Oscillation (NAO): the NAO index and the North Atlantic Center of Action (NACOA) indices. This influence has far-reaching implications for the ecology of the bays because freshwater flow controls physical and biological processes that affect a suite of organisms, including blue crabs. Significant correlations were found between the NAO and NACOA indices and freshwater flow in multiple river systems (Susquehanna River, Delaware River, Potomac River, James River, and the Chesapeake basin) during selected months (March - May, July - October). Five variable regression models had adjusted R² values that ranged from 0.10 to 0.61 with highest predictive ability for total Chesapeake flow in April. NACOA variables with lags of 18-22 and 33-35 months were significant terms in several regression models of flow in May and July-September, indicating that freshwater flow may be influenced by coupled ocean-atmospheric processes that occurred ~2.8 and ~1.5 years prior to these months. Results support the use of parameters that describe the intensity and location of the Icelandic Low and Azores High pressure systems (i.e., the NACOA indices) in addition to, or instead of, the NAO index (which is the difference between the normalized pressures of the two systems). The predictive ability of the regression equations was tested by comparing observed versus modeled values from 1992 to the present. R² values of observed versus modeled ranged from nearly zero to 0.74 and suggest that in some months and river systems these large-scale climate indices could be used to reasonably predict freshwater flow.

The diets of larval and age-0 juvenile striped bass (Morone saxatilis) were evaluated with respect to the estuarine turbidity maximum zone (ETM) in upper Chesapeake Bay. Stomachs of larval and juvenile M. saxatilis collected on NSF sponsored BITMAX cruises (2001-2003) were analyzed. The three years in which samples were collected had experienced different hydrological conditions that affect hydrography of the upper Bay and its ETM. Precipitation and freshwater flow were near average in 2001, low in 2002, but well above average in 2003. Prey items in striped bass stomachs were quantified and identified to the lowest feasible taxonomic level. Prey compositions and amounts consumed were compared among the three years as well as among locations relative to the ETM (above or within). Feeding incidence, fullness index ([Stomach Content Weight] + [Stomach Weight + Content Weight] X 100), and weight of prey items per fish stomach were used as measures of feeding success. Ontogenetic shifts in feeding and feeding success were determined. Feeding and feeding success were analyzed with respect to salinity gradients and turbidity that define the ETM. Diets of larval striped bass in upper Chesapeake Bay consisted mainly of copepods (Eurytemora affinis and other calanoid copepods) and cladocerans (Bosmina longirostris). The analysis revealed that larval striped bass fed more successfully in the wet year 2003 than in the average year 2001. The most common prey items in juvenile striped bass diets were amphipods in three genera (Gammarus, Monoculodes, and Corophium) and shrimps in two genera (Neomysis and Crangon). Juveniles fed better in the dry year 2002, when their numbers were low, than in either 2001 or 2003 when they were more abundant. Spatial feeding success differed between years. In 2001 juvenile striped bass fed more successfully above the ETM whereas in 2002 and 2003 they fed better within the ETM. The ontogenetic analysis revealed that larval striped bass became increasingly successful feeders as they grew. Complex combinations of physical and biological factors, including retention of prey and its visibility under variable turbidity and salinity conditions in the ETM region, can control prey consumption, growth and survival of young striped bass in estuarine nurseries.

Recruitment, defined as the number of individuals that reach a specific stage of the life cycle, is highly variable and largely unpredictable in fish populations. Although many studies have focused on the early life stages of fish, the mechanisms causing mortality or affecting growth, which contribute to recruitment variability, still are not clearly understood. Cohort analysis of larval white perch, Morone americana, was used to investigate the effects of environmental conditions and zooplankton density on cohort-specific vital rates and recruitment in the Patuxent River estuary (Maryland). Cohort-specific vital rates and abundances were expected to reflect the biotic and abiotic factors experienced by the cohorts. More specifically, early spawned cohorts were expected to experience lower growth rates and higher mortality due primarily to lower temperatures and storm events. Additionally, we expected cohort-specific abundance to be positively correlated with zooplankton density. Ichthyoplankton and zooplankton were collected from two stations in the Patuxent River on five dates (April to May) in 2004. Modal progression analysis based on larval length frequency distributions indicated that only two cohorts dominated the young of the year in 2004, one of which only appeared on the last two sampling dates. A comparison of continuous records of environmental variables (temperature, precipitation, dissolved oxygen, and salinity) with larval white perch abundance suggested an early season storm event, associated with a drop in temperature to approximately 10°C and substantial rainfall, resulted in high larval mortality and influenced larval white perch spatial distribution. In addition, microzooplankton densities remained sub-optimal to support white perch growth and development throughout the sampling period, which may also have been a contributing factor in cohort-specific mortality. The results of this study emphasize the importance of sampling across a wide geographical range when tracking cohorts, as weather events advecting cohorts downriver can bias mortality estimates in a narrow geographical sampling range. The uncharacteristically low number of cohorts observed in the 2004 year class was reflected in the historically low juvenile white perch abundance in 2004. This study builds on the foundation of research investigating the effects of environmental conditions and zooplankton density on larval recruitment, and contributes to our understanding of the influence of storm events on recruitment variability in the early life history of white perch.

The release of PCBs into the upper Hudson River during the mid 20th century has potentially had a detrimental effect on the river's biota. To examine some of these effects, snapping turtles were used as a model species to examine several behavioral endpoints. Turtles were chosen for study because they are long lived, accumulate high PCB concentrations due to their high lipid compounds, and are at a high trophic status. Turtles were collected as eggs in the upper Hudson River from both contaminated and reference sites. High PCB load in eggs is due to maternal transport during vitellogenesis. Once hatched, turtles from each treatment group received either contaminated or reference food. To test the sublethal effects of PCBs, assays of four behaviors having implication for survivorship were conducted; righting response (time to reorient body after being inverted), stimulus response (recognition and response to a physical stimulus), foraging efficiency (ability to located and consume food) and predator response (recognition to a visual response representing a predator). The only test with significant results was the latter. Turtles from uncontaminated areas had a greater response to a simulated avian predator than turtles fed contaminated food or from contaminated areas of the Hudson River. Because of this result, PCBs in the upper Hudson River appear to remain a threat to the health of the Hudson River biota, even decades after PCB inputs have ceased.

Estuaries are important nursery grounds for many economically and ecologically important species including Atlantic menhaden, Brevoortia tryannus (Clupeidae). The Chesapeake Bay serves as one of the most important nursery grounds for this species, with nearly 80% of the annual recruitment of this species thought to be derived from the Chesapeake Bay. Recently, recruitments of menhaden in Chesapeake Bay have declined. One potential cause is reduced production associated with declines in habitat quality. There are multiple ways to measure habitat quality, but due to the rapid response of cellular RNA concentrations to variation in resource availability to menhaden, RNA:DNA ratios represent an ideal metric to measure habitat quality. Our objectives were to quantify the relationship between food ration, RNA:DNA ratio, and growth of menhaden in the laboratory and then use these data to infer habitat quality from RNA:DNA ratios measured from menhaden collected in the Patuxent River. Laboratory RNA:DNA ratios were measured by spectroflourometric techniques on fish from a 14 day experiment with three varying food rations based on body weight. We detected a significant change in RNA:DNA ratio among treatments (F_2,2 = 21.54,p = 0.0444), days (F_1,26 = 54.05, p= 0.0001), and treatment-day interactions (F_1,26 = 39.52, p = 0.0001). A change in weight and length was also observed with significance for day (F_1,29 = 200.44, p = 0.0001; F _1,29 = 91.73, p = 0.0001), and treatment-day interaction (F _2,29 = 156.8, p = 0.0001; F_2,29 = 31.96, p = 0.0001). These laboratory results represent the first measurements of RNA:DNA ratios for menhaden and will aid in future studies. Field work took place by seining in the Patuxent River on a weekly basis at multiple sites. Field results observed for CBL Pier were a decline in RNA:DNA ratio over the course of the summer months and based on laboratory results, show a RNA:DNA ratio equivalent to low treatment fish.

Marine sedimentary environments are biogeochemically active and face regular disturbance events. The topmost centimeters of the sediment are characterized by steep chemical gradients, and by disrupting the sediment surface, disturbance can create chemical patchiness on local scales. This has important implications for primary productivity, as well as recruitment and growth of benthic larvae and juveniles. The results of many laboratory studies focusing on the effects of increased concentrations of sedimentary ammonium on habitat selection and productivity are significant to the understanding of the processes by which larval and juvenile infauna colonize new environments. This experiment examined the effects of manipulated ammonium levels on primary productivity in the field. While ammonium levels were successfully spiked, chlorophyll A concentrations were not stimulated by the experimental manipulation. This is likely due to the fact that the environment in which the study was conducted did not seem to be nitrogen limited, and in fact, inhibition may have occurred in the face of excess nitrogen. Future studies involving altered surface chemistry are very important to understanding the biogeochemical processes that influence recruitment, growth, and primary productivity.

Cnidarian-algal symbioses exist as a sensitive balance between the two partners. This symbiosis may be disrupted upon exposure to stressors including organic contaminants such as crude oil. Symbiosis breakdown can lead to algal cell loss i.e. bleaching which may result in death of the species. This is particularly problematic in tropical regions where corals form the trophic and structural foundation of the ecosystem. Following an oil spill responders must decide upon the appropriate action, often leading to trade off decisions regarding different species and habitats. Therefore, using chemical dispersants would protect mangroves but potentially impact the sub-tidal coral reefs that previously would have received little or no impact from an overlying spill. Therefore, these decisions must be made using sound scientific data. We used the temperate sea anemone Anthopleura elegantissima as a model of this sensitive cnidarian-algal symbiosis. Anemones were exposed to realistic short-term (8 hour) acute doses of various dilutions of water accommodated fractions (WAF) and chemically enhanced WAF (CEWAF) of weathered Arabian Light crude oil using standard CROSERF techniques. Injury was determined using a multi-tiered approach employing an array of metrics from acute endpoints (e.g. mortality) to behavioral and biochemical assays. Mortality was not observed even at the highest dose (100% stock solution, using 25g oil / L water) after 8 hours of exposure in either the WAF or CEWAF exposures. However, tentacle and column expansions were significantly reduced in the higher CEWAF (not WAF) exposures. In addition visual and quantified production of mucus increased with dose, particularly in the CEWAF exposures. Further analyses of water chemistry (for determination of PAH and TPH levels) and molecular endpoints are underway. This work has provided the information required to base a detailed long-term anemone exposure on and will assess the sensitivity of this species to crude oil and whether it serves as a good model of cnidarian-algal symbiosis.

Recruitment variability causes fluctuations in fish populations and is mostly attributed to variable survival rates during early life. High mortality rates in egg and larval stages prevent dramatic increases in numbers, but mortality rates significantly higher than the average cause poor recruitments of year classes. Factors that control feeding and growth at the larval stage are important in determining survival and thus recruitment success or failure. In this study, feeding success and growth rates of white perch larvae in designated areas of the upper Chesapeake Bay were evaluated based upon ichthyoplankton surveys conducted during the May 2002 spawning season. A particular objective was to determine if the salt-fresh transition zone, i.e., the Estuarine Turbidity Maximum (ETM), was an area of enhanced feeding or larval growth. Comparisons of larval feeding (based on gut analysis) and growth rate (based on otolith increment analysis) encompassed four broad areas in the upper Bay, including the 1) shoal and 2) channel regions 3) above and 4) within the ETM. Planktonic copepods were the most common prey in larval white perch guts in all areas. The mean number of copepods per gut was significantly higher in larvae from channel stations within the ETM compared to channel stations up-estuary of the ETM, supporting the hypothesis that the ETM serves as an advantageous larval nursery. However, feeding incidence (percent of larvae with prey in guts) and mean number of total prey per larval gut did not differ among areas. Larvae >4.5mm in length had a higher feeding incidence and mean number of prey per gut than first-feeding larvae <4.5mm. The low feeding incidence and mean number of prey per larval gut in the smaller larvae suggested poor feeding conditions for the smaller larvae, which may have contributed to poor recruitment of white perch in 2002. No significant differences in growth rates were detected among locations. Larvae in the channel, on the shoal, above the ETM, or below the ETM were growing at similar rates (0.20 to 0.30 mm/d). In addition, no significant differences were detected in recent (past 48 hours) growth rates. One interpretation is that there is little variability in larval habitat quality among the areas evaluated in this study. However, high variability in growth rates among individual larvae suggests that small sample sizes may have reduced the power of statistical inference to detect differences. A potential confounding factor is the possibility that undernourished or starving larvae, which had been feeding and growing poorly, were dying at above average rates and were poorly sampled, making them unavailable for inclusion in the growth-rate analysis. A future study focusing on spatial variability in abundance of microzooplankton that serve as prey for white perch larvae would be important to better define relative habitat quality of areas occupied by larval white perch.