GPMB September 2011 Colloquium:
Abstracts of Oral Presentations
TOXIC DINOGLAGELLATES AND THEIR ASSOCIATED MICROBIAL COMMUNITY: PRELIMINARY EVIDENCE THAT ALGICIDAL BACTERIA INTERFERE WITH THE AL-2 QUORUM SENSING SYSTEM
Blair, W. (GPMB, College of Charleston) and Doucette, G. (Marine Biotoxins Program, NOAA/NOS)
The development of harmful algal blooms that impact coastal zones worldwide is regulated by a wide range of biophysical factors. Marine bacteria, although often overlooked, serve important ecological functions in a variety of microhabitats, including delivery of trace metals, vitamins, and limiting nutrients, as well as algicidal assaults. How the complex microbial community coordinates to conduct these influential activities is a rapidly growing research area. Discovered only in the last few decades, quorum sensing (QS) systems allow synchronization of density-dependent behaviors, such as biofilm formation and bioluminescence, sometimes eliciting chemical interference from eukaryotic hosts. Involvement of Karenia spp. and their associated bacteria, including algicidal taxa, in QS systems would introduce a new paradigm for understanding biotic regulation of bloom dynamics. The Autoinducer-2 (AI-2) QS system in particular is utilized by a wide variety of bacterial taxa and is inhibited by a number of biological and synthetic antimicrobial compounds. Preliminary data suggest that several algicidal bacterial strains isolated from harmful algal blooms may interfere with this important AI-2 system. Using a bioluminescent reporter strain of Vibrio harveyi stimulated by exogenous AI-2, algicidal and non-algicidal bacterial strains were screened for inhibitory activity. Incubation with sterile filtrate taken from late-exponential algicidal cultures significantly reduced sensor luminescence (ANOVA, p < 0.05), whereas incubation with non-algicidal filtrate did not reduce luminescence. While additional work will be required, this preliminary report provides the first evidence that the widespread AI-2 quorum sensing system may be inhibited by bacterial activity.
MYXOZOAN PARASITES OF THE SPOTTED SEATROUT: DO THEY WEAKEN THE HEALTH OF THEIR HOSTS?
Cosmann, P. (GPMB, College of Charleston), Arnott, S. (SCDNR), de Buron, I. (College of Charleston), Roumillat, W. A. (SCDNR) and Strand, A. (College of Charleston)
Preliminary studies into the parasite community in the spotted seatrout, Cynoscion nebulosus, have revealed infection by particularly pathogenic parasites: Kudoa inornata in the skeletal muscle, and Henneguya sp. in the bulbus arteriosus. I hypothesized that these two parasite species negatively affect of the health of infected spotted seatrout. To examine this, I quantified the intensity of parasite infection in wild-caught seatrout via histology and assessed fish health in terms of somatic, splenosomatic and hepatosomatic indices. After controlling for variation in fish size (using an analysis of covariance), fish that were infected with K. inornata were shown to have significantly lower liver and spleen weights compared to those that were not infected. Preliminary results show liver weight but not spleen weight correlated negatively with the intensity of K. inornata infection. There was no correlation detected between the presence of Henneguya sp. and liver and spleen weights. Since liver mass is a proxy for energy storage, these results imply that K. inornata imposes a substantial energy burden on seatrout, and that infected fish have less energy available for metabolically expensive processes, such as overwinter survival and reproduction.
RECOVERY FROM HYPOXIA AND HYPERCAPNIC HYPOXIA: IMPACTS ON THE TRANSCRIPTION OF KEY ANTIOXIDANT GENES IN THE SHRIMP LITOPENAEUS VANNAMEI
Darling, C. (GPMB, College of Charleston), Burnett, K (College of Charleston) and Burnett, L. (College of Charleston)
The Pacific whiteleg shrimp, Litopenaeus vannamei, inhabits coastal estuarine waters which are prone to intermittent bouts of low oxygen (hypoxia) and high CO2 (hypercapnia) followed by a return, or recovery, to fully air-saturated levels of oxygen and CO2 (normoxia). Hypoxia (H) and hypercapnic hypoxia (HH) can cause oxidative stress which induces production of reactive oxygen species (ROS) that damage surrounding cells and tissues. In vertebrates, sudden rises in oxygen levels also increase ROS production. Cellular production of antioxidants, which convert ROS into non-toxic molecules, can prevent cell damage. Here we examine antioxidant production in the hepatopancreas of L. vannamei after recovery from H or HH. Prior studies revealed that transcription of antioxidants thioredoxin-2 (TRX-2) and glutathione-s-transferase (GST) was significantly upregulated (2.7; 2.9-fold, respectively) after 4 h exposure to H. Only GST was significantly upregulated (3.9-fold) after 4 h in HH; neither antioxidant was upregulated after 24 h in H or HH. In the current study we exposed shrimp to H or HH for 4 or 24 h. Shrimp subsequently recovered in normoxia for 1, 6, or 24 h. Transcriptional changes of TRX-2, GST, and other antioxidant-related genes are currently being quantified by qRT-PCR. Based on prior studies detailed above, we expect that transcription of antioxidant-related genes will further increase in response to increases in oxygen during recovery from 4 h H or HH, but that this increase will not occur during recovery from 24 h H or HH, leaving these animals susceptible to oxidative damage as tissue oxygenation returns to normal levels.
DETERMINING FACTORS THAT INFLUENCE THE MOLECULAR QUANTIFICATION OF THE HARMFUL RAPHIDOPHYTE, HETEROSIGMA AKASHIWO, USING A SANDWICH HYBRIDIZATION ASSAY (SHA)
Doll, C. (GPMB, College of Charleston) and Greenfield, D. (University of South Carolina & SCDNR)
Sandwich Hybridization Assay (SHA) is a molecular technique that enables direct detection of a particular species or taxa using large subunit rRNA-targeted DNA probes. SHA has been adapted to detect and quantify bacteria, invertebrate larva and several harmful algal bloom (HAB) species. In each case, an optical density directly proportional to the amount of rRNA in a sample is used to approximate organism abundance. The extent to which variability in cellular rRNA content may influence SHA results for HAB species, however, remains unclear. This study examines the influence of various factors on the SHA response of Heterosigma akashiwo; a globally distributed HAB species known to produce ichthyotoxic blooms for which SHA capabilities have previously been developed and assessed. Experiments in this study demonstrate variable SHA responses among geographically distinct isolates and changing responses throughout the algae’s growth phases and diel cycle. Additional experiments will test the influence of Lugol’s iodine preservation and nutrient limitation on SHA response. Results will provide new insights on the influence of these factors on SHA responses and whether observable variations pose important considerations for resource managers interpreting results. Additionally, SHA results will be compared to quantitative PCR data as part of a broader four-year NOAA-MERHAB award to assess the two using criteria such as cost, accuracy, range of detection, speed, etc.
QUANTIFICATION OF THE INTERACTION BETWEEN BOTTLENOSE DOLPHINS AND THE ATLANTIC BLUE CRAB FISHERY IN CHARLESTON, SC
Duquette, A. (GPMB, College of Charleston), Kracker, L. (NOAA/NOS/CCEHBR) and McFee W. (NOAA/NOS/CCEHBR)
Entanglement in commercial fishery equipment is the number one anthropogenic cause of marine mammal death worldwide. Scientists turned to acoustic methods for monitoring and deterring marine mammals from fishing gear. Despite research efforts, there remains a lack of knowledge about the methods of entanglement. Until those methods are understood, sufficient management is near impossible. The Atlantic blue crab fishery is the number one cause of fishery-related incidents for bottlenose dolphins (Tursiops truncatus) in South Carolina. It is hypothesized that the dolphins are attracted to the pots by the bait and possibly the other marine life drawn to the pots. Hydrophones deployed in baited and unbaited crab pots will record dolphin sounds and allow for a quantification of the interaction. If dolphins are attracted by the bait, then they will approach and spend more time around the baited pots than the unbaited pots. There appears to be little difference between the numbers of times dolphins vocalize in the vicinity of the crab pots between the two treatments. There is also no significant difference between the amounts of time dolphins spend around the traps based on treatment. Thus, the presence of bait does not appear to affect how often and for how long dolphins are in the vicinity of crab pots.
THE ROLE OF NANOG IN MAINTAINING LUNG HEALTH IN THE BOTTLENOSE DOLPHIN, TURSIOPS TRUNCATUS
Glade, L. (GPMB, College of Charleston) and Baatz, J.E. (Department of Pediatrics, MBES, MUSC)
Diving marine mammals experience decreased oxygen supplies during periods of breath holding but are well adapted to managing their oxygen stores to survive extended periods of submergence. One of the most important organs that aids in diving is the lung, which is involved in buoyancy control and undergoes progressive collapse with increases in pressure, causing the alveoli to empty gas into the small airways. Although the physiological, anatomical, and behavioral adaptations of marine mammals to limited oxygen supplies have been thoroughly researched, the processes involved in the maintenance and repair of the lung remain to be investigated. This study examines the role of Nanog, a protein that is associated with pluripotency of stem cells, in directing replenishment of lung cells. Bottlenose dolphin (Tursiops truncatus) lung cells were grown in 6-well plates in an incubator for 72 hours under conditions of normoxia (21% O2) or hypoxia (1.5% O2). Cells from both treatment groups were collected via lysis buffer and stored at -80°C. Total RNA was extracted from thawed cells and RNA was converted to cDNA. Real-time PCR was then used to analyze cDNA products, and preliminary results indicate that Nanog expression is considerably higher in lung cells exposed to hypoxic conditions relative to lung cells under normoxia. Future research will focus on protein changes in lung cells under hypoxia, and inhibitory RNA will also be used to initiate downregulation of Nanog and determine if lung cells are able to differentiate.
ANTAGONISTIC INTERACTIONS AMONG BACTERIAL POPULATIONS OF MARINE INTERTIDAL SEDIMENTS
Hook, W. (GPMB, College of Charleston) and Plante, C. (College of Charleston)
Most sedimentary bacteria live in biofilms attached to sediment grains. High densities and diversity of these bacteria implicate inter-specific competition as a likely force in structuring communities. A common means of competition exhibited by both free-living and substrate-associated bacteria is the secretion of antimicrobial compounds, serving to inhibit growth or cause cell death of nearby bacteria. Samples collected from the intertidal zone at Breach Inlet, South Carolina were separated into sediment and porewater fractions. A disc-diffusion assay was use to detect antagonistic activity in bacterial isolates. Of the sediment-derived isolates, 40% displayed the ability to produce inhibitory compounds whereas significantly fewer porewater-associated bacteria (25%) produced antimicrobials. The higher frequency of antagonism assumed by surface-attached bacteria likely reflects a more effective means of antimicrobial conveyance by attached bacteria. Compounds secreted by free-living bacteria are dispersed into the water column whereas secretions of surface-associated producers are sequestered in biofilms and on surfaces, where the antimicrobials remain and can affect surrounding microbes. In order to establish relative in situ abundances of the isolated antimicrobial-producing bacteria and designated target strains, denaturating gradient gel electrophoresis (DGGE) was used to compare DNA bands from whole-community environmental samples to the banding patterns of individual isolates. Sequencing of a region of the 16S rRNA gene was used to confirm presence/absence of the model bacteria within the sediment communities. These environmentally relevant bacteria will be employed in microcosm experiments to elucidate the role of pre-emptive and interference competition in benthic microbial communities.
POPULATION STRUCTURE OF SPOT (LEIOSTOMUS XANTHURUS) IN SOUTH CAROLINA
Johnson, J. (MES, College of Charleston), Arnott, S. (SCDNR), Roumillat, W.A. (SCDNR), Whitaker, D. (SCDNR), Ballenger, J. (SCDNR) and McDonough, C. (SCDNR)
Spot is a commercially and recreationally important fish species along the Atlantic and Gulf of Mexico coastlines. To date, no coast-wide stock assessment has been performed for the species due to a lack of biological and fisheries data. In particular, poor information exists on the age structure of the population, the size at which fish mature, and the age and size structure of harvested fish. The purpose of this study is to address these needs by, first, validating the timing and frequency of annulus deposition in spot otoliths, which are used for ageing them. With this information, I will then age spot caught in a long-term fishery-independent survey (SCDNR trammel net survey) and a fishery-dependent survey (recreationally harvested fish). I will also determine the sex of each fish and histologically examine their gonads to determine their maturity. These data will be used to model the relationships between size and age, size and maturity and age and maturity. The models will then be applied to the size data that are routinely collected during the surveys in order to infer the age and maturity structure of the population. The findings from this study will provide essential life history information that is needed for stock assessment purposes.
IMPACT OF TEMPERATURE-INDUCED VIRAL RESISTANCE ON DIMETHYLATED SULFUR COMPOUNDS IN THE COCCOLITHOPHORID, EMILIANIA HUXLEYI
Kendrick, J.B. (College of Charleston, GPMB) and DiTullio, J. (College of Charleston)
Emiliania huxleyi (Ehux) is a cosmopolitan coccolithophorid which forms very dense blooms annually that impact the global cycles of carbon and sulfur. The blooms are known to be terminated by host-specific viral infection. Until recently the effect of temperature on the success of viral infection has not been addressed. In preliminary work it was found that Ehux strain CCMP 374 is resistant to viral infection if incubated at 21°C but susceptible to infection at 18°C. Given that viral infection is known to cause oxidative stress in Ehux and that dimethylated sulfur compounds (e.g. DMS, DMSP, and DMSO) are involved in Ehux’s natural antioxidant cascade, we hypothesized that these compounds helped impart resistance by enhancing the cell’s antioxidant defense. The results of this experiment will be presented outlining the interactive effects of elevated temperature and viral infection on several host physiological factors, including the production and release of environmentally important sulfur compounds such as dimethylsulfoniopropionate (DMSP), dimethylsulfoxide (DMSO), and DMS. These findings could indicate significant changes in global biogeochemical cycles if the Earth’s temperature continues to rise..
A LATITUDINAL BODY SIZE CLINE IN A MARINE ISOPOD REFLECTS LOCAL ADAPTATION TO SEAWATER TEMPERATURE AND PREDATOR RISK
Manyak, A. (GPMB, College of Charleston), Bell, T. (College of Charleston) and Sotka E. (College of Charleston)
Organismal body size strongly affects individual fitness, with larger body sizes generally being positively correlated to mating success and fecundity. It has been widely observed that organisms from higher latitudes tend to be larger than their lower latitude counterparts (termed Bergmann’s Rule). For most body size clines, however, it remains unclear whether this reflects a genetic or phenotypically-plastic response, nor what evolutionary mechanism(s) maintain the cline. Consistent with Bergmann’s rule, field-collected individuals of the marine isopod Idotea balthica from northern (Massachusetts) sites are larger than individuals from southern (Virginia) sites. We reared juvenile I. balthica from field-collected mothers at a range of temperatures (6°, 12°, 18°, 24°, and 30°C), and measured growth and survivorship for 15 weeks, or until female sexual maturity. Overall, juvenile growth rate increased and time to female maturity decreased with temperature among all populations. Females from the south, however, became mature in less time and at a smaller size than did northern females. For example, southern and northern females became reproductive at ~52 and 71 days (respectively) at 18ºC. Earlier maturation of southern females is predicted by life-history theory to occur within predator-rich environments. We confirmed this prediction using field-tethering experiments in July and August 2011: tethered isopods within southern seagrass beds were 2-3 times more likely to be consumed than were isopods at northern sites. We conclude that the body size cline in I. balthica is adaptive, and that both predation risk and temperature may play an important evolutionary force in the development of this cline.
THE INFLUENCE OF ENVIRONMENTAL pH/HYPERCAPNIA ON THE SHELL STRENGTH OF THE EASTERN OYSTER, CRASSOSTREA VIRGINICA
Sherer, E. (MES, College of Charleston) and Burnett, L. (College of Charleston)
Estuaries are highly productive areas that, as a result, are subject to large fluctuations in CO2 and pH. Respired CO2 results in decreased pH and drives carbonate chemistry reactions away from CO32-, the carbon species used by bivalves to form their CaCO3 shells. The result could lead to weaker shells and higher metabolic demands to compensate for acid-base regulation. The threats faced by bivalves in these areas are compounded by “Ocean Acidification”, the process by which the anthropogenic CO2from the burning of fossil fuels sinks into the water and drives pH even lower. Estuaries are particularly prone to ocean acidification due to their limited buffering capacity. Understanding the threats faced by estuarine bivalves under present day hypercapnia can help us understand what may happen under future ocean acidification regimes. One possibility is that pH induced weakening of shells could result in making them easier for predators, such as crabs, to prey on. In this study I examine the relative shell strengths of Eastern oysters collected from various pH regimes in Charleston estuaries. In addition I am collecting shell size, mass, thickness and density for each organism tested. If oysters collected from lower pH waters are weaker then it could result in higher predation on these keystone species and dampen the ecosystem services they provide. This field approach also takes into account any long-term adaptation or acclimation to ambient water conditions that cannot be obtained in lab studies.
EFFECT OF SEDIMENT ON THE ANTIMICROBIAL ACTIVITY OF MELANOCHLAMYS DIOMEDEA AMONG DIFFERENT HABITATS
Smoot, S. (GPMB, College of Charleston), Plante, C. (College of Charleston) and Podolsky, R.D. (College of Charleston)
Gelatinous egg masses are used by several marine invertebrates as a reproductive mode that encapsulates embryos until hatching. The absence of a hard outer covering makes these egg masses particularly susceptible to microbial infection, biofouling, and predation. The mucus and gel matrix surrounding the egg capsules may contain compounds that deter microbial infection. If adults could adjust the level of protection in response to risk, then the amount of antimicrobial activity found within an egg mass should be correlated with the bacterial load of the local environment. We are comparing antimicrobial activity among sites of Melanochlamys diomedea egg masses on San Juan Island, WA with the bacterial density and sediment grain size. Egg masses were collected from the field, lyophilized, and extracted with non-polar ethyl acetate (EtOAc) and polar methanol (MeOH). The extracts were then tested and quantified for antimicrobial activity against a marine type culture (Bacillus subtilis) in a Burkholder petri dish and 96-well plate assay. Bacterial density was determined using a general bacteria stain and sediment grain size with a RoTap. Differences among sites have been found and continue to be investigated.y.
PERFORMANCE CHANGES WHEN EXPOSED TO VARYING OXYGEN LEVELS IN THE ATLANTIC BLUE CRAB, CALLINECTES SAPIDUS RATHBUN
Stover, K. (GPMB, College of Charleston), Burnett, L. (College of Charleston), McElroy, E. (College of Charleston) and Burnett, K. (College of Charleston)
The ability to perform continuous activity and resist fatigue is essential to completing tasks such as obtaining food, avoiding predators and finding a mate, but these abilities may be compromised by environmental stressors. Estuarine organisms face daily and seasonal fluctuations in salinity, temperature, dissolved oxygen and pH. The Atlantic blue crab, Callinectes sapidus (Rathbun) is an important commercial and recreational fishing species that resides in the estuarine waters of the Atlantic Ocean and Gulf of Mexico. In this study we tested the effects of 2 levels of hypoxia (4 kPa, 20% air saturation; 10.4 kPa, 50% air saturation) on fatigue during sustained continuous exercise. Fatigue was induced by an exercise trial that entailed continuous sideways hexapedal walking on an underwater treadmill. Fatigue was quantified as the percent decrease in holding performance, which was assessed with a repeated hold force test that mimics the way a male holds a female during mate guarding. Fatigue was defined as a 33% decrease in hold force from pre-exercise values. Fatigue was reached after 6 h of walking for crabs in normoxic seawater, 4 h in 50% air saturation and 2 h in hypoxia. Fatigue-resisting behaviors (180˚ turns, stopping and riding to the end) increased from the initial time point by 0.910 behaviors per h in normoxia, 4.075 in 50% air saturation, and 13.821 in 20% air saturation. The force and behavioral results indicate that under low oxygen conditions performance is decreased and fatigue is reached more quickly as the level of hypoxia intensifies.
COMPARATIVE EFFECTS OF IN VITRO PFOS AND DE-71 EXPOSURE ON BOTTLENOSE DOLPHIN AND MURINE IMMUNE FUNCTION
Wirth, J.R. (GPMB, College of Charleston), Peden-Adams, M.M. (University of Nevada), Henry, N. (NOAA/NOS/CCEHBR) and Fair, P.A. (NOAA/NOS/CCEHBR)
The effects of emerging contaminants on the immune system of mammals are poorly understood, with limited information available about the immunotoxicological effects of perfluorooctane sulfonate (PFOS) and DE-71, a polybrominated diphenyl ether (PBDE) mixture, at environmentally relevant exposures. Due to the challenges of working with dolphins, this study assessed the effects of in vitro exposures of PFOS and DE-71 on immune functions in dolphins and mice to determine the predictability of in vitro effects to in vivo observations as part of a parallelogram approach to assessing risk. Bottlenose dolphin peripheral blood leukocytes and B6C3F1 mouse splenocytes were exposed in culture to PFOS at 0, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 μg/mL concentrations or DE-71 at 0, 0.025, 0.05, 0.25, 0.5, 2.5, 5.0, 25 or 50 μg/mL concentrations. The innate and adaptive immune systems were assessed using natural killer (NK) cell activity and lymphocyte proliferation assays. PFOS modulated NK cell activity with significant decreases at 0.01, 0.05, 0.5, and 1.0 μg/mL concentrations and significant increases at 5.0 μg/mL in both species. Mitogen-induced lymphocyte proliferation was significantly decreased in murine cells, but not in dolphin cells exposed to PFOS. DE-71 significantly increased T cell proliferation in bottlenose dolphins at concentrations of 0.5, 2.5, and 5.0 μg/mL, but did not alter B cell proliferation or NK cell activity. DE-71 in vitro exposure is currently ongoing with murine cells. Using both dolphin and murine cells provides a comparative approach for evaluating the effects of PFOS and DE-71 on immune functions.
KEYNOTE SPEAKER OPENING ADDRESS ABSTRACT:
CHANGES IN CARIBBEAN REEF COMMUNITIES: PATTERNS, CAUSES AND MITIGATION
Bruno, John (University of North Carolina, Dept. of Marine Sciences)
Caribbean coral reefs have changed dramatically since I was in high school. One thing that fascinates me about Caribbean reef change is how quickly an ecosystem can be transformed across a huge regional scale. I also constantly wonder how much of this change can be attributed to people and how much is natural. I will discuss the spatio-temporal patterns in coral reef change, primarily in terms of coral and fish loss and macroalgal increase. Despite broad agreement by reef scientists about the degree of change, there is an ongoing debate about when, where, and why reefs have changed, and even about whether they are currently locked in an “Alternative Stable State”. I will also address some of the main explanations for changes in coral reef benthic communities and the potential for local management actions, such as the implementation of marine reserves, to mitigate coral loss by increasing “reef resilience”.
KEYNOTE SPEAKER CLOSING ADDRESS ABSTRACT:
IMPACTS OF CLIMATE CHANGE ON OCEAN ECOSYSTEMS
Bruno, John (University of North Carolina, Dept. of Marine Sciences)
That Anthropogenic Climate Change (ACC) is changing ocean ecosystems will surprise nobody at an ocean sciences conference. Yet there are still some interesting surprises about the realized and predicted impacts of ACC. For example, in some systems, warming could drive an increase in species richness via range shifts. In other systems, warming will facilitate the development of new biogenic habitats or change species composition to such a degree that we have no idea what the resulting community will look like or how it will function. There are also underappreciated temperature-dependent biological and ecological processes (e.g., metabolism) that will be affected, greatly altering how ecosystems function. Finally, the seemingly obvious negative impacts of warming can be exceedingly difficult to reliably attribute to ACC when placed in a rigorous empirical context, particularly when following the guidelines of the IPCC. Take home message: there are still plenty of surprises out there and studying the effects of ACC is a lot more interesting – and challenging – than simply documenting animals dying by overheating.