GPMB September 2010 Colloquium:
Abstracts of Poster Presentations
TOXIC DINOFLAGELLATES AND THEIR ASSOCIATED MICROBIAL COMMUNITY: INTERACTIONS OF KARENIA SPP. WITH ALGICIDAL BACTERIA AND THE POTENTIAL ROLE OF QUORUM SENSING
Blair, W. (GPMB, College of Charleston) and Doucette, G. (Marine Biotoxins Program, NOAA/National Ocean Service)
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/or their associated bacteria, including algicidal taxa, in QS systems would introduce a new paradigm for understanding biotic regulation of bloom dynamics. This project aims to detect microbial QS signal production and identify QS influences on Karenia spp., algicidal bacteria, and algicidal activity. Bioinformatic techniques will be used to scan relevant genomes for QS homologues, and bioassays will be used to screen both Karenia spp. and bacterial cultures for QS signals. Compounds that are detected will be characterized using LC-MS. QS influences on microbial activity will be identified by monitoring changes in growth, morphology, toxin production, and selected gene expression after in vitro additions of autoinducers and their inhibitors. QS modulation of microbial activity will demonstrate that intraspecific and inter-kingdom QS signaling may be a key feature of natural bloom communities, and manipulation of algicidal activity using QS compounds may lead to new strategies for control and mitigation of harmful algal blooms.
EVALUATION OF THE POPULATION GENETIC STRUCTURE OF LEIOSTOMUS XANTHURUS ALONG THE WESTERN NORTH ATLANTIC COAST AND THE GULF OF MEXICO
Bryant, S. (GPMB, College of Charleston) and Greig, T. (Center for Coastal Environmental Health and Biomolecular Research)
The observation has been made that population genetic structure has been documented more commonly in freshwater fish species than in marine species. However, empirical evidence suggests that spot, (Leiostomus xanthurus), may be a species which exhibits population genetic structure. Spot is a commercially and environmentally important fish which ranges from Maine to Florida and throughout the Gulf of Mexico. This study aims to examine the degree of genetic variation among spot and to evaluate whether Cape Hatteras and the Florida peninsula serve as barriers to gene flow and influence the genetic structure. With the help of NEAMAP, SEAMAP, and SCDNR, spot will be received from multiple discrete locations north and south of Cape Hatteras, as well as throughout the Gulf of Mexico. Population structure will be determined using DNA sequence analysis of cytochrome B of the mitochondrial DNA and potentially the control region. These data, in conjunction with age data obtained through otolith extractions, will be used to assess the magnitude and spatial distribution of mtDNA variation in L. xanthurus throughout the eastern Atlantic coast and eastern Gulf of Mexico. In doing so, it will be possible to determine the extent that Cape Hatteras and the Florida peninsula serve as phylogeographic barriers, and to appraise the amount of gene flow among localities. Data obtained through this study may be used to improve future decisions regarding management of this fishery resource, as relatively information about the spot population structure is available to management entities at this time.
PARASITES OF THE SPOTTED SEATROUT: DO THEY WEAKEN THE HEALTH OF THE FISH AND THEIR TOLERANCE TO COLD WATER TEMPERATURE?
Cosmann, P. (GPMB, College of Charleston), and de Buron, I. (College of Charleston)
The spotted seatrout, Cynoscion nebulosus (Cuvier, 1830) is an economically important estuarine species that inhabits the coastal waters off of the southeastern United States. In March 2010, the spotted seatrout population off of South Carolina reached near-record low numbers. Below average water temperatures experienced in January 2010 have been hypothesized as the causative agent in the population decline. However, preliminary studies into the parasite community in spotted seatrout have revealed the presence of particularly pathogenic parasites that could also be a factor in the fish population decline. Three species of myxosporeans infect the skeletal muscle, the heart, and the urinary system respectively. A digenean infects the vascular system, and a protist like fungus infects the kidney. In this study, presence of parasites and health of the fish will be determined using histology and gross health indices, respectively. Impact of the parasites on the fish health will then be evaluated using a stepwise multiple regression model. Further, we hypothesize that these parasitic infections are an added stress that decreases the fish tolerance to cold-water temperatures. This hypothesis will be tested experimentally via cold tolerance experiments in which reflex impairment of the fish will be determined and analyzed according to histological parasite burden.
CHARACTERIZING THE ROLE OF KEY ANTIOXIDANTS AND ROS PRODUCTION IN THE PACIFIC WHITELEG SHRIMP, LITOPENAEUS VANNAMEI, DURING RECOVERY FROM HYPOXIA AND HYPERCAPNIC HYPOXIA
Darling, C. (GPMB, College of Charleston), Burnett, K. and Burnett, L. (College of Charleston, Hollings Marine Laboratory)
The Pacific whiteleg shrimp, Litopenaeus vannamei, inhabits coastal and estuarine waters which are prone to intermittent bouts of low oxygen (hypoxia) and high carbon dioxide (hypercapnia). In the current study we will profile the responses of key antioxidants in shrimp during recovery from hypoxia (H) and hypercapnic hypoxia (HH) by measuring changes in gene transcripts (qRT-PCR) as well as at the protein/activity level through enzyme assays. Episodic H and HH (4-6 hours) are followed by recovery to normal oxygen (normoxia) and carbon dioxide levels (8-12 hours) in a typical semidiurnal tidal cycle. Similar recovery scenarios are known to produce a large number of reactive oxygen species (ROS) in vertebrates as a result of respiratory burst, and thereby ischemia/reperfusion injury can occur. While ROS can be detrimental to cells, they also play an important role in crustacean immune function and are eventually remedied through the production of antioxidant enzymes. Microarray analysis of shrimp exposed to H and HH for 4 and 24 hours provides evidence that changes in gene transcripts occur for antioxidant enzymes and pathways associated with a response to ROS production. The powerful antioxidants superoxide dismutase, thioredoxin-2, and genes involved in glutathione production via the transsulferation pathway such as cystathione beta synthase, betaine-homocysteine methyltransferase and glutathione s-transferase will be assessed in this study. Differences among short (4 hours), intermediate (8 hours) and extended exposure times (24 hours) will be evaluated as they may indicate that adaptations involving impaired immune function and/or decreased protein synthesis occur after sustained oxidative stress.
DETERMINING FACTORS THAT INFLUENCE 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, MRRI)
Sandwich hybridization assay (SHA) directly detects a target sequence using large subunit rRNA-targeted DNA oligonucleotides. It entails two taxa or species-specific probes that bind to different rRNA regions thus “sandwiching” the sequence. A conjugate detects the second probe that subsequently reacts with a substrate producing a colorimetric response; the intensity of which is directly proportional to sample rRNA. The resultant optical density can be related to approximate cell density. SHA has been adapted to detect and quantify bacteria, invertebrate larva and several harmful algal bloom (HAB) species. The extent to which variability in rRNA may influence SHA results, however, remains unclear. This study will examine various factors that may influence cellular rRNA and associated SHA results using the harmful raphidophyte Heterosigma akashiwo as a model organism. H. akashiwo was chosen because it occurs globally, produces ichthyotoxic blooms and SHA capabilities have previously been developed and assessed for it. Specifically, this study will examine whether geographically distinct populations exhibit different SHA responses; to what extent sample preservation affects quantification by SHA; how SHA results change over algal growth cycles; and the influence of inorganic nitrogen and phosphorus limitation. Results will provide new insights to the factors that may influence SHA responses and whether any observable variations pose important considerations for resource managers when interpreting SHA results for H. akashiwo. This project is one part of a broader, four-year NOAA-MERHAB award that compares SHA with QPCR for the detection and quantification of harmful algae, using H. akashiwo as a model organism.
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. Cartilaginous reinforcement of the peripheral airways in the lungs also allows for rapid tidal ventilation and gas exchange when these animals surface to breathe. Other strategies for slowing the rate of oxygen consumption and prolonging dive duration include switching from active stroking to sustained gliding during dive descent and maintaining high concentrations of myoglobin in the skeletal muscles. 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 will examine 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 will be exposed to ambient and low oxygen levels, and the expression of Nanog will be validated using gene array and protein analyses. Inhibitory RNA will also be used to initiate downregulation of Nanog and determine if lung cells are able to differentiate. The results of this study could be used for further investigation of other diving marine mammals and human respiratory diseases.
EFFECT OF BACTERIAL ANTAGONISM IN STRUCTURING COMMUNITIES OF INTERTIDAL MICROBES
Hook, W. (GPMB, College of Charleston), and Plante, C. (College of Charleston)
The majority of sedimentary bacteria live in biofilms attached to sediment grains. The high densities (~109/g) and diversity of these bacteria implicate inter-specific competition as a likely force in structuring communities. A 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 adjacent bacteria. This research aims to 1) identify sedimentary bacteria that produce anti-microbial compounds and 2) determine whether bacterial antagonisms play a significant role in structuring microbial sediment communities. Bacteria displaying inhibitory capabilities and susceptible strains were isolated from intertidal sediments in Breach Inlet, South Carolina in 2008. Producers were identified using the Burkholder agar diffusion assay and sequenced at the 16S rRNA gene region. Additional screening of antimicrobial producers from intertidal sediment samples is underway. The effects of bacterial growth conditions and culture age on antimicrobial production will be determined in order to optimize assays and characterize inhibitory compounds. Denaturing gradient gel electrophoresis (DGGE) and DNA sequencing will be used to determine which producers are found in relatively high abundance in situ. After model antagonistic bacteria are selected, in vitro and microcosm experiments will be used to examine competitive interactions between producer strains and various targets. Experiments will follow specific target populations and complete communities, after addition of both producer cultures and cell-free extracts. Emphasis will be placed on characterizing the final bacterial communities resultant of antagonistic interaction. Findings should elucidate the structuring effects of antimicrobials on bacterial assemblages of intertidal sediments.
INTERACTIONS OF VIRAL INFECTION, OXIDATIVE STRESS, AND INTRACELLULAR SULFUR COMPOUNDS IN THE COCCOLITHOPHORID, EMILIANIA HUXLEYI: A PUTATIVE MECHANISM FOR ACTIVE VIRAL RESISTANCE
Kendrick, B.J. (GPMB, College of Charleston) and DiTullio, G.R. (College of Charleston)
Emiliania huxleyi (Lohman) Hay & Mohler (Ehux) is a cosmopolitan coccolithophorid which forms very dense blooms annually and impacts the global ocean and climate via several mechanisms including: calcium carbonate export and increased CO2 uptake from the atmosphere, increased albedo via elevated turbidity, and dimethylsulfide (DMS) production leading to cloud condensation nuclei. Through lysis of host cells Emiliania huxleyi Virus (EhV, Phycodnaviridae; Coccolithovirus) has been demonstrated to play an important ecological role in population structure as well as biogeochemical cycling of carbon and sulfur by E. huxleyi. Viral infection is known to cause oxidative stress in Ehux via the accumulation of reactive oxygen species (ROS). Dimethylated sulfur compounds (e.g. DMS, DMSP, and DMSO) have been shown to play an antioxidant role in these cells, of which Evans et al. demonstrated that DMS reduces infectious titers of EhV. Virally resistant Ehux strains demonstrate high DMSP Lyase activity which leads to increased production of these antioxidant compounds. The proposed research will investigate the hypothesis that the primary mechanism of host lysis due to viral infection is oxidative stress and that the antioxidant function of dimethylated sulfur compounds mitigates this stress and therefore imparts viral resistance in high DMSP Lyase Activity (DLA) Ehux strains.
THE EVOLUTION OF A LATITUDINAL BODY SIZE CLINE IN THE SUBTIDAL ISOPOD IDOTEA BALTICA
Manyak, A. (GPMB, College of Charleston)
Variable environmental conditions induce changes in phenotypic characters during development, a phenomenon known as phenotypic plasticity. Organisms that span broad latitudinal ranges are confronted with a gradient of biotic (predation, food quantity/quality) and abiotic (temperature, seasonality) conditions. We have little information on whether organisms tend to respond to these environmental gradients either through phenotypic plasticity (environmentally-driven), local adaptation (genetically-driven), or an interaction between the two. Bergmann’s rule is one such latitudinal pattern, in which clinal environmental conditions along a latitudinal gradient lead to shifts in body size, with organisms from higher latitudes possessing larger body sizes than their lower latitude counterparts. As an example, the subtidal isopod, Idotea baltica, ranges from Virginia to Maine, and preliminary data suggests that a distinct body size difference exists between the northern (Massachusetts) and southern (Virginia) populations, following a Bergmann’s rule pattern. This project aims to shed light on both the mechanisms which may have lead to this size difference and the ecological consequences of variable size. Specifically, I hope to answer the following questions: 1) Do northern and southern populations of I. baltica differ in their survivorship, growth and fecundity across a range of developmental temperatures (6º, 12º, 18º, 24º and 30º)? 2) Are these geographic patterns in fitness due to differences in the sensitivities of respiration and consumption rates to temperature? 3) Are smaller bodies of I. baltica in southern regions an adaptive response to increased predation pressure?
IDENTIFICATION AND DISTRIBUTION OF ANISAKID PARASITES OF PYGMY SPERM WHALES (KOGIA BREVICEPS)
Rein, R. (MES, College of Charleston), Greig, T. (NOAA/NOS), McFee, W. (NOAA/NOS) de Buron, I. (College of Charleston) and Arnott, S. (SCDNR)
Pygmy sperm whales (Kogia breviceps) are a poorly understood species as live sightings are rare and most of our knowledge comes from stranded animals. Currently populations from the northwestern Atlantic Ocean and Gulf of Mexico are managed as separate stocks, despite the lack of evidence to support this designation. Preliminary observations showed that K. breviceps stranded on our coast are infected with anisakids, which are parasitic nematodes commonly found in the digestive tract of marine mammals. The aim of this study is to identify these anisakids at the species level and determine their distribution in the three chambers of the whale stomach. Scanning electron microscopy allowed us to identify a sample of 14 worms as belonging to the genus Anisakis, but did not allow lower taxon identification. Therefore, DNA sequencing of the mitochondrial DNA cox2 gene and ITS region of the 18S ribosomal gene was implemented in an attempt to identify species. Preliminary results of sequencing of the cox2 gene of 10 specimens allowed us to identify 6 as Anisakis brevispiculata (4 from the forestomach and 2 in the fundic chamber), 1 as A. physeteris (from the fundic chamber), 2 as A. simplex s.s. (from the pyloric stomach), and 1 gave ambiguous results. Possible further directions for this study include using these parasites, which have been used to assess stock structures of fish, as markers to infer stock structure of pygmy sperm whales in the Gulf of Mexico and northwestern Atlantic Ocean.
ANTIMICROBIAL ACTIVITY OF MOLLUSCAN EGG MASSES IN THE SAN JUAN ISLANDS, WASHINGTON
Smoot, S. (GPMB, College of Charleston), Plante, C. (College of Charleston), and Podolsky, R. (College of Charleston)
Gelatinous egg masses have evolved several times in invertebrates as a reproductive mode that encapsulates embryos until hatching. The absence of a mechanical outer covering makes these egg masses particularly susceptible to microbial infection, biofouling, and predation. The mucus and gel matrix surrounding the egg capsules can contain compounds that deter microbial infection. Furthermore, if adults can adjust the level of protection, then the amount of antimicrobial activity found within an egg mass should be correlated with the bacterial load found in the local environment. We are comparing the antimicrobial activity of egg masses among 15 molluscan species, among the components of egg masses (gel vs. embryos) of Haminoea vesicula, and among sites where the sea slugs H. vesicula and Melanochlamys diomedea are located. Egg masses were collected from the field, lyophilized and extracted with ethyl acetate and methanol to isolate non-polar and polar compounds. The extracts were then tested and quantified for antimicrobial activity against marine type cultures (Bacillus subtillis, Vibrio harveyi, and Pseudoaltermonas atlantica), as well as two environmental bacterial isolates, in a Burkholder petri dish and 96-well plate assay. We have observed antimicrobial activity in non-polar and polar extracts of Haminoea and Melanochlamys against Bacillus subtillis and Pseudoaltermonas atlantica, and are continuing with our other comparisons.
PERFORMANCE CHANGES FOLLOWING BACTERIAL CHALLENGE AND VARYING OXYGEN LEVELS IN THE ATLANTIC BLUE CRAB, CALLINECTES SAPIDUS RATHBUN
Stover, K. (GPMB, College of Charleston), Burnett, L. (College of Charleston, Hollings Marine Laboratory), McElroy, E. (College of Charleston) and Burnett, K. (College of Charleston, Hollings Marine Laboratory)
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 this ability may be compromised by environmental stressors. Estuarine organisms face daily and seasonal fluctuations in salinity, temperature, dissolved oxygen and pH, along with a large microbial load that is characteristic of marine environments. The Atlantic blue crab, Callinectes sapidus (Rathbun), an important commercial and recreational fishing species, resides in the estuarine waters of the Atlantic Ocean and Gulf of Mexico. Blue crabs survive these conditions using a variety of physiological and behavioral mechanisms such as osmoregulation or migration. Maintaining an immune response against environmental microbes is also necessary for survival but, as previously documented, respiration decreases dramatically in crustaceans that receive a sublethal dose of bacteria. In the proposed study we will test whether impaired respiration following bacterial exposure alters performance, as well as describe the effect of hypoxia (4 kPa) and hyperoxia (41 kPa) on the same performance challenge. Performance will be evaluated by an exercise trial that entails sideways hexapedal walking on a treadmill until fatigued and then analyzing the three dimensional kinematics. In addition, the reproductive ability of each crab will be assessed as a measure of fatigue by means of a hold test that mimics a male mate guarding a female. The time to fatigue and kinematics will be compared statistically to determine differences between saline injected, bacteria injected, hypoxic and hyperoxic crabs.
DIMETHYLSULFONIOPROPIONATE (DMSP) PRODUCTION AND PHYSIOLOGICAL RESPONSE OF THE ANTARCTIC DIATOM, FRAGILARIOPSIS CYLINDRUS, TO TEMPERATURE AND pCO2 STRESS
Vied, S.B. (GPMB, College of Charleston), Boroujerdi, A.F.B. (NIST), Bearden, D.W. (NIST), DiTullio, G.R. (College of Charleston), Janech, M.G. (Dept. of Medicine, MUSC), and Lee, P.A. (College of Charleston)
Dimethylsulfoniopropionate (DMSP) is a major component of the global sulfur cycle. DMSP is synthesized by phytoplankton, serving several known and suggested cellular functions such as osmolyte, cryoprotectant, antioxidant, and grazing deterrent. DMSP production by marine algae not only varies by species, but also in response to various environmental stressors including temperature and pCO2. Polar phytoplankton have not been studied as thoroughly as more temperate species, yet results suggest polar algae may produce more DMSP. DMSP production by the Antarctic diatom, Fragilariopsis cylindrus, will be examined as a function of changing temperature and pCO2. For the temperature stress experiments, axenic cultures will be grown at 0°C, 4°C (control), and 8°C. For the CO2 stress experiments, axenic cultures will be grown at 150 ppm, approximately 380 ppm (control), and 760 ppm CO2. DMSP will be quantified using gas chromatography. Physiological parameters, including Fv/Fm, GSH/GSSG assay, and accessory pigments, will be measured as a proxy of oxidative stress. Alkalinity and pH will be measured to verify the correct pCO2 levels in each incubation. Examining the effect of these stressors on DMSP production in F. cylindrus provides the opportunity to discover how this ecologically-relevant polar alga responds to conditions found in sea-ice and conditions projected to occur due to climate change. Additional samples will be collected for analysis by Nuclear Magnetic Resonance to determine the metabolic changes that result from these stressors, providing an innovative application for this technique in polar marine phytoplankton.
EMERGING CONTAMINANTS IN THE MARINE ENVIRONMENT" AN IN VITRO STUDY OF THE IMMUNOLOGICAL EFFECTS OF PERFLUOROOCTANE SULFONIC ACID (PFOS) AND POLYBROMINATED DIPHENYLETHERS (PBDEs) ON DOLPHIN AND MURINE IMMUNE CELLS
Wirth, J. (GPMB, College of Charleston), Peden-Adams, M. (MUSC), Henry, N. (NOAA), Owens, D. (College of Charleston), and Strand, A. (College of Charleston), Fair, P. (NOAA)
Of all the determinants of health in an organism, immune function is one of the most crucial. The effects of emerging contaminants on the immune system of mammals are also poorly understood, with limited information about the immunosuppressive and toxicological effects of PBDE congeners and mixtures and PFOS salt. Marine mammals are protected species so directly assessing the effects of contaminant exposure in dolphins creates ethical challenges, similar to studying exposure in humans; in vitro laboratory studies provide a valuable tool to assess susceptibility to contaminants. This research project will investigate immunological and cytotoxicological effects of the DE-71 (PBDE) mixture and PFOS on mouse (Mus musculus) and dolphin cellular systems. Bottlenose dolphins (Tursiops truncatus) are a good sentinel species as they are exposed to similar levels of PFOS and PBDEs through diet and environment as the local human population. In vitro studies will determine the thresholds for cytotoxicity and immune alterations following exposure to a commercial PBDE mixtures and PFOS salt. Using PBDE and PFOS concentrations previously documented in bottlenose dolphins, immune status and function will be assessed by immunological assays including natural killer cell (NK) activity and lymphocyte proliferation.Preliminary data indicates that NK cells are significantly affected by PFOS concentrations in both species. Using both murine and dolphin cells will provide a comprehensive and comparative approach in evaluating PBDE and PFOS effects at environmentally relevant concentrations.