Saline-saturated DMSO-EDTA as a storage medium for microbial DNA analysis from coral mucus swab samples

The mucus surface layer of corals plays a number of integral roles in their overall health and fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier against physical invasions and trauma, and serves as a medium to host a community of microorganisms distinct from the surrounding seawater. In healthy corals the associated microbial communities are known to provide antibiotics that contribute to the coral’s innate immunity and function metabolic activities such as biogeochemical cycling. Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change with changes in the environment and health condition of the coral. These changes may suggest that changes in the microbial associates not only reflect health status but also may assist corals in acclimating to changing environmental conditions. With the increasing availability of molecular biology tools, culture-independent methods are being used more frequently for evaluating the health of the animal host. Although culture-independent methods are able to provide more in-depth insights into the constituents of the coral surface mucus layer’s microbial community, their reliability and reproducibility rely on the initial sample collection maintaining sample integrity. In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry shipper. The samples are then sent to a laboratory for analysis. After the initial collection and preservation of the sample, the duration of the sample voyage to a recipient laboratory is often another critical part of the sampling process, as unanticipated delays may exceed the length of time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust prematurely. In remote areas, service by international shipping companies may be non-existent, which requires the use of an alternative preservation medium. Other methods for preserving environmental samples for microbial DNA analysis include drying on various matrices (DNA cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, however, they add expense and permitting requirements for hazardous liquid components, and the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et al., 2010). A method to preserve coral mucus samples without cold storage or use of hazardous solvents, while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a means of storing tissue of marine invertebrates at ambient temperatures without significant loss of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the coral microbiota DNA would be adversely affected by this storage medium either by degradation of the DNA, or a bias in the DNA recovered during the extraction process created by variations in extraction efficiencies among the various community members. Tests to determine the efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are presented here.

Genetic engineering of microorganisms: free release into the environment

Genetic engineering now makes possible the insertion of DNA from many organisms into other prokaryotic, eukaryotic and viral hosts. This technology has been used to construct a variety of such genetically engineered microorganisms (GEMs). The possibility of accidental or deliberate release of GEMs into the natural environment has recently raised much public concern. The prospect of deliberate release of these microorganisms has prompted an increased need to understand the processes of survival, expression, transfer and rearrangement of recombinant DNA molecules in microbial communities. The methodology which is being developed to investigate these processes will greatly enhance our ability to study microbial population ecology.

Threatened corals provide underexplored microbial habitats

Contemporary in-depth sequencing of environmental samples has provided novel insights into microbial community structures, revealing that their diversity had been previously underestimated. Communities in marine environments are commonly composed of a few dominant taxa and a high number of taxonomically diverse, low-abundance organisms. However, studying the roles and genomic information of these “rare” organisms remains challenging, because little is known about their ecological niches and the environmental conditions to which they respond. Given the current threat to coral reef ecosystems, we investigated the potential of corals to provide highly specialized habitats for bacterial taxa including those that are rarely detected or absent in surrounding reef waters. The analysis of more than 350,000 small subunit ribosomal RNA (16S rRNA) sequence tags and almost 2,000 nearly full-length 16S rRNA gene sequences revealed that rare seawater biosphere members are highly abundant or even dominant in diverse Caribbean corals. Closely related corals (in the same genus/family) harbored similar bacterial communities. At higher taxonomic levels, however, the similarities of these communities did not correlate with the phylogenetic relationships among corals, opening novel questions about the evolutionary stability of coral-microbial associations. Large proportions of OTUs (28.7–49.1%) were unique to the coral species of origin. Analysis of the most dominant ribotypes suggests that many uncovered bacterial taxa exist in coral habitats and await future exploration. Our results indicate that coral species, and by extension other animal hosts, act as specialized habitats of otherwise rare microbes in marine ecosystems. Here, deep sequencing provided insights into coral microbiota at an unparalleled resolution and revealed that corals harbor many bacterial taxa previously not known. Given that two of the coral species investigated are listed as threatened under the U.S. Endangered Species Act, our results add an important microbial diversity-based perspective to the significance of conserving coral reefs.

Observations of the biological communities at Bolsa Chica artificial reef

Bolsa Chica Artificial Reef (BCAR) was constructed in November 1986 with 10,400 tons of concrete rubble and eight concrete and steel barges. Prior to any additional augmentation of BCAR, the u.s. Army Corps of Engineers and the California Coastal Commission required the California Department of Fish and Game (CDFG) to survey the bioloqical communities on and around BCAR. In April 1992, qualitative surveys of the biological communities were conducted on one of the eight modules at BCAR and at a nearby sand-only site. One of the modules, Module D, located in 90 feet of water (MLLW), was surveyed for fish, macroinvertebrates, and turf community organisms (small plants and sessile animals). Twelve species of fish were observed, including kelp bass (Paralabrax clathratus) and barred sand bass (P. nebulifer). Eight macroinvertebrate species were observed, rock scallops (Crassedoma giganteum) being the most abundant. The turf community was comprised of thirteen invertebrate taxa, among which erect ectoprocts (Bugula spp.) were the most numerous. Two species of foliose red algae (Rhodymenia pacifica and Anisocladella pacifica) were also observed. The reef has reached an advanced stage of successional development with fish and invertebrate communities diverse and well established. However, due,.to its depth and the turbidity of surrounding waters, this reef is not likely to ever support a diverse algal community. The diversity and abundance of fish and macroinvertebrates were, as to be expected, much lower in the nearby sand-only site. Only two species of fish and seven macroinvertebrate species were observed. Of these, only the sea pen, Stylatula elongata, was common. Overall, when compared to nearby sand-only habitats, Bolsa Chica Artificial Reef appears to contribute substantially to the local biological productivity. In addition, the concrete rubble used in BCAR' s construction appears to be performing as well as the quarry rock used in all of CDFG's experimental reefs. (Document pdf contains 22 pages)

Redefining Stewardship: Public Lands and Rural Communities in the Pacific Northwest

pdf contains 24 pages

Catch-22: conservation, communities and the privatization of B.C. fisheries. An economic, social and ecological impact study

pdf contains 47 pages

The India MPA Workshop Proceedings. Social Dimensions of Marine Protected Areas Implementation in India: do Fishing Communities Benefit? 21-22 January 2009, IMAGE Auditorium, Chennai, India

In the current context of natural resource management, marine protected areas (MPAs) are being widely propagated as an important tool for the conservation of marine and fisheries resources. The International Collective in Support of Fishworkers (ICSF) recently undertook a series of studies on MPAs in India to highlight the various legal, institutional, policy and livelihoods issues that confront fishing and coastal communities. In order to discuss the findings of these case studies and to suggest proposals for livelihood-sensitive conservation and management of coastal and fisheries resources through participatory processes, ICSF organized a two-day workshop on ‘Social Dimensions of Marine Protected Area Implementation in India: Do Fishing Communities Benefit?’ at Chennai on 21-22 January 2009. This publication—the India MPA Workshop Proceedings—contains the prospectus of the workshop, a report of the proceedings and the consensus statement that was reached by organizations and individuals who particapated in the workshop. This publication will be useful for fishworkers, non-governmental organizations, policymakers, trade unions, researchers and others interested in natural resource management and coastal and fishing communities.

Comparative Microbial Dynamics in Crassostrea virginica and Crassostrea ariakensis

Considerations to introduce the Suminoe or Asian oyster Crassostrea ariakensis along the East Coast have raised many questions regarding ecology, economics, and human health. To date, research has focused primarily on the ecological and socioeconomic implications of this initiative, yet few studies have assessed its potential impact on public health. Our work compares the rates of bioaccumulation, depuration and post harvest decay of indicator organisms (such as E. coli) and Vibrio sp. between Crassostrea virginica and Crassostrea ariakensis in the laboratory. Preliminary results suggest that the rates of bioaccumulation of E. coli in Crassostrea ariakensis were significantly lower than those for Crassostrea virginica, depuration of E. coli was variable between the two species, and Crassostrea ariakensis post harvest decay rates of Vibrio sp. were significantly lower than Crassostrea virginica. This research provides coastal managers with insight into the response of Crassostrea ariakensis to bacteria, an important consideration for determining appropriate management strategies for this species. Further field-based studies will be necessary to elucidate the mechanisms responsible for the differences in rates of bioaccumulation and depuration. (PDF contains 40 pages)

Environmental impact of hydraulic escalator dredging on oyster communities

A study to assess the effects of hydraulic escalator dredging on an oyster community was initiated at the request of the Department of Natural Resources, Mary1and Tidewater Fisheries Administration. The purpose of the work was to reevaluate a study done by Manning (1957). A site in the Patuxent River north of Broome Island classified as oyster bottom was selected to conduct an in - situ intensive experiment using the Manning study as a framework for project design. Clams and oysters were present in the study area,although oyster populations were limited in number. (PDFs consists of pp 1-97 and additional Sect II: Middle Bay proposed reclassification sites)

Cruise Report NOAA Ship McARTHUR II Cruise AR-04-04: Leg 2 (June 1-12, 2004): A pilot survey of deepwater coral/sponge assemblages and their susceptibility to fishing/harvest impacts at the Olympic Coast National Marine Sanctuary (OCNMS)

Summary: The offshore shelf and canyon habitats of the OCNMS (Fig. 1) are areas of high primary productivity and biodiversity that support extensive groundfish fisheries. Recent acoustic surveys conducted in these waters have indicated the presence of hard-bottom substrates believed to harbor unique deep-sea coral and sponge assemblages. Such fauna are often associated with shallow tropical waters, however an increasing number of studies around the world have recorded them in deeper, cold-water habitats in both northern and southern latitudes. These habitats are of tremendous value as sites of recruitment for commercially important fishes. Yet, ironically, studies have shown how the gear used in offshore demersal fishing, as well as other commercial operations on the seafloor, can cause severe physical disturbances to resident benthic fauna. Due to their exposed structure, slow growth and recruitment rates, and long life spans, deep-sea corals and sponges may be especially vulnerable to such disturbances, requiring very long periods to recover. Potential effects of fishing and other commercial operations in such critical habitats, and the need to define appropriate strategies for the protection of these resources, have been identified as a high-priority management issue for the sanctuary. To begin addressing this issue, an initial pilot survey was conducted June 1-12, 2004 at six sites in offshore waters of the OCNMS (Fig. 2, average depths of 147-265 m) to explore for the presence of deep-sea coral/sponge assemblages and to look for evidence of potential anthropogenic impacts in these critical habitats. The survey was conducted on the NOAA Ship McARTHUR-II using the Navy’s Phantom DHD2+2 remotely operated vehicle (ROV), which was equipped with a video camera, lasers, and a manipulator arm for the collection of voucher specimens. At each site, a 0.1-m2 grab sampler also was used to collect samples of sediments for the analysis of macroinfauna (> 1.0 mm), total organic carbon (TOC), grain size, and chemical contaminants. Vertical profiles of salinity, dissolved oxygen (DO), temperature, and pressure were recorded at each site with a small SeaCat conductivity-temperature-depth (CTD) profiler. Niskin bottles attached to the CTD also obtained near-bottom water samples in support of a companion study of microbial indicators of coral health and general ecological condition across these sites. All samples except the sediment-contaminant samples are being analyzed with present project funds. Original cruise plans included a total of 12 candidate stations to investigate (Fig. 3). However, inclement weather and equipment failures restricted the sampling to half of these sites. In spite of the limited sampling, the work completed was sufficient to address key project objectives and included several significant scientific observations. Foremost, the cruise was successful in demonstrating the presence of target deepwater coral species in these waters. Patches of the rare stony coral Lophelia pertusa, more characteristic of deepwater coral/sponge assemblages in the North Atlantic, were observed for the first time in OCNMS at a site in 271 meters of water. A large proportion of these corals consisted of dead and broken skeletal remains, and a broken gorgonian (soft coral) also was observed nearby. The source of these disturbances is not known. However, observations from several sites included evidence of bottom trawl marks in the sediment and derelict fishing gear (long lines). Preliminary results also support the view that these areas are important reservoirs of marine biodiversity and of value as habitat for demersal fishes. For example, onboard examination of 18 bottom-sediment grabs revealed benthic infaunal species representative of 14 different invertebrate phyla. Twenty-eight species of fishes from 11 families, including 11 (possibly 12) species of ommercially important rockfishes, also were identified from ROV video footage. These initial discoveries have sparked considerable interests in follow-up studies to learn more about the spatial extent of these assemblages and magnitude of potential impacts from commercial-fishing and other anthropogenic activities in the area. It is essential to expand our knowledge of these deep-sea communities and their vulnerability to potential environmental risks in order to determine the most appropriate management strategies. The survey was conducted under a partnership between NOAA’s National Centers for Coastal Ocean Science (NCCOS) and National Marine Sanctuary Program (NMSP) and included scientists from NCCOS, OCNMS, and several other west-coast State, academic, private, and tribal research institutions (see Section 4 for a complete listing of participating scientists). (PDF contains 20 pages)

Self-recruiting species (SRS) from farmer managed aquatic systems: are they important to the livelihoods of rural communities?

Why are SRS important? The answer is to be found in this well-structured survey under: SRS as food source; SRS as additional source of cash income; Role of SRS in social capital. An analysis of the threats to SRS and the potential management options for farmer managed aquatic systems are also available in this survey along with the following definition of SRS: SRS are defined as aquatic animals that can be harvested from farmer managed aquatic systems without regular stocking. (PDF contains 4 pages)

Developing alternatives for optimal representation of seafloor habitats and associated communities in Stellwagen Bank National Marine Sanctuary

The implementation of various types of marine protected areas is one of several management tools available for conserving representative examples of the biological diversity within marine ecosystems in general and National Marine Sanctuaries in particular. However, deciding where and how many sites to establish within a given area is frequently hampered by incomplete knowledge of the distribution of organisms and an understanding of the potential tradeoffs that would allow planners to address frequently competing interests in an objective manner. Fortunately, this is beginning to change. Recent studies on the continental shelf of the northeastern United States suggest that substrate and water mass characteristics are highly correlated with the composition of benthic communities and may therefore, serve as proxies for the distribution of biological biodiversity. A detailed geo-referenced interpretative map of major sediment types within Stellwagen Bank National Marine Sanctuary (SBNMS) has recently been developed, and computer-aided decision support tools have reached new levels of sophistication. We demonstrate the use of simulated annealing, a type of mathematical optimization, to identify suites of potential conservation sites within SBNMS that equally represent 1) all major sediment types and 2) derived habitat types based on both sediment and depth in the smallest amount of space. The Sanctuary was divided into 3610 0.5 min2 sampling units. Simulations incorporated constraints on the physical dispersion of sampling units to varying degrees such that solutions included between one and four site clusters. Target representation goals were set at 5, 10, 15, 20, and 25 percent of each sediment type, and 10 and 20 percent of each habitat type. Simulations consisted of 100 runs, from which we identified the best solution (i.e., smallest total area) and four nearoptimal alternates. We also plotted total instances in which each sampling unit occurred in solution sets of the 100 runs as a means of gauging the variety of spatial configurations available under each scenario. Results suggested that the total combined area needed to represent each of the sediment types in equal proportions was equal to the percent representation level sought. Slightly larger areas were required to represent all habitat types at the same representation levels. Total boundary length increased in direct proportion to the number of sites at all levels of representation for simulations involving sediment and habitat classes, but increased more rapidly with number of sites at higher representation levels. There were a large number of alternate spatial configurations at all representation levels, although generally fewer among one and two versus three- and four-site solutions. These differences were less pronounced among simulations targeting habitat representation, suggesting that a similar degree of flexibility is inherent in the spatial arrangement of potential protected area systems containing one versus several sites for similar levels of habitat representation. We attribute these results to the distribution of sediment and depth zones within the Sanctuary, and to the fact that even levels of representation were sought in each scenario. (PDF contains 33 pages.)

Structure and diversity in Mytilus californianus (Mollusca: Bivalvia) communities

Mytilus californianus (Mollusca: Bivalvia), the California marine mussel, occurs in intertidal populations so derise that they are referred to as "Mussel beds." The mussel beds range in physical complexity from structurally simple, essentially mono-layered assemblages, to structurally complex, multi-layered assemblages. The internal environment within the bed varies accordingly. The mussel bed provides either directly or indirectly, habitat, food and shelter for a large community of associated invertebrates. This study examines the relationship between physical complexity of the mussel bed habitat and composition of the associated community.

Patterns of succession in benthic infaunal communities following dredging and dredged material disposal in Monterey Bay

This report is the final product of a two-year study conducted for the Office, Chief of Engineers, by the Moss Landing Marine Laboratories, Moss Landing, California, under Contract No. DACW39-74-C-OI51 with the Environmental Effects Laboratory (EEL), U. S. Army Engineer Waterways Experiment Station (WES), Yicksburg, Mississippi. (PDF contains 192 pages)