Microbial community analysis of Acropora palamata mucus swabs, water and sediment samples from Hawksnest Bay, St. John, U.S. Virgin Islands

Colonies of the scleractinian coral Acropora palmata, listed as threatened under the US Endangered Species Act in 2006, have been monitored in Hawksnest Bay, within Virgin Islands National Park, St. John, from 2004 through 2010 by scientists with the US Geological Survey, National Park Service, and the University of the Virgin Islands. The focus has been on documenting the prevalence of disease, including white band, white pox (also called patchy necrosis and white patches), and unidentified diseases (Rogers et al., 2008; Muller et al., 2008). In an effort to learn more about the pathologies that might be involved with the diseases that were observed, samples were collected from apparently healthy and diseased colonies in July 2009 for analysis. Two different microbial assays were performed on Epicentre Biotechnologies DNA swabs containing A. palmata coral mucus, and on water and sediment samples collected in Hawksnest Bay. Both assays are based on polymerase chain reaction (PCR) amplification of portions of the small rRNA gene (16S). The objectives were to determine 1) if known coral bacterial pathogens Serratia marcescens (Acroporid Serratiosis), Vibrio coralliilyticus (temperature-dependent bleaching, White Syndrome), Vibrio shiloi (bleaching, necrosis), and Aurantimonas coralicida (White Plague Type II) were present in any samples, and 2) if there were any differences in microbial community profiles of each healthy, unaffected or diseased coral mucus swab. In addition to coral mucus, water and sediment samples were included to show ambient microbial populations. In the first test, PCR was used to separately amplify the unique and diagnostic region of the 16S rRNA gene for each of the coral pathogens being screened. Each pathogen test was designed so that an amplified DNA fragment could be seen only if the specific pathogen was present in a sample. A positive result was indicated by bands of DNA of the appropriate size on an agarose gel, which separates DNA fragments based on the size of the molecule. DNA from pure cultures of each of the pathogens was used as a positive control for each assay.

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.

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.

Influence of microbial interactions on the susceptibility of Karenia spp. to algicidal bacteria

A bacterial strain (D38BY) belonging to the family Flavobacteriaceae and antagonistic towards an algicidal bacterium (strain S03; Flavobacteriaceae) was isolated from a culture of the red tide dinoflagellate Karenia brevis that had previously been characterized as resistant to attack by strain S03. This antagonistic bacterium increased the survival time of otherwise susceptible, bacteriafree K. brevis cultures in a concentration-dependent manner during exposure to the algicidal bacterium. Experimental evidence indicated that direct contact was required in order for strain D38BY to inhibit the killing activity of algicidal strain S03. While further work is needed to determine its precise mode of action, the antagonistic properties of strain D38BY provide further evidence that the resistance or susceptibility of certain algal taxa to algicidal attack can be more a function of interactions within the ambient microbial community than an intrinsic property of the alga.

The coastal environment and human health: microbial indicators, pathogens, sentinels and reservoirs

Innovative research relating oceans and human health is advancing our understanding of disease-causing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to disease-causing organisms from the oceans.

Report of the 2005 Workshop on Ocean Ecodynamics Comparison in the Subarctic Pacific

I. Scientific Issues Posed by OECOS II. Participant Contributions to the OECOS Workshop A. ASPECTS OF PHYTOPLANKTON ECOLOGY IN THE SUBARCTIC PACIFIC Microbial community compositions by Karen E. Selph Subarctic Pacific lower trophic interactions: Production-based grazing rates and grazing-corrected production rates by Nicholas Welschmeyer Phytoplankton bloom dynamics and their physiological status in the western subarctic Pacific by Ken Furuya Temporal and spatial variability of phytoplankton biomass and productivity in the northwestern Pacific by Sei-ichi Saitoh, Suguru Okamoto, Hiroki Takemura and Kosei Sasaoka The use of molecular indicators of phytoplankton iron limitation by Deana Erdner B. IRON CONCENTRATION AND CHEMICAL SPECIATION Iron measurements during OECOS by Zanna Chase and Jay Cullen 25 The measurement of iron, nutrients and other chemical components in the northwestern North Pacific Ocean by Kenshi Kuma The measurement of iron, nutrients and other chemical components in the northwestern North Pacific Ocean by Kenshi Kuma C. PHYSICAL OCEANOGRAPHY, FINE-SCALE DISTRIBUTION PATTERNS AND AUTONOMOUS DRIFTERS The use of drifters in Lagrangian experiments: Positives, negatives and what can really be measured by Peter Strutton The interaction between plankton distribution patterns and vertical and horizontal physical processes in the eastern subarctic North Pacific by Timothy J. Cowles D. MICROZOOPLANKTON Microzooplankton processes in oceanic waters of the eastern subarctic Pacific: Project OECOS by Suzanne Strom Functional role of microzooplankton in the pelagic marine ecosystem during phytoplankton blooms in the western subarctic Pacific by Takashi Ota and Akiyoshi Shinada E. MESOZOOPLANKTON Vertical zonation of mesozooplankton, and its variability in response to food availability, density stratification, and turbulence by David L. Mackas and Moira Galbraith Marine ecosystem characteristics and seasonal abundance of dominant calanoid copepods in the Oyashio region by Atsushi Yamaguchi, Tsutomu Ikeda and Naonobu Shiga OECOS: Proposed mesozooplankton research in the Oyashio region, western subarctic Pacific by Tsutomu Ikeda Some background on Neocalanus feeding by Michael Dagg Size and growth of interzonally migrating copepods by Charles B. Miller Growth of large interzonal migrating copepods by Toru Kobari F. MODELING Ecosystem and population dynamics modeling by Harold P. Batchelder III. Reports from Workshop Breakout Groups A. PHYSICAL AND CHEMICAL ASPECTS WITH EMPHASIS ON IRON AND IRON SPECIATION B. PHYTOPLANKTON/MICROZOOPLANKTON STUDIES C. MESOZOOPLANKTON STUDIES IV. Issues arising during the workshop A. PHYTOPLANKTON STOCK VARIATIONS IN HNLC SYSTEMS AND TROPHIC CASCADES IN THE NANO AND MICRO REGIMES B. DIFFERENCES BETWEEN EAST AND WEST IN SITE SELECTION FOR OECOS TIME SERIES C. TIMING OF OECOS EXPEDITIONS D. CHARACTERIZATION OF PHYSICAL OCEANOGRAPHY V. Concluding Remarks VI. References (109 page document)

The microbiological quality of water: the nature of the problem

Improvements in methods for the detection and enumeration of microbes in water, particularly the application of techniques of molecular biology, have highlighted shortcomings in the ”standard methods” for assessing water quality. Higher expectations from the consumer and increased publicity associated with pollution incidents can lead to an uncoupling of the cycle which links methodological development with standard-setting and legislation. The new methodology has also highlighted problems within the water cycle, related to the introduction, growth and metabolism of microbes. A greater understanding of the true diversity of the microbial community and the ability to transmit genetic information within aquatic systems ensures that the subject of this symposium and volume provides an ideal forum to discuss the problems encountered by both researcher and practitioner.