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.

Der Fischei-Test: Ein Toxizitätstest für ökotoxikologische Untersuchungen

Zebrafish (Danio rerio) embryos have been used to quantify the teratogenic potential of environmental samples and harmful substances respectively. The short spawning interval renders this species a good test organism in toxicological research. Due to the transparency of the eggs several lethal and non-lethal endpoints can be detected in parallel after 48 h of embryonic development. Zebrafishembryos have been shown to be sensitive to a number of environmental relevant contaminants, as well as to ex-tracts from polluted sediments

Anwendung von Fischzellkulturen in der Meeresforschung

A large number of chemical pollutants can be found in the marine environment. So it is necessary to obtain informations about the toxic effects of this contaminant mixtures in general and especially on single cell level. We used an organic extract of a marine sediment from the North Sea to investigate its cyto- and genotoxicity with an in vitro system, the comet assay or single cell gel electrophoresis (SCGE). The comet assay can be applied for estimating genotoxic effects of chemicals on single cell level. First results confirm the sensitivity of this assay and its applicability in assessing genotoxic load in environmental samples. A permant cell line, the EPC (Ephithelioma papulosum cyprini) was used for the experiments. It was possible to demonstrate the suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells.

Analysis of soils and sediments for organic pollutants

20 samples of soil or sediment (7 of which were predominantly sand) from various locations were received for analysis of their content of organic pollutants. These analyses were performed using a capillary column gas chromatograph equipped with an electron impact (E.I.) mass spectrometer as detector and using computerised data storage. In addition to the target compounds, the full scan data were examined to determine the composition of natural organic products and a series of diagnostic fragment ions was used to search for additional anthropogenic products. Organic-rich environmental samples are notoriously difficult to analyse for pollutant organics owing to the presence of high concentrations of many natural organic compounds. A single procedure for extraction and clean-up was adopted. It was designed for chlorinated aliphatic and aromatic hydrocarbons and other pesticides containing acidic functional groups and was based on published methods for the determination of organic pollutants in soils and sediments. 4 soils and 2 sands showed levels of one or two groups of PCBs slightly in excess of the detection limit, one sample showed a similar level of 2,4-D and 3 samples contained dieldrin at or just above the detection limit.

Production of positive controls for calcivirus-specific PCR using recombinant baculiovirus technology

Recent advances in our knowledge of the genetic structure of human caliciviruses (HuCVs) and small round-structured viruses (SRSVs) have led to the development of polymerase chain reaction (PCR)-based molecular tests specific for these viruses. These methods have been developed to detect a number of human pathogenic viruses in environmental samples including water, sewage and shellfish. HuCVs and SRSVs are not culturable, and no animal model is currently available. Therefore there is no convenient method of preparing viruses for study or for reagent production. One problem facing those attempting to use PCR-based methods for the detection of HuCVs and SRSVs is the lack of a suitable positive control substrate. This is particularly important when screening complex samples in which the levels of inhibitors present may significantly interfere with amplificiation. Regions within the RNA polymerase regions of two genetically distinct human caliciviruses have been amplified and used to produce recombinant baculoviruses which express RNA corresponding to the calicivirus polymerase. This RNA is being investigated as a positive control substrate for PCR testing, using current diagnostic primer sets. Recombinant baculovirus technology will enable efficient and cost-effective production of large quantities of positive control RNA with a specific known genotype. We consider the development of these systems as essential for successful screening and monitoring applications.

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.

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.

Environmental quality and aquaculture systems: Proceedings of the Thirteenth U.S.-Japan Meeting on Aquaculture, Mie, Japan, October 24-25, 1984

The United States and Japanese counterpart panels on aquaculture were formed in 1969 under the United States-Japan Cooperative Program in Natural Resources (UJNR). The panels currently include specialists drawn from the federal departments most concerned with aquaculture. Charged with exploring and developing bilateral cooperation, the panels have focused their efforts on exchanging information related to aquaculture which could be of benefit to both countries. The UJNR was begun during the Third Cabinet-Level Meeting of the Joint United States-Japan Committee on Trade and Economic Affairs in January 1964. In addition to aquaculture, current subjects in the program include desalination of seawater, toxic microorganisms, air population, energy, forage crops, national park management, mycoplasmosis, wind and seismic effects, protein resources, forestry, and several joint panels and committees in marine resources research, development, and utilization. Accomplishments include: Increased communication and cooperation among tecbnical specialists; exchanges of information, data, and research findings; annual meetings of the panels, a· policy-coordinative body; administrative staff meetings; exchanges of equipment, materials, and samples; several major technical conferences; and beneficial effects on international relations. (PDF file contains 56 pages.)

Report on the physical, bacteriological and biochemical analyses of seawater samples collected off existing sewage outfalls in the Sierra Leone River Estuary

The studies reported were undertaken as part of a wide environmental feasibility study for the establishment of a modern sewage system in Freetown. The aim of this part of the study was to determine whether the hydrological regime of the Sierra Leone River Estuary would permit the large-scale introduction of sewage into the estuary without damaging the environment. The important factors were whether: 1) there would be sufficient dilution of the sewage; 2) fleatable particles or other substances would create significant adverse effects in the estuarine ecosystem. The outfall sites are described together with the sampling stations, methods and analyses. Results include: 1) T/S profiles; 2) chemical analysis of the water. A review of literature on the Sierra Leone River Estuary is included which provides information on the plankton, benthos and fisheries. Results suggest that at certain points where local circulations occur it would be inadvisable to locate untreated sewage outfalls. Such points are frequently observed in small embayments. These studies have been of short duration but the data can serve as baseline for more extended investigations which would give a more complete picture of the seasonal patterns in the estuary.

Environmental variables and fisheries diversity of the Naaf River Estuary, Bangladesh

The Naaf River estuary is one of the large estuaries in the Bangladesh coastal region not to have been affected by extensive human disturbance. This research provides information about the fisheries diversity status by Estuarine Set Bag Net (ESBN) sampling relation to physicochemical variables in both spatio-temporal scales. About 25 km of the lower estuary was divided into six zones for sample collection by considering the accessibility and availability of the ESBN operation, fish landing centers and location of the fishing villages. In total 48 samples have been analyzed which were taken throughout March to October 2006. To quantify the species diversity, all fisheries data were analyzed by using EstimateS and EcoSim software which accounts the different diversity indices viz., species richness, Shannon–Wiener diversity Index, Dominance and Evenness index. The research results demonstrate that the Naaf River estuary is a habitat of 161 (species richness, Sobs=161, Choa 1=162±2.34, ACE=161.73) different species which belong to 98 fin fishes, 23 shrimps and prawns,13 crabs, 11 molluscs, 3 echinoderms, 4 other crustaceans;while 9 remain unidentified. Results on the aquatic environment,mainly salinity and turbidity were found to have a major influence on their occurrence and distribution. All the findings indicated that the Naaf River estuary is a highly productive system and provides a favourable environment for large variety of estuarine species assemblages.

Interpreting the environmental record in the sediments of Blelham Tarn

Two aspects of the environmental record in the sediments of Blelham Tarn have been investigated: (1) the ecological history of the catchment, and lake-catchment relationships, by detailed analysis of the preserved pollen (as an indication of vegetation) and sediment composition with respect to a range of inorganic and organic geochemical variables and (2) a detailed investigation of the manner in which sediment is being formed today, including the way in which microfossils (pollen and diatoms) are being recruited and incorporated into sediments. When the record was examined by biological and geochemical analysis, together with radionuclide dating, of closely spaced samples, it was found that the changes of the last 30 yrs represented only the most recent episode in a long history of modification of the lake by man. To find an approach to this it is necessary to go back for at least 2500 yrs.

Detection of cryptosporidium oocysts in water and environmental concentrates

Whilst current methods for the isolation and enumeration of Cryptosporidium spp. oocysts in water have provided some insight into their occurrence and significance, they are regarded as being inefficient, variable and time-consuming, with much of the interpretation being left to the expertise of the analyst. Two expectations of novel developments are to reduce the variability and subjectivity associated with the isolation and identification of oocysts. Flocculation, immunomagnetisable and flow cytometric techniques, for concentrating oocysts from water samples, should prove more reliable than current methods, whilst the development of more avid and specific monoclonal antibodies in conjunction with the use of nuclear fluorochromes will aid identification. Further insight into the viability, taxonomy, species identification, infectivity and virulence of the parasite should be forthcoming through the use of techniques such as the polymerase chain reaction, in situ hybridisation and non-uniform alternating current electrical fields. Such information is necessary in order to enable microbiologists, epidemiologists, engineers, utility operators and regulators to assess the safety of a water supply, with respect to Cryptosporidium contamination, more effectively.

Mersey Estuary baseline survey. Analysis of macroinfaunal samples, literature review and database production

This is the Mersey Estuary baseline survey: Analysis of macroinfaunal samples, literature review and database production report produced by the Environment Agency North West in 2002. This report presents an ecological review of the Mersey estuary along with an extensive bibliography of the available environmental literature for this system. The central objective of this programme has been to provide the information necessary to support the Environment Agency's review of existing and future consents (for discharges, abstractions etc) in the Mersey estuary. This review of consents was required because the Mersey had been designated as a Special Protection Area (SPA) under the EC Birds Directive. Therefore under Regulation 50 of the Conservation, the Environment Agency was responsible for reviewing any extant consent, or future applications, which may directly or indirectly, affected this SPA.

Technical report on the environmental monitoring of the cage area at the Source of the Nile (SON) Fish Farm for quarter 1: January-March 2015

Source of the Nile (SON) fish farm is located at Bugungu in Napoleon Gulf, northern Lake Victoria. The proprietors of the farm have a collaborative arrangement with NaFIRRI, a lead agency in fisheries research and innovations, to undertake quarterly environment monitoring surveys at the farm. The agreed areas for monitoring are: selected physico-chemical parameters (i.e. temperature, dissolved oxygen, pH, conductivity, secchi depth); total suspended solids (TSS); nutrient status; BOD5) and biological parameters (i.e. algae, zooplankton, macro-benthos and fish). Water and biological samples as well as field measurements were taken at 3 sites: within the fish cage rows (WIC/experimental), upstream (USC/control) and downstream (DSC) of the fish cages. The key research question was: Does fish cage operations have impacts on the water quality and aquatic biota in and around the SON cage fish farm? The environment monitoring surveys were projected to cover a full calendar year (i.e. from January to December). The first surveys were undertaken in 2011 and have continued on an annual basis since then. The present report presents field observations made for the fourth quarter survey undertaken in November 2014 and provides a scientific interpretation and discussion of the results with reference to possible impacts of the cage facilities to the water environment and the different aquatic biota in and around the fish cage site.

Impacts of environmental conditions on macrobenthic distribution along the Suez Gulf, Egypt

Increases in coastal development and human activities leading to sedimentation degrade the quality of water; disturb the natural structure and functions of aquatic communities. The Suez Gulf is a large semi-closed area (~625 km long coastline).The assemblages of bottom fauna were studied qualitatively and quantitatively in the shallow intertidal waters along the western coast of the Suez Gulf. The quality of seawater and sediment structures were analyzed. The distribution of macro-benthos included a total of 38 species of Gastropoda and 9 Bivalvia; and 25 species from the other invertebrates included 7 groups namely, Rhizostoma, Polychaeta, Cirripedia, Amphipoda, Isopoda, Decapoda and Echinodermata. The most dominant group among invertebrate groups was the Polychaeta which included 4 species: Hydroides elegans, Perinereis cultilifera, Perinereis nuntia and Ophelina acuminata. The Cirripedia were represented by 3 species namely, Balanus amphitrite, Chithamalus challengeri and Tetraclita squamosa. The variations in the numerical abundance and biomass of bottom fauna studied between the observation periods and at sampling sites. There was a marked increase in benthos biomass at St. IV (Ras Gharib) yielding an average of 318.8 g/m² in which the gastropod community represented the dominant species in collected samples reaching 270.28 g/m² (84.4% of the total biomass) and numerically numbered 116 ind./m². Veliger larvae of bivalves and gastropods appeared to be present in the plankton for long periods and their production seems to be continuous throughout the year. In the intertidal zone of the Suez Gulf, the values of pH varied within narrow limits. Water temperature and salinity seemed to be important in the distribution and abundance of the macro-benthos communities in the study areas. The organic content in shallow intertidal waters and sediments indicated high values in the central part of the Gulf of Suez.