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.

Letter from the Editor: special issue of papers on “The Use of Native Insect Herbivores for the Management of Eurasian Watermilfoil," presented at a special session of the Annual Meeting, Aquatic Plant Management Society, July 1998, Memphis, TN

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Letter and Recollections of Alexander Agassiz: The 1891 Albatross Expedition

Presented here is another in the list of historic accounts of iconic research cruises of the USFC Steamer Albatross, this a reminiscence of the renowned scientist Alexander Agassiz edited by his son G. R. Agassiz, a chapter from the volume “Letters and Recollections of Alexander Agassiz,” published in 1913. Agassiz made three major cruises in the Albatross in 1891, 1899–1900, and 1904–05, adding greatly to the world’s store of specimens and knowledge of thalasography, his favored term for oceangraphy, and specifically of the Pacific Ocean. Having made important cruises and studies with the Blake in the Caribbean, he sought to do comparable research in the Pacific. His opportunity came in 1890, and with the consent of President Benjamin Harrison, he took charge of this Albatross research cruise, paying much of the expense himself. In contrast with the other ships he had been on, he found the laboratories, equipment, and furnishings to be comparatively luxurious and extremely well appointed for his work. Further, the Albatross was then captained by Lieutenant Commander Zera Luther Tanner who seemed to take as much interest in the oceanographic research as did the scientists, and Agassiz appreciated working with him, too. Little of the original text has been altered, and readers are cautioned that some of the views expressed may reflect unfortunate prejudices of that era toward individuals, nationalities, etc.

Plan of the Lune Fishery District. Map of District sealed by the Board of agriculture & fisheries

Historic map of the River Lune Fishery District.