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.

Developing alternative shoreline armoring strategies: the living shoreline approach in North Carolina

This paper reviews the scientific data on the ecosystem services provided by shoreline habitats, the evidence for adverse impacts from bulkheading on those habitats and services, and describes alternative approaches to shoreline stabilization, which minimize adverse impacts to the shoreline ecosystem. Alternative shoreline stabilization structures that incorporate natural habitats, also known as living shorelines, have been popularized by environmental groups and state regulatory agencies in the mid-Atlantic. Recent data on living shoreline projects in North Carolina that include a stone sill demonstrate that the sills increase sedimentation rates, that after 3 years marshes behind the sills have slightly reduced biomass, and that the living shoreline projects exhibit similar rates of fishery utilization as nearby natural fringing marshes. Although the current emphasis on shoreline armoring in Puget Sound is on steeper, higher-energy shorelines, armoring of lower-energy shorelines may become an issue in the future with expansion of residential development and projected rates of sea level rise. The implementation of regulatory policy on estuarine shoreline stabilization in North Carolina and elsewhere is presented. The regulatory and public education issues experienced in North Carolina, which have made changes in estuarine shoreline stabilization policy difficult, may inform efforts to adopt a sustainable shoreline armoring strategy in Puget Sound. A necessary foundation for regulatory change in shoreline armoring policy, and public support for that change, is rigorous scientific assessment of the variety of services that natural shoreline habitats provide both to the ecosystem and to coastal communities, and evidence demonstrating that shoreline armoring can adversely impact the provision of those services.

Application of remote sensing to marine fisheries: alternative approaches

A discussion is presented on the applications of remote sensing to fisheries. The measurement of temperature, wind stress, and ocean colour using remote sensing techniques is considered.

Alternative phosphorus sources for formulated fish feed

An experiment was conducted to evaluate the possibility of using inorganic fertilizer triple super phosphate (TSP), inorganic fertilizer 16:20 (a 16:20 grade fertilizer contains 16 percent N and 20 percent P20 5), rice-bran and duck-manure as phosphorus sources in formulated fish feed for Nile tilapia ( Oreochromis niloticus). Experiment was conducted for a period of 2 months in net-cages suspended in fertilized earthen ponds and all male sex-reversed Nile tilapia (9.39- 10.37 g) were used in the experiment. Seven treatments including one non-feed treatment were used in this experiment. Treatment 1 (non-feed), treatment 2 (-P) where fish fed with phosphorus non-supplemented diet acted as control 1 and treatment 3, 4, 5, 6 and 7 where fish fed with 3% di-calcium phosphate (DCP), 3% triple supper phosphate (TSP), 7% 16:20 inorganic fertilizer, 30% rice-bran and 30% duck-manure supplemented diet, respectively. Results showed that the TSP and 16:20 grade inorganic fertilizer supplementation in diets as phosphorus sources were equivalent to DCP (Di-calcium phosphate) supplementation in terms of growth performance, feed utilization efficiency and final body composition of Nile tilapia. Ricebran and duck-manure were not found as good phosphorus sources.