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.

Estimation of RNA/DNA ratio in two north-Indian natural populations of mahseer (Tor tor) and its relationship with growth and hydrobiology

Samples of Tor tor were collected from Bari Reservoir of Udaipur and Narmada River at Hoshangabad (India), in the months of July and November 2005, respectively. Twenty-five samples were collected from each location. Bari Reservoir samples ranged from 17.0 to 24.5 cm in total length and from 75 to 155 g in weight, while Narmada samples ranged from 20.0 to 42.0 cm in length and 90 to 425 g in weight. The nucleic acid content in body muscle of Tor tor and the RNA/DNA ratio were estimated. The age of fishes was estimated by the scale study method and specimens were classified into four age groups. RNA/DNA ratio showed significant linear increase with increase in weight and age till the age of three years after which, the growth rate reduced. The 1-2 year group was the only one common between the two water bodies and a comparison of RNA/DNA ratios showed higher growth rate in Bari Reservoir. The gross primary productivity was also higher in Bari Reservoir being 551 mg cmˉ³ dˉ¹ compared to 404 mg cmˉ³ dˉ¹ observed for Narmada River. The condition factor (K) was found to be higher (1.21) in the fish from the Bari Reservoir compared to those of Narmada River (1.14). The growth rate was higher in females compared to males in >100 g specimens.

Nutritional evaluation of texturised meat from Nemipterus japonicus

Evaluation of the nutritional quality of texturised fish meat from threadfin bream (Nemipterus japonicus) is reported. Proximate composition and amino acid analysis showed that its protein content is very high with all the essential amino acids present in higher levels than those prescribed for man in the 1973 FAO/WHO amino acid scoring pattern. Lysine amounted to 11.0 g per 16 g N of which 75 percent is biologically available. Studies on PER and NPU also corroborate the excellent nutritional quality of the texturised meat.

Studies on two unexploited fish species: Peristedion adeni and Peristedion weberi

The chemical composition and nutritional quality of Peristedion adeni and Peristedion weberi, two unexploited fish species available in abundance in the Exclusive Economic Zone of India are reported. Size of the fish is small and separation of edible meat is difficult. Best method of utilization of the fish is conversion into meal. Meals having crude protein content varying from 55.0 to 57.0%, ash content from 17.0 to 19.6% and fat content from 17.7 to 18.1% can be prepared from the two species of fish. PER values of the meals are lower than that of casein. Amino acid analysis showed that isoleucine, leucine and valine are not present in the meals in adequate quantities compared to the FAO/WHO (1973) prescribed pattern.

New insights into the diets of harbor seals (Phoca vitulina) in the Salish Sea revealed by analysis of fatty acid signatures

Harbor seals (Phoca fvitulina) are an abundant predator along the west coast of North America, and there is considerable interest in their diet composition, especially in regard to predation on valued fish stocks. Available informationon harbor seal diets, primarily derived from scat analysis, suggests that adult salmon (Oncorhynchus spp.), Pacific Herring (Clupea pallasii), and gadids predominate. Because diet assessments based on scat analysis may be biased, we investigated diet composition through quantitative analysis of fatty acid signatures. Blubber samples from 49 harbor seals captured in western North America from haul-outs within the area of the San Juan Islands and southern Strait of Georgia in the Salish Sea were analyzed for fatty acid composition, along with 269 fish and squid specimens representing 27 potential prey classes. Diet estimates varied spatially, demographically, and among individual harbor seals. Findings confirmed the prevalence of previously identified prey species in harbor seal diets, but other species also contributed significantly. In particular, Black (Sebastes melanops) and Yellowtail (S. flavidus) Rockfish were estimated to compose up to 50% of some individual seal diets. Specialization and high predation rates on Black and Yellowtail Rockfish by a subset of harbor seals may play a role in the population dynamics of these regional rockfish stocks that is greater than previously realized.