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.

Studies on radiation pasteurisation of medium fatty fish 2. Precursors of yellow discolouration in irradiated white pomfret (Stromateus cinereus)

Triglycerides, phospholipids and sarcoplasmic proteins fractions of white pomfret produced considerable amounts of thiobarbituric acid reactive substances (TBRS) on irradiation. Incubation of malonaldehyde with pomfret skin under aseptic conditions developed yellow pigmentation of the skin tissues, similar in spectral characteristics to those produced on irradiation of the skin.

Ascorbic acid turnover in the ocular tissues of some fishes

The ascorbic acid turnover from the ocular tissues of 11 species of fishes from a culture pond and the river Godavari (Andhra Pradesh, India) has been studied. The free ascorbic acid and ascorbigen contents were more in the case of bottom or deep dwelling fishes and the least in the case of surface living forms, depending upon the light penetration in the area that each species inhabits. The enzymic utilization and ascorbic acid-macromolecule complex varied among the fishes possibly depending upon individual energy requirements and not upon light intensity. No size-related or sex-related variation was observed. No variation was observed between riverine and pond-reared fishes of the same species.

The study of Indian shrimp larvae feeding by enriched rotifer with vitamin C and unsaturated fatty acid (epa, dha) and its effects on growth factors, survival rate and environmental stresses

In this experiment, the feeding of Indian white shrimp larvae by unenriched rotifers (treatment 1) and enriched with highly unsaturated fatty acid (treatment 2) and highly unsaturated fatty acid along with vitamin C (treatment 3) on the growth factors, survival and resistance against salinity and formalin stress tests were studied and their differences with control treatment including newly hatched Artemia nauplii is compared. In this the study four treatments in a completely randomized design with 3 replicates per treatment were used. Farming of shrimp larvae of Zoea II to postlarvae 5 was done in 20 liter plastic bucket. Present results indicated that growth factors and survival rate of stage Zoea II to postlarvae 1 in treatments 1, 2 and 3 improve rather than control in which this case was due to optimal size rotifer rather than Artemia nauplii. Also, treatments 2 and 3 feeding with oil liver cod emulsion enriched rotifer have the highest concentration of DHA (mg/g DW) and the ratio DHA/EPA in which due to have shown the highest growth factors and a significant difference (P<0.05) with treatments 1 and control. The highest survival at stage PL1 were observed in treatment 3 that was enriched with ascorbyl palmitate in which have to the synergistic properties of vitamin C rather than treatments 2, 1 and control and showed a significant difference (P<0.05). But in stage PL5 the highest amount of growth and survival rates were related to control treatment which showed a significant difference (P<0.05) with other treatments that control has higher size rather than treatments 1, 2 and 3. Also, among experiment treatments that the two treatments 2 and 3 due to enrichment had higher growth and survival rates compared with treatment 1 in which their differences have also been significant (P<0.05). In the case of stress tests, results indicated that the highest survival rate has been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids with vitamin C. So that in stage PL1 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3. As for the second, treatment 2 showed a significant difference (P<0.05) with treatment 3. It is worth mentioning that treatment 3 showed a higher survival rate compared to treatment 2 due to the synergistic properties of vitamin C. The difference between these two treatments with treatment 1 and control was also significant. No significant difference was observed in formalin stress test 100 ppm in this stage between treatments 3 and 2 which shows the highest survival rate. But their difference with treatments 1 and control was significant (P<0.05). Also, in stage PL5 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3 which showed no significant difference (P<0.05) with control treatment. While their difference in the amount of survival rate with treatment 1 and 2 was significant (P<0.05). In this stage, the highest observed survival rate in formalin stress test 100 ppm included treatments control, 3 and 2 among which there were no significant differences (P<0.05). While the difference between these three treatments with treatment 1 was significant.