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.

Saline water intrusion from deep artesian sources in McGregor Isles area of Lee County, Florida

Upward leakage of saline water from an artesian aquifer below 1,500 feet has caused an increase in chloride concentration in the lower Hawthorn aquifer from less than 1,000 mg/1 (milligrams per liter) to values ranging from about 1,300 to 15,000 mg/1. Similarly the higher temperatures of the intruding water has caused an increase in water temperatures in the aquifer from 82"F to values ranging from 83 to 93"F. The intruding water moves upward either through the open bore hole of deep wells or test holes, or along a fault or fracture system, which has been identified in the area. From these points of entry into the lower Hawthorn aquifer, the saline water spreads laterally toward the south and southeast, but is generally confined to components of the fault system. The saline water moves upward from the lower Hawthorn aquifer into the upper Hawthorn aquifer through the open bore hole of wells, which connect the aquifers. This movement has resulted in an increase in chloride from less than 200 mg/1 in the unaffected parts of the upper Hawthorn aquifer to values commonly ranging from about 300 to more than 3,000 mg/1 in parts of the aquifer affected by upward leakage. The upper Hawthorn aquifer is the principal source of ground-water supply for public water-supply systems in western Lee County. Similar effects have been noted in the water-table aquifer, where chloride increased from less than 100 to concentrations ranging from about 500 to more than 5,000 mg/1. This was caused by the downward infiltration of water discharged at land surface from wells tapping the lower Hawthorn aquifer. The spread of saline water throughout most of the McGregor Isles area is continuing as of 1971. (40 page document)

A Comparison of Two Methods for Enhancing the Recovery of Seagrasses into Propellor Scars: Mechanical Injection of a Nutrient and Growth Hormone Solution vs. Defecation by Roosting Seabirds: Final Report.

Based on the recovery rates for Thalassia testudinum measured in this study for scars of these excavation depths and assuming a linear recovery horizon, we estimate that it would take ~ 6.9 years (95% CI. = 5.4 to 9.6 years) for T. testudinum to return to the same density as recorded for the adjacent undisturbed population. The application of water soluble fertilizers and plant growth hormones by mechanical injection into the sediments adjacent to ten propellor scars at Lignumvitae State Botanical Site did not significantly increase the recovery rate of Thalassia testudinum or Halodule wrightii. An alternative method of fertilization and restoration of propellor scars was also tested by a using a method of “compressed succession” where Halodule wrightii is substituted for T. testudinum in the initial stages of restoration. Bird roosting stakes were placed among H.wrightii bare root plantings in prop scars to facilitate the defecation of nitrogen and phosphorus enriched feces. In contrast to the fertilizer injection method, the bird stakes produced extremely high recovery rates of transplanted H. wrightii. We conclude that use of a fertilizer/hormone injection machine in the manner described here is not a feasible means of enhancing T. testudinum recovery in propellor scars on soft bottom carbonate sediments. Existing techniques such as the bird stake approach provide a reliable, and inexpensive alternative method that should be considered for application to restoration of seagrasses in these environments. Document contains 40 pages)

Control of insect infestation in smoked West African sardines Sardinella maderensis 'Sawa' using actellic 50 EC solution

Fish which has been cured or are in the process of being cured by traditional methods are usually infested by insects, posing a real problem to traders and processors, especially in Nigeria. The effects of 0.03% actellic 50 EC solution and vegetable oil on insect infestion were studied using West African sardines, Sardinella maderensis. Actellic solution was more effective in combating insect infestation than vegetable oil. Appearance and perceived smoked fish flavour of fish treated with Actellic and vegetable oil differed (P<0.05), while taste was unaffected by treatment. Actellic 50 EC solution though effective, could be subject to abuse

Shoal manipulation as an effective solution to site specific beach management, examples from three South Carolina beaches

Soft engineering solutions are the current standard for addressing coastal erosion in the US. In South Carolina, beach nourishment from offshore sand deposits and navigation channels has mostly replaced construction of seawalls and groins, which were common occurrences in earlier decades. Soft engineering solutions typically provide a more natural product than hard solutions, and also eliminate negative impacts to adjacent areas which are often associated with hard solutions. A soft engineering solution which may be underutilized in certain areas is shoal manipulation. (PDF contains 4 pages)

Subspecies, morphs and clines in the amphipod Gammarus duebeni from fresh and saline waters

The nature and extent of morphological variation within populations of Gammarus duebeni are examined. The exceptional tolerance of G. duebeni to salinities that encompass three orders of magnitude was known in the 19th Century, and has attracted considerable attention from physiologists and ecologists in the 20th Century, including the likelihood that populations in freshwater are distinct from those living in more saline environments. It is concluded that gradual evolution into discrete and readily distinguished subspecies is currently underway, eventually producing several new species in freshwater and saline habitats.