Molecular probes and polymerase chain reaction (PCR) -based kits for diagnosis of shrimp diseases

Technology for effective and fast diagnosis of animal diseases is essential for developing aquaculture management strategies. This paper reviews the conventional techniques for shrimp disease diagnosis and discusses the emergence of nuclei acid probes and polymerase chain reaction (PCR)-based kits as powerful tools for rapid and accurate detection of shrimp diseases.

Recent Developments in In Situ Nutrient Sensors: applications and future directions, Savannah, Georgia, December 11-13, 2006: workshop proceedings

The Alliance for Coastal Technologies (ACT) convened a Workshop on "Recent Developments in In Situ Nutrient Sensors: Applications and Future Directions" from 11-13 December, 2006. The workshop was held at the Georgia Coastal Center in Savannah, Georgia, with local coordination provided by the ACT partner at the Skidaway Institute of Oceanography (University System of Georgia). Since its formation in 2000, ACT partners have been conducting workshops on various sensor technologies and supporting infrastructure for sensor systems. This was the first workshop to revisit a topic area addressed previously by ACT. An earlier workshop on the "State of Technology in the Development and Application of Nutrient Sensors" was held in Savannah, Georgia from 10-12 March, 2003. Participants in the first workshop included representatives from management, industry, and research sectors. Among the topics addressed at the first workshop were characteristics of "ideal" in situ nutrient sensors, particularly with regard to applications in coastal marine waters. In contrast, the present workshop focused on the existing commercial solutions. The in situ nutrient sensor technologies that appear likely to remain the dominant commercial options for the next decade are reagent-based in situ auto-analyzers (or fluidics systems) and an optical approach (spectrophotometric measurement of nitrate). The number of available commercial systems has expanded since 2003, and community support for expanded application and further development of these technologies appears warranted. Application in coastal observing systems, including freshwater as well as estuarine and marine environments, was a focus of the present workshop. This included discussion of possible refinements for sustained deployments as part of integrated instrument packages and means to better promote broader use of nutrient sensors in observing system and management applications. The present workshop also made a number of specific recommendations concerning plans for a demonstration of in situ nutrient sensor technologies that ACT will be conducting in coordination with sensor manufacturers.[PDF contains 40 pages]

A rapid and simple method for the determination of Formaldehyde in Fishery Products

The content of free formaldehyde (FA) in minced fish muscle was measured by the following procedure: A mixture of minced fish muscle and water was deproteinized by means of the Carrez reagent. The FA content of the filtrate was determined reflectometrically using the Reflectoquant test strips and the RQflex. The results agreed well with the colorimetrically (Nash test) measured FA content. Der Gehalt an freiem Formaldehyd (FA)in Fischerzeugnissen wurde mit folgender Methode bestimmt: Zerkleinertes Fischfleisch wurde mit Wasser homogenisiert und mit Carrez-Reagenz enteiweißt. Der Formaldehydgehalt des Filtrates wurde reflektometrisch unter Verwendung von Reflectoquant-Teststätbchen und des RQflex ermittelt. Die Ergebnisse stimmten gut mit kolorimetrisch (Nash Test) gemessenen FA-Gehalten überein.

Production of positive controls for calcivirus-specific PCR using recombinant baculiovirus technology

Recent advances in our knowledge of the genetic structure of human caliciviruses (HuCVs) and small round-structured viruses (SRSVs) have led to the development of polymerase chain reaction (PCR)-based molecular tests specific for these viruses. These methods have been developed to detect a number of human pathogenic viruses in environmental samples including water, sewage and shellfish. HuCVs and SRSVs are not culturable, and no animal model is currently available. Therefore there is no convenient method of preparing viruses for study or for reagent production. One problem facing those attempting to use PCR-based methods for the detection of HuCVs and SRSVs is the lack of a suitable positive control substrate. This is particularly important when screening complex samples in which the levels of inhibitors present may significantly interfere with amplificiation. Regions within the RNA polymerase regions of two genetically distinct human caliciviruses have been amplified and used to produce recombinant baculoviruses which express RNA corresponding to the calicivirus polymerase. This RNA is being investigated as a positive control substrate for PCR testing, using current diagnostic primer sets. Recombinant baculovirus technology will enable efficient and cost-effective production of large quantities of positive control RNA with a specific known genotype. We consider the development of these systems as essential for successful screening and monitoring applications.

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.

Cruise report: regional assessment of ecosystem condition and stressor impacts along the northeastern Gulf of Mexico shelf; NOAA Ship Nancy Foster NF-10-09-RACOW (August 13-21, 2010)

This cruise report is a summary of a field survey conducted along the continental shelf of the northeastern Gulf of Mexico (GOM), encompassing 70,062 square kilometers of productive marine habitats located between the Mississippi Delta and Tampa Bay, August 13–21, 2010 on NOAA Ship Nancy Foster Cruise NF-10-09-RACOW. Synoptic sampling of multiple ecological indicators was conducted at each of 50 stations throughout these waters using a random probabilistic sampling design. At each station samples were collected for the analysis of benthic community structure and composition; concentrations of chemical contaminants (metals, pesticides, TPHs, PAHs, PCBs, PBDEs) in sediments and target demersal biota; sediment toxicity; nutrient and chlorophyll levels in the water column; and other basic habitat characteristics such as depth, salinity, temperature, dissolved oxygen, turbidity, pH, CDOM fluorescence, sediment grain size, and organic carbon content. Discrete water samples were collected just below the sea surface, in addition to any deeper subsurface depths where there was an occurrence of suspicious CDOM fluorescence signals, and analyzed for total BTEX/TPH and carcinogenic PAHs using immunoassay test kits. Other indicators of potential value from a human-dimension perspective were also recorded, including presence of any vessels, oil rigs, surface trash, visual oil sheens in sediments or water, marine mammals, or noxious/oily sediment odors. The overall purpose of the survey was to collect data to assess the status of ecosystem condition and potential stressor impacts throughout the region, based on these various indicators and corresponding management thresholds, and to provide this information as a baseline for determining how such conditions may be changing with time. In addition to the original project goals, both the scientific scope and general location of this project are relevant to addressing potential ecological impacts of the Deepwater Horizon oil spill. While sample analysis is still ongoing, a few preliminary results and observations are reported here. A final report will be completed once all data have been processed.

Marine bacteria as indicators of upwelling in the Sea

The “oxidase reaction” (using p-amino-dimethyl-aniline oxalate as the reagent) has been used to distinguish oxidase-negative from oxidase-positive bacteria from the sea, when grown on membrane filters. By this means, it has been shown (a) that under conditions of stable stratification of the sea as in the tropics, a relationship exists between the percentage incidence of oxidase negative bacteria in the flora and the depth of the water; (b) that the maximum value for this percentage incidence (100) is reached at or immediately below the upper limit of the oxygen minimum layer; (c) that this percentage value (expressed as Oxⁿvalues) may be used to demonstrate the movements of water masses during upwelling. Such upwelling as indicated by theoretical findings and by temperature determinations along two transects off the west coast of Ceylon during the north east monsoon, has been confirmed by the distribution of Oxⁿvalues at these transects.

Acute toxicity of unionized ammonia to milkfish (Chanos chanos Forsskal) fry

The study was conducted to determine the effects of varying concentrations of ammonia to milkfish fry. Two runs of static 96h bioassays were conducted to determine the median lethal concentration (LC 50) of unionized ammonia (NH₃) to milkfish fry. Test concentrations were based on exploratory 24h and 48h bioassays and were made in three replicates. Reagent grade ammonium chloride (NH₄Cl) was used to adjust the level of unionized ammonia. The 96h median lethal concentration, determined by the Reed Muench method was calculated at 28.029 ppm NH₃ 29.69 ppm. Even at high concentrations of unionized ammonia, most of the fry mortality occurred after 48 to 96 hours exposure. Severe gill damage occurs only at concentrations above 20 ppm, especially above the LC 50. The high LC 50 value obtain shows that milkfish fry has great tolerance to ammonia, that even fry with severely-damaged gills can still recover days after it is returned to favorable culture condition. The result suggest that observed mortalities of milkfish fry under culture conditions are not due to ammonia toxicity.