DNA extraction from formalin-fixed Franciscana tissues

The present paper reports the extraction of DNA from formalin-fixed Pontoporia blainvillei tissues. Following the Vachot and Monerot (1996) protocol, fragmented DNA (300-700bp) was extracted from more than 95% of liver and muscle samples. DNA yield in liver samples was significantly higher than in muscle samples (4.574 ± 1.169mg DNA/mg versus 0.808 ± 0.297mg DNA/mg). Similar results were obtained from nine other species of cetaceans and five species of pinnipeds. It is of special interest to have a method that allows the utilisation of museum specimens not originally preserved for genetic studies, which may include rarely available, declining or extinct species. SPANISH: El presente trabajo reporta la extracción de ADN a partir de tejidos formolizados de Pontoporia blainvillei. Siguiendo el protocolo de Vachot y Monerot (1996) se pudo extraer ADN degradado (300-700pb) en más del 95% de las muestras de hígado y músculo analizadas. El rendimiento en ADN fue significativamente mayor en muestras de hígado que en muestras de músculo (4.574 ± 1.169mg DNA/mg tejido húmedo versus 0.808 ± 0.297mg DNA/mg tejido húmedo). Resultados similares se obtuvieron en otras nueve especies de Cetáceos y cinco de Pinnípedos. Resulta de gran interés contar con un método que permita la utilización de especímenes depositados en museos y que no hayan sido originalmente colectados para estudios genéticos, incluyendo especies de difícil obtención, en franca declinación o extintas.

Assessment of the hydrogeological impacts of aggregate extraction in the Delamere area, Cheshire

This is a technical report on the assessment of the hydrogeological impacts of aggregate extraction activities in the Delamere Area, Cheshire. The first aim of the study was to carry out Stage 3-appropriate assessment, under the EU Habitats Directive (92/43/EEC), of the possible hydrogeological impacts of aggregate extraction activities authorised by the Cheshire CC on candidate Special Areas of Conservation (cSAC) on the Delamere sandsheet, Cheshire. Identifying possible impacts if these activities on the hydrogeological environment, construction of a numerical groundwater flow model of the groundwater system to investigate and quantify impacts and to produce a report as required under Stage 3 of the Habitats Regulations. Secondly, to identify the future potential impacts of the continued extraction of sand and gravel reserves from above and below the water tables from within the Delamere sandsheet, thus releasing reserves identified within the Area of Search of the Cheshire Replacement Minerals Local Plan 1999. This aspect of the study should assist in identifying the implications of further working within Delamere for North West sub-regional apportionment.

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.

Effects of blood extraction on horseshoe crabs (Limulus polyphemus)

Horseshoe crabs (Limulus polyphemus) are caught by commercial fishermen for use as bait in eel and whelk fisheries (Berkson and Shuster, 1999)—fisheries with an annual economic value of $13 to $17 million (Manion et al.1). Horse-shoe crabs are ecologically important, as well (Walls et al., 2002). Migratory shorebirds rely on horseshoe crab eggs for food as they journey from South American wintering grounds to Arctic breeding grounds (Clark, 1996). Horse-shoe crabs are also essential for public health (Berkson and Shuster, 1999). Biomedical companies bleed horse-shoe crabs to extract a chemical used to detect the presence of endotoxins pathogenic to humans in injectable and implantable medical devices (Novitsky, 1984; Mikkelsen, 1988). Bled horseshoe crabs are returned to the wild, subject to the possibility of postbleeding mortality. Recent concerns of overharvesting have led to conflicts among commercial fishermen, environmentalists acting on behalf of the shorebirds, and biomedical companies (Berkson and Shuster, 1999; Walls et al., 2002).

Improved chemical methods for extraction of fin rays from shark fins

A survey was undertaken of methods available for the extraction of fin rays from shark fins. The development of new, quicker and easier methods of processing is presented.

A new method for venom extraction from venomous fish, green scat

Scatophagus argus argus (Green Scat) is a pretty aquarium fish. Its hard spines are venomous and can cause painful injury. In this study 60 specimens of Green Scat were collected periodically from coastal waters of Boushehr (south of Iran) from May 2011 to April 2012. Anatomical features of venomous spines were investigated. Scat venom was extracted from the spines in a new manner for keeping the specimens alive. The nature of venom was tested by SDS-PAGE. Ethical issues and animal welfare principles such as rapid and instantaneous anesthetizing, post operation disinfection and fast recovery of the specimens was practiced in order to minimize the complications. This method enhanced the purity and quantity of venom as demonstrated by 12 separated proteins in electrophoresis. New ethical issues were developed to surviving the specimens and prolong viability as well.

Shark fin rays: technology of extraction

A simple process is described for extraction of rays from shark fins. The process consists in treating the rays with acetic acid to soften the tissue, separation of the rays by hand and drying. White fins yield almost double the quantity of rays compared to black fins.

The extraction of mimosine from ipil-ipil (Leucaena leucocephala) by soaking in water

The study aimed to find a cheap and practical method of extracting mimosine from Leucaena leucocephala, otherwise known as ipil-ipil in the Philippines. L. leucocephala leaves are used in cattle, poultry and swine feed and have been tried as a food ingredient in some fish diets. While it contains relatively high amount of protein, its use as feed has been limited because of the presence of toxic substance, mimosine. Findings revealed that soaking the leaves in water was highly efficient for the extraction of mimosine, the longer the duration of soaking the more mimosine was extracted. On the other hand, 87 % of the juveniles Penaeus monodon fed with diets containing L. leucocephala leaves soaked for 24 hours survived, much higher compared to those that were fed with unsoaked leaves for eight weeks.

Extraction, purification and analysis of conotoxin of Conus textile captured from Persian Gulf and the investigation of analgesic effects of conotoxins in an animal model

Identification of venomous species of Persian Gulf cone snails and characterization of venom composition and their features is so important from the point of medical importance. Marine cone snails from the genus Conus are estimated to consist of up to 700 species. The venom of cone snails has yielded a rich source of novel neuroactive peptides or conotoxins. The present study was aimed to study the analgesic effect of Persian Gulf Conus textile and its comparison with morphine in mouse model. The specimens of Conus textile were collected of Larak Island from depth of 7 m. The collected samples were transferred to laboratory alive and were stored at -700 c. he veno s ducts were separated and ho ogenized with deionized water he ixture centrifuged at rp for inutes upernatant was considered as extracted veno and stored at - C after lyophylization. The protein profile of venom determined by using SDS-PAGE and HPLC used to investigate the extracted venom and to evaluate the analgesic activity, formalin test was carried out. SDS-PAGE indicated several bands ranged between 6 and 250 kDa. Chromatogram of the venom demonstrated more than 44 large and small fractions. The amount of 10 ng of Conus crude venom and analgesic peptide showed the best anti-pain activity in formalin test. No death observed up to 100 mg/kg, which is 250,000 times higher than the effective dose.Venom characterization of Persian Gulf Conus textile may be of medical importance and potential for new pharmaceutical drugs as well.

Venomics and biological analysis of Scatophagus argus argus (Spotted scat) venom isolated in Persian Gulf, extraction and purification of hemolytic protein

Green scat namely as Scatophagus argus argus is a venomous aquarium fish belonging to Scatophagidae family. It can induce painful wounds in injured hand with partial paralysis to whom that touch the spines. Dorsal and ventral rough spines contain cells that produce venom with toxic activities. According to unpublished data collected from local hospitals in southern coastal region of Iran, S. argus is reported as a venomous fish. Envenomation induces clinical symptoms such as local pain, partial paralysis, erythema and itching. In the present study green scat (spotted scat) was collected from Persian Gulf coastal waters. SDS-PAGE indicated 12 distinct bands in the venom ranged between 10-250 KDa. The crude venom had hemolytic activity on human erythrocytes (1%) with an LC100 (Lytic Concentration) of about 1.7 μg. The crude venom can release 813 μg proteins from 0.5% casein. Phospholipase C activity was recorded at 3.125 μg of total venom. Our findings showed that the edematic activity remained over 48 h after injection. The purification of the venom was done by HPLC and 30 peaks were obtained within 80 min but only one peak in 68 min retention time showed hemolytic activity at 90% acetonitril was isolated. The area percentage of the hemolytic protein showed that this hemolytic protein consist of 32 percent of total proteins and its molecular weight was 72 KDa in SDS_PAGE. The results demonstrated that crude venom extracted from Iranian coastal border has different toxic and enzymatic activities.

Extraction and structure elucidation of natural compounds of some gorgonians of the Persian Gulf and also physiological and pharmacological effects of gorgonian extract on some selected organisms

In the present research, investigations were carried out for structure elucidation of natural compounds and also for studing biological and teratogenical effects of two Genus of soft corals named as " Echinogorgia cf. indica" and "Sinularia erecta" in Persian Gulf. First, 350 gr Echinogorgia was extracted by Acetone, then, the extract was separated by ether from aqueos phase to give 4.5 gr oil. The oil eluted with Petrol - ether Et2 o (9:1) which was recovered Linderazulene and it's derivative as purple Cristals (350 mg/ca 0.1 %). In order to determine molecular structure, the Samples were used for spectroscopic method as: H1- NMR , C13- NMR and 2D NMR. Also, for extraction and structure elucidation of natural compounds, the soft coral " sinularia erecta " were used 1187/37 gr and extracted by Aceton. The extract was concentrated and resulting aqueous suspension and extracted by using ether to give 8.41 gr oil. The oil , was Chromatographed on a column of silica gel and some different fractions were gathered. Initial fraction (1-11) which were nonpolar compounds were seprated by GC/MS. Mass spectrum were prepared and much compounds were recognized.