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Studies on molluscan conservation genetics, phylogenetics and evolution provide important data to access the diversity, populations' structure and dispersal patterns of these organisms (). Current environmental changes associated to anthropogenic pressures may have several negative ecological impacts on molluscs (e.g.; ), involving the reduction in their habitat suitability, which is particularly damaging for terrestrial slugs requirements of persistence (e.g.; ). The literature on slugs is greatly biased towards those that are agricultural pests (), while there is little information about benign native species such as Geomalacus maculosus (). Therefore, it is mandatory to develop multidisciplinary studies that integrate genetic data, biological and behaviour features to understand the adaptive, evolutionary and population dynamics of these species.

The improvement of non-invasive DNA-based methodologies is essential to minimize the potential harmful effects of these approaches, particularly in studies of rare or endangered molluscs. Although most of methods to collect biological materials for DNA isolation in molluscs are intrusive (e.g.;; ), some protocols have been proposed based on non-invasive sources of DNA in terrestrial and marine snails and slugs (;;; ). All these successful strategies were developed considering the DNA isolation from foot mucus and specific procedures for sample collection. Nevertheless, the sampling protocols are time-consuming and require the handling of the individuals.

The development of simple and efficient protocols to improve these characteristics can significantly enhance its field applicability. Thus, the main goal of this study is to demonstrate and report a non-invasive, rapid, efficient and cost-effective method based on DNA isolation from body swabs of terrestrial slugs. In this perspective, the hypothesis under study is that slugs' body surface epithelial cells provide an alternative good DNA source for genetic analysis. Body swabs of Geomalacus maculosus ( n = 12) and Arion spp.

( n = 12) were collected in Vila Real (Northern Portugal) in their natural habitats. This procedure was accomplished by carefully scraping a sterile cotton swab against each individuals' body 10 times (Fig. ). Swabs were directly placed in sterile 1.5 ml eppendorf tubes and stored at −20°C until DNA extraction. Samples from three different and independent specimens of G. Maculosus and Arion spp.

Were processed following the DNA extraction methods described below. Body swab method performed in the collection of biological samples from Geomalacus maculosus in its natural habitat. To demonstrate the flexibility of the proposed protocol four methods of DNA isolation were tested and optimized: salting out extraction, Quick gDNA MiniPrep Kit (Zymo Research), QIAamp DNA Micro Kit (Qiagen) and DNA IQ Reference Sample Kit for Maxwell 16 (Promega). In the conventional salting out protocol, samples were incubated at 55°C for 2 h on a thermal-shaker with 300 μl of lysis buffer [10 mM Tris (pH 7.5), 400 mM NaCl, 2 mM EDTA (pH 8.0)] (pH 7.3–7.5), 15 μl of 20% sodium dodecyl sulphate (SDS) and 20 μl of 20 mg/ml proteinase K. Download windows vista home premium.

The swabs were then removed with sterile tweezers and 50 μl of 6 M NaCl (saturated solution) was added to the extraction mixture, samples were mixed thoroughly by vortexing for 10 s, followed by centrifugation at 8000 g for 10 min to precipitate the residual cellular debris. The supernatant was transferred to a clean eppendorf tube and 500 μl of 100% ethanol was added to each sample, mixed thoroughly by vortexing for 10 s, and centrifuged at 8000 g for 5 min to pellet the DNA. The DNA pellets were washed with 250 μl of 70% ethanol, followed by centrifugation at 8000 g for 5 min.

The pellets were completely air dried and resuspended in 100 μl of sterile nuclease-free water. The Quick gDNA MiniPrep Kit was used according to manufacturer's instructions, with the following optimizations: the samples were digested at 56°C for 2 h in a solution containing 500 μl of genomic lysis buffer, 15 μl of 20% sodium dodecyl sulphate (SDS) and 20 μl of 20 mg/ml proteinase K; the washing step was performed twice with 500 μl of g-DNA wash buffer, followed by recentrifugation at 10 000 g for 1 min; and DNA was collected by two sequential elutions with 50 μl of elution buffer (incubation time was extended to 15 min at room temperature). The QIAamp DNA Micro Kit was used following the standard protocol recommended by the manufacturer, performing the initial incubation for 2 h and recovering DNA samples with the same final procedure of the previous protocol. In the automated DNA extraction carried out using the Maxwell 16 System (Promega) the protocol presented in the DNA IQ Reference Sample Kit was followed, modifying only the initial incubation time (extended to 2 h).