Sample Collection and Extraction
Sharks will be caught and released once samples and measurements are taken. The sharks are fished for using a rig that contains a fifty pound weight and a line with a buoy on the top, labelled with “GHRI” (Guy Harvey Research Institute) and the license number that allows us to fish with such gear. Gear is set in groups of 10, with two at each of the following depths: 25ft, 40ft, 60ft, 80ft, and 100ft. Attached to the weight is a 100ft 900lb microfilament line with a circle hook and bait on the end so that the shark can swim and maintain water filtration while we are picking up the gear. When the line is picked up, it is pulled up by hand and the shark is secured with a tail loop and the line from the hook so that measurements and samples can be taken.
Samples will be collected from as many species as possible in hopes of achieving a wide range of habitats and diets among sharks and will be taken using dual sterile swabs (Henry Schein, Cat. 1228715) which will be transported in a cooler to the Microbiology and Genetics lab at the Halmos College of Natural Science and Oceanography. For each species that is considered, I intend to sample a minimum of five individuals of each species (with a goal of 10 individuals per species). For each individual, four samples will be taken: mouth, gills, skin, and cloaca. Additionally, swabs of bait, Atlantic Bonito ( Sarda sarda ), will be taken on each trip, and water samples for each individual if possible, which are filtered after each trip, so that any environmental microbes can be characterized and possibly discounted. This would lead to 5 samples per individual and 1 bait sample per trip. I intend to examine at least 3 species with 5 or 6 individuals of each species. This would yield 75-90 samples just from the organisms, with the bait sample total depending on the number of trips to collect the samples needed. Experimental design will follow the tenets of (Knight et al., 2012) for the minimum number of samples required, as well as including all possible “metadata” associated with each sample. Microbial community differences will be examined between sampling areas on individuals, as well as compared to other individuals in order to determine species or area specific microbes which could provide insight to be utilized when identifying shark species involved in bites. Additionally, mouthspecific microbes could be identified to determine better treatments for shark bite victims. The community differences will be determined by sequencing the samples taken and determining what OTUs are present in the microbial community. These OTUs could then be used to identify which bacteria are present at a particular sampling area on an individual. The composition of each sample can be compared to determine significant differences and similarities between individuals.
Spatial examination of the microbial communities of individuals will be accomplished by mapping where each individual was sampled and comparing the community data for individuals in a common area versus different geographical locations. If significant differences are found based on location, it could lead to further study to determine the cause of the spatial microbial differences. If differences in microbial communities are found between species by the data analysis in this study, a protocol could be proposed utilizing the MiSeq technology to further characterize microbial communities, potentially allowing for the accurate identification of the species involved in a shark attack.
DNA will be extracted with the Biostic and Powersiol DNA isolation kits (MoBio Laboratories Inc.). The extracted DNA will then be amplified using PCR while targeting the V4 region of the 16S rRNA gene. Amplicons will be sequenced with an Illumina MiSeq sequencing platform equipped with a V2 chemistry 500 cycle cartridge. Data will be processed to remove all reads which have a quality score that is less than 30, as well as any chimeric sequences. The sequences which remain will be clustered to determine Operational taxonomic units (OTUs).