Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success

Abstract

Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing.

Immune relevant molecules identified in the skin mucus of fish using -omics technologies

“…52 Host DNA in mucus could be used in selective breeding as a noninvasive method for PCR based studies to select disease resistant broodstock. In mantra rays skin mucus samples have been used successfully for genotyping by PCR, however the yields were not, with the present methods, enough to do next generation sequencing studies 53 .…”

The importance of considering genetic diversity in shark and ray conservation policies

“…For example, Lieber et al (2013) tested the potential of mucus swabs from a vulnerable species, the basking shark (Cetorhinus maximus, Cetorhinidae), at three molecular markers (cytochrome oxidase I (COI), CR mtDNA, and ITS2). Similarly, Kashiwagi et al (2015) evaluated the PCR success of mtDNA ND5 and nuclear DNA RAG1 for manta rays, as well as microsatellite loci from manta ray mucus collected underwater using toothbrushes. Such collection methods combined with new DNA technology, which require less representative sampling, show promise as a solution for more sustainable and less invasive genetics studies.…”

A new non-invasive in situ underwater DNA sampling method for estimating genetic diversity

“…Inserted map shows the locations of the five populations aware of one published attempt. Kashiwagi et al (2015) used a toothbrush to collect mucus from Manta rays (Manta birostris) in situ and then preserved the sample on dry land. Later, they successfully used the samples to amplify mtDNA, nuclear DNA and microsatellite loci.…”