High-Throughput Genetic Identification of Larval Fishes: A Fast, Cheap, Non-Tissue Destructive Methodology

Species identification from ithyoplanton assemblages is often difficult, tedious, and time consuming, particularly when working with early larval stages.  In addition to the difficulties associated with meristic or morphometric identification, in early larval stages there is little tissue available for DNA analysis when intact specimen preservation is desired.  Here we describe a rapid non-destructive alternative to isolate DNA from an individual fish larva, based on the suspension of epithelial cells through vortex forces, and the release of DNA in a heated alkaline solution. DNA from >6056 fish larvae isolated using this protocol has yielded a high PCR amplification success rate (>93%), suggesting its applicability to other taxonomic groups or sources when tissue amount is the limiting factor.  In addition genetic identification assays for Gulf of Mexico flying fish, tuna, and billfish species were developed using unlabeled probe High-resolution melting (UP-HRM) by targeting diagnostic regions of the mitochondrial genes 16S, ND4, and ND2 respecitively.  We found UP-HRMA to be an inexpensive, low error, genotyping assay that is easiliy amenable for high-throughput.