A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use.

Introduction to animal DNA barcoding protocols.

Procedures and protocols common to many DNA barcoding projects are summarized. Planning for any project should emphasize front-end procedures, especially the "genetic lockdown" of collected materials for downstream genetic procedures. Steps further into the DNA barcoding process chain, such as sequencing, data processing, and other back-end functions vary slightly, if at all, among projects and are presented elsewhere in the volume. Point-of-collection sample and tissue handling and data/metadata handling are stressed. Specific predictions of the future workflows and mechanics of DNA barcoding are difficult, so focus is on that which most or all future methods and technologies will surely share.

DNA barcoding fishes.

This chapter is an overview of the techniques for DNA barcoding of fishes from field collection to DNA sequence analysis. Recommendations for modifications of field protocols and best tissue sampling practices are made. A variety of DNA extraction protocols is provided, including high-throughput robot-assisted methods. A pair of well-tested forward and reverse primers for PCR amplification and sequencing are presented. These primers have been successfully used for DNA barcode on a wide array of marine fish taxa and also work well in most freshwater and cartilaginous fishes. Recipes and cycling protocols for both PCR amplification and sequencing and cleanup methods for the reaction products are provided. A method for the consistent production of high-quality DNA barcodes from DNA sequence data is given and stringent guidelines for judging the quality of raw sequence data are laid out.

Using DNA barcoding to assess Caribbean reef fish biodiversity: expanding taxonomic and geographic coverage.

This paper represents a DNA barcode data release for 3,400 specimens representing 521 species of fishes from 6 areas across the Caribbean and western central Atlantic regions (FAO Region 31). Merged with our prior published data, the combined efforts result in 3,964 specimens representing 572 species of marine fishes and constitute one of the most comprehensive DNA barcoding "coverages" for a region reported to date. The barcode data are providing new insights into Caribbean shorefish diversity, allowing for more and more accurate DNA-based identifications of larvae, juveniles, and unknown specimens. Examples are given correcting previous work that was erroneous due to database incompleteness.

An unprecedented aggregation of whale sharks, Rhincodon typus, in Mexican coastal waters of the Caribbean Sea.

Whale sharks, Rhincodon typus, are often perceived as solitary behemoths that live and feed in the open ocean. To the contrary, evidence is accumulating that they are gregarious and form seasonal aggregations in some coastal waters. One such aggregation occurs annually north of Cabo Catoche, off Isla Holbox on the Yucatán Peninsula of Mexico. Here we report a second, much denser aggregation of whale sharks (dubbed "the Afuera") that occurs east of the tip of the Yucatán Peninsula in the Caribbean Sea. The 2009 Afuera event comprised the largest aggregation of whale sharks ever reported, with up to 420 whale sharks observed in a single aerial survey, all gathered in an elliptical patch of ocean approximately 18 km(2). Plankton studies indicated that the sharks were feeding on dense homogenous patches of fish eggs, which DNA barcoding analysis identified as belonging to little tunny, Euthynnus alletteratus. This contrasts with the annual Cabo Catoche aggregation nearby, where prey consists mostly of copepods and sergestid shrimp. Increased sightings at the Afuera coincide with decreased sightings at Cabo Catoche, and both groups have the same sex ratio, implying that the same animals are likely involved in both aggregations; tagging data support this idea. With two whale shark aggregation areas, high coastal productivity and a previously-unknown scombrid spawning ground, the northeastern Yucatán marine region is a critical habitat that deserves more concerted conservation efforts.