Shipboard identification of fish eggs and larvae by multiplex PCR, and description of fertilized eggs of blue marlin, shortbill spearfish, and wahoo

The study of the early life history of large, open-ocean pelagic fishes such as tunas and billfish, and the identification of spawning and nursery habitats, has been extremely difficult as these animals are intrinsically rare, highly migratory, and difficult to study in captivity. Traditional methods such as the assembling of a developmental series of life stages, or the culturing of unknown eggs and larvae to a point where they can be identified, has not been easy or fruitful for many pelagic species. The discovery of a putative spawning 'hot spot' off the Kona coast of Hawaii, coupled with the devel- opment of shipboard approaches to real time identification and adaptive sampling of eggs, may pro- vide new approaches and insights into the spawning ecology and reproductive biology of these highly valuable but poorly known species. Here we report the use of a shipboard PCR based assay to differentiate species of istiophorid billfish larvae and identify eggs of istiophorid and xiphiid billfish, coryphaenid dolphinfish, and wahoo. A species-specific multiplex PCR assay was designed to amplify a single, unique size fragment of the mitochondrial cytochrome b gene for all 6 species of Indo-Pacific billfish, both dolphinfish, and the monospecific wahoo. A boiling technique used to extract DNA from larval eye tissue or an individual egg, combined with a single-step PCR assay and agarose electrophoresis, allowed species identification within 3 h of sample acquisition. This nearly real-time identification method for morphologically indistinguishable eggs and larvae provides an opportunity to employ adaptive sampling methods to increase sampling efficiency and will help in determining the spatial and temporal dimensions of spawning and nursery habitats offshore. This study describes the occurrence of blue marlin, dolphinfish, shortbill spearfish, swordfish and wahoo off the Kona coast by molecular approaches, and it provides the first description of the eggs of blue marlin, shortbill spearfish and wahoo.