NextGen molecular barcoding of larval grouper diet in an extensive green-water pond system

Molecular barcoding and next generation amplicon sequencing were used to investigate the diet of the larvae of two species: tiger (Epinephelus fuscoguttatus) and hybrid (E. lanceolatus × E. fuscoguttatus) grouper. Larvae were reared in open green-water ponds. These ponds had natural populations of wild zooplankton, and were also seeded with additional zooplankton. Zooplankton from water samples and larvae were collected daily for the first 12 days post hatch (dph) for hybrid grouper and the first 6 dph for tiger grouper. Next generation (NextGen) sequencing with degenerate cytochrome oxidase 1 oligonucleotide primers was performed on all samples. Resulting sequences suggest that both grouper species predated heavily on calanoid copepods – especially at first feeding and while still drawing energy from endogenous supplies. In addition, tiger grouper consumed mussel trochophores as a first feed. Grouper larvae diversified their diets at 6 dph and hybrid grouper were found to be consuming an unidentified insect as their primary food source from this point. Diets further diversified at 10 dph and the first substantial representation of ‘traditional’ aquaculture live feed was seen at 12 dph with the appearance of Artemia. Rotifers (Brachionus spp.) were consumed at a low level by tiger grouper larvae and at a negligible level by hybrid grouper despite daily addition to the pond systems. This study confirms that within the first 12 dph calanoid copepods are the key prey zooplankton for grouper larvae, and demonstrates that NextGen sequencing is a powerful tool for diet assessment in aquaculture.