PCR-based techniques to determine diet of the Australian sea lion (Neophoca cinerea): a comparison with morphological analysis

Comprehensive dietary information for the endangered Australian sea lion (Neophoca cinerea) is currently limited by the deficiency and poor quality of identifiable prey remains recovered from regurgitate and faeces and the difficulty of observing feeding in the wild. In this study, we investigated DNA-based prey detection methods using conventional (end-point) and quantitative real-time PCR (qPCR) on faeces collected from two captive Australian sea lions fed experimental diets of whole teleost fish, squid and shark tissue. PCR prey detection methods using the mitochondrial cytochrome oxidase subunit I (COI) and 16S genes combined with clone sequencing were compared with prey identified using traditional hard part analysis. The molecular results indicated that prey DNA was degraded. However, prey amplification was successful by targeting short (71 bp) DNA fragments. Both conventional PCR and qPCR techniques significantly increased prey detection compared with analysis of hard parts. For both sea lions, the hard part analysis was constrained by sporadic and extremely low recovery of fish otoliths (<2%), and cephalopod beaks were not recovered from the 116 squid fed. Comparisons between PCR techniques indicated comparable prey detection frequencies for all species tested; however, the sensitivity and greater resolution of qPCR improved prey detection by ~25% in one sea lion fed the experimental squid and perch. The detection of squid DNA ≤ 6 day post-ingestion by qPCR further exhibits the ability and potential of this method to detect low concentrations of infrequent or pulse prey. This study highlights the use of DNA-based analysis to detect prey taxa in the absence of identifiable hard prey remains