Development of NGS molecular tools for Dietary Analysis

1. Development of NGS molecular tools for Dietary Analysis Tina Berry1, Dáithí Murray1, Megan Coghlan1, Sylvia Osterrieder2, Lars Bejder3, James Haile1, Nicole White1 and Michael Bunce1 1TrEnD Lab, Curtin University, 2Centre for Marine Science and Technology, Curtin University, 3Cetacean Research Unit, Murdoch University Introduction. The health of an ecosystem is essential for its conservation and for the life it supports. This is nowhere more important than where that environment supports an endangered species. However the ecosystems where marine species are found are difficult to monitor. Dietary analysis of the scat an apex predator can deliver a non-invasive insight in to the ecosystems that maintain them, as well as delivering information pertaining to the animal itself. Problem Statement. The Australian sea lion (Neophoca cinerea) is an endangered apex predator with a distribution across much of southern Australia. However, despite several morphological studies, the diet of the sea lion remains relatively uncharacterised. Procedures. This novel research applied next generation meta barcoding to study the species composition contained within Australian sea lion scat. The objectives of the study were to investigate and develop a variety of metabarcoding assays to characterise DNA isolated from scat and other marine substrates such as water and gastrointestinal samples. Extracts from 36 sea lion scat samples (from five sites across southwest WA) yielded sufficient DNA to be analysed using NGS metabarcoding. Results. Over a million sequences were successfully amplified and sequenced from the 36 samples using several existing and novel primer sets; for the fish mitochondrial markers alone 26 taxa were identified to a genus or species level. Other primers identified many more taxa including other fish, cephalopods, birds, and crustaceans. Vertebrates.Of the 36 samples collected 34 were confirmed as originating from Neophoca Cinerea. A pied cormorant (right) was detected in 3 samples using dedicated bird primers targeting the 12S rRNA gene. While at least one sea lion haul out beach being very close to a little penguin colony, no penguins were detected in the sea lion scat. At least 66 different fish were detected with primers targeting the 16S rRNA gene of the mitochondrion; 26 of these were identified to a genus or species level. Another primer set targeting the cytochrome oxidase I gene identified another 23 fish mainly to species level some which confirmed the 16S findings and others identifying species not identified using the 16S primers, such as Urolophuspaucimaculatus (the Sparsely-spotted Stingaree – left) Invertebrates. Several primer pairs were trialled and developed to target the 16S rRNA gene in both cephalopods and crustaceans. A dozen cephalopods were identified to genus or species level using 16S primers. The results showed that the sea lions tended to favour two species in particular; Sepia apama (the Giant Cuttlefish – below right) and Sepioteuthis australis (the Southern Calamari squid – below), as well as several species of Octopus The results from the 16S crustacean primers were limited, only two species were identified the commercially important Panuliruscygnus (the Western Rock Lobster – below right) and Thalamitadanae (a species of blue swimming crab – below left) The shorter COI DNA sequenced further confirmed the sea lions’ dietary selection of the Western Rock lobster as well as their preference for Sepia apama. 1 Future implications. It is anticipated that the techniques developed for sea lion diet could be applied in a number of marine dietary applications. Additionally the molecular tool kit acquired during this study has been used successfully in a small pilot study targeting the identification of local species from sea water samples. Conclusions. Despite the limitations encountered in the reference database, the data demonstrates that NGS is a powerful approach to dietary analysis. Given the somewhat small number of samples a large amount of information was gained both in regards to both the diet of the sea lion and diversity of their environments. The genus and species identified from the scat provided confirmation of some dietary items but also offered several surprises, both in what was commonly detected and what wasn’t. Illustrations: Sea lion sketch - http://drawingforkids.org/images/15319-animal-sketch-i-sea-lion-by-oryza.jpgb; Sparsely spotted stigaree, Mark Norman/Museum Victoria http://images.ala.org.au/image/proxyImageThumbnailLarge?imageId=74b7c278-11d8-4111-836a-171cc44c2cfe; Pied cormorant, Ian Montgomery, http://bie.ala.org.au/repo/1101/167/1678686/raw.jpg; Southern Calamari squid, Julian Finn/Museum Victoria http://images.ala.org.au/image/proxyImageThumbnailLarge?imageId=716192d5-332a-4b5e-8757-98a5ff5dcad4; The giant Cuttlefish, Mark Norman; Museum of Victoria http://images.ala.org.au/image/proxyImageThumbnailLarge?imageId=932b3534-5b40-4e8a-9f99-724bd92ea86b; Thalamitadanae,Anne Hoggett; Lizard Island Research Station http://images.ala.org.au/image/proxyImageThumbnailLarge?imageId=94c85235-3169-4d4d-8468-db2998454013; Western Rock Lobster, Reef life survey images http://images.ala.org.au/image/proxyImageThumbnailLarge?imageId=24189c96-94c3-4ed5-91e9-4ac2ba5e3413