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Combining the use of predators as biological samplers together with measurements of the stable isotopic ratios (d13CBulk and d15NBulk) of their sclerotized beaks help investigate foraging ecology of poorly known oceanic cephalopods. However, high chitin content (an amino-sugar macromolecule) lowers beak d15NBulk values, thus precluding direct isotopic comparison with other tissues and organisms. To overcome the chitin effect, compound-specific isotopic analysis of amino acids (CSIA-AA) was performed on squid beaks. The method was applied on beaks and muscle, and the resulting d13CAA and d15NAA values compared between tissues. The usefulness of CSIA was tested by defining the habitat and trophic position (TPCSIA) of squids using their d13CAA and d15NAA values. Beak d13CAA values were reliably measured on 12 AA that included 5 essential and 7 non-essential AA, and d15NAA values were quantified on at least 7 AA that included 2 source and 4 trophic AA. Importantly, d13CAA and d15NAA varied little between muscle and lower and upper beaks, and TPCSIA estimates were identical regardless of the tissue considered. Tissue d13CAA values of both essential and non-essential AA reflected the latitudinal baseline d13C gradient that occurs in the Southern Indian Ocean, while beak d15NAA from source and trophic AA allowed the disentangling of the baseline effect from the trophic effect, and thus better calculations of squid TP estimates than from d15NBulk values. Beak d13CAA and d15NAA defined isotopic niches of colossal and giant squids, the 2 largest living invertebrates. In subantarctic waters, they segregate by having species-specific foraging habitats (using d13CGly or d15NPhe) and TPCSIA (using d15NGlx and d15NPhe). TPCSIA is higher in colossal (4.7) than giant (4.3) squids, and both values compare well with those of myctophid-eaters, suggesting very large squids prey primarily upon small zoo planktivorous fishes. As expected, CSIA-AA overcomes the chitin effect on beaks and it is a powerful tool to investigate trophic interactions of cephalopods. The method has a great potential with arthropods, because chitin is a main component of their exoskeleton but the deleterious effect of chitin is overlooked in isotopic studies focusing on crustaceans and insects.

Amino acid δ13C and δ15N from sclerotized beaks: a new tool to investigate the foraging ecology of cephalopods, including giant and colossal squids