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The contents of the digestive tracts of 21 loggerhead turtles (20 juveniles and 1 adult) collected along the French Mediterranean coast were extracted during necropsies. Hard and soft items were collected and identified by visual observation. A 15-mL sample of the liquid from the rinsed contents of each digestive tract was collected and dietary DNA (dDNA) was extracted and amplified with a 18S universal eukaryotic primers. The obtained reads were compared with taxonomic DNA reference database. Ten taxonomic groups (annelids, cnidarians, crustaceans, echinoderms, fish, insects, molluscs, plants, poriferans and tunicates) were identified with visual observation and eight (ctenophores, cnidarians, crustaceans, fish, molluscs, poriferans, tunicates and plants) with dDNA metabarcoding. Annelids, echinoderms and insects were detected only with visual observation, whereas ctenophores were detected only with dDNA. The two methods were complementary to cover the range of prey, with more soft-bodied organisms detected by dDNA and more hard-shelled organisms detected by visual observation. The increased use of the dDNA metabarcoding method will help compare sea turtle diets of the different stages such as juveniles and adults, or specimens living in different areas, despite the limitations and complexity associated with its use. [ABSTRACT FROM AUTHOR]

The following protocol is intended to respond to the requirements set by the European Union's Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: "There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region", where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: "There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region".