A Novel Ex Vivo Approach Based on Proteomics and Biomarkers to Evaluate the Effects of Chrysene, MEHP, and PBDE-47 on Loggerhead Sea Turtles (Caretta caretta)

The principal aim of the present study was to develop and apply novel ex vivo tests as an alternative to cell cultures able to evaluate the possible effects of emerging and legacy contaminants in Caretta caretta. To this end, we performed ex vivo experiments on non-invasively collected whole-blood and skin-biopsy slices treated with chrysene, MEHP, or PBDE-47. Blood samples were tested by oxidative stress (TAS), immune system (respiratory burst, lysozyme, and complement system), and genotoxicity (ENA assay) biomarkers, and genotoxic and immune system effects were observed. Skin slices were analyzed by applying a 2D-PAGE/MS proteomic approach, and specific contaminant signatures were delineated on the skin proteomic profile. These reflect biochemical effects induced by each treatment and allowed to identify glutathione S-transferase P, peptidyl-prolyl cis-trans isomerase A, mimecan, and protein S100-A6 as potential biomarkers of the health-threatening impact the texted toxicants have on C. caretta. Obtained results confirm the suitability of the ex vivo system and indicate the potential risk the loggerhead sea turtle is undergoing in the natural environment. In conclusion, this work proved the relevance that the applied ex vivo models may have in testing the toxicity of other compounds and mixtures and in biomarker discovery

Haplotype and biometric patterns in loggerhead turtles from the Adriatic foraging ground

The Adriatic Sea is an important foraging area for the loggerhead turtle Caretta caretta. Due to the increase of anthropogenic impacts, a growing number of individuals are captured as by-catch, found stranded or drifting at sea. Turtles mortality within the Adriatic Sea can affect Mediterranean populations, increasing the risk of losing genetic diversity. Biometric data on 93 loggerheads from three Adriatic areas (northwestern, northeastern and southern Adriatic) and patterns of sequence polymorphism of the mtDNA Control Region long fragment were used to investigate the sizes/age distribution of the collected individuals and to assess their geographic origins by using a Bayesian Mixed Stock Analysis (MSA). The biometric data revealed that collected turtles belonged to a wide range of sizes, although juveniles and subadults were the most abundant classes. Five haplotypes were detected. Two are endemic of Mediterranean rookeries and one is a rare haplotype found once in a Floridian rookery and in two Mediterranean nests. The other two haplotypes were found both in Atlantic and Mediterranean populations. The Mixed Stock Analysis revealed the presence of individuals from Greek and Turkish rookeries, this latter group was found in the southern Adriatic. Individuals from Libya and Cyprus rookeries were also found in the Adriatic stock. Current patterns, juvenile natal homing and imprinting of ontogenetic habitats could be potential drivers of the stock composition in the three Adriatic areas. Variations of turtle migration patterns, recruitment and mortality may drive differential contributions of rookeries to the foraging grounds. In this study, a substructuring within the Adriatic basin is hypothesized, consisting of differential distribution of stocks among three areas. However, these results might be biased by the uneven sample size among areas. Further studies with consistent sample sizes are recommended to quantify potential genetic differentiation within the Adriatic Sea. Nevertheless, present results recommend preservation action to be taken both at the nesting sites and in the foraging grounds were genetically isolated Caretta caretta populations, originating from different Mediterranean rookeries, mix together

First multicenter coprological survey on helminth parasite communities of free-living loggerhead sea turtles Caretta caretta (Linnaeus, 1758) from the Adriatic Sea and Northern Ionian Sea

The prevalence of endoparasite infections in 83 free-living specimens of Caretta caretta, classified as vulnerable species, from the Adriatic Sea and Northern Ionian Sea was investigated by coprological examination. Thirty-seven (44.6%) turtles were found to be infected with helminths. The helminth infections found were: Rhytidodes gelatinosus and Sulcascaris sulcata (18.1% each), Hapalotrema mistroides (13.2%), Cymatocarpus solearis (9.6%), Eniodotrema megachondrus (7.2%), Kathlania/Tonaudia sp. (3.6%), Neospirorchis sp., Octangium sagitta and Plesiochorus cymbiformis (1.2% each). There were no significant differences in the total prevalence of helminth infections between sexes, size classes, and seasonal periods. Conversely, the prevalence of helminth infections was significantly higher (P < 0.01) in accidentally caught turtles than in stranded turtles. Highly significant differences in prevalence of helminthiases were also seen among marine sampling areas. This report provides important baseline information about the helminth fauna of free-living C. caretta in the examined geographical region. This is also the first report of O. sagitta infection in C. caretta thus broadening the host range of the parasite

Growth rates and age at maturity of Mediterranean loggerhead sea turtles estimated from a single-population foraging ground

In sea turtles, somatic growth rates and Age at Sexual Maturity (ASM) are important parameters for investigating population dynamics. Moreover, ASM informs on the time lag needed to observe the effects of past environmental variables, threats and conservation measures acting at nesting beaches on future numbers of clutches or nesting females, that are the most monitored indices. Assessing ASM of mixed populations that share the same foraging areas is difficult, as in the case of the Mediterranean Sea that is frequented by turtles originating from both the Mediterranean and the Atlantic. Here we investigated growth rates of loggerhead sea turtles (Caretta caretta) foraging in the Adriatic Sea, known to be frequented only by individuals of Mediterranean origin. Capture-mark-recapture records of 79 turtles provided growth rates ranging from - 1.1 to 9.6 cm yr(-1) which were analyzed through Generalized Additive Models. When integrated with previous data on growth rates in the first years of life, results indicate a non-monotonic growth curve, likely due to an ontogenetic shift from oceanic to neritic areas. Using an integration equation, we estimated mean ASM to be 29.5 or 25.0 year based on mean nester size in Greece (83.4 cm curved carapace length, CCL) or across the Mediterranean (79.7 cm CCL), respectively. Growth rates and size-at-age are similar to the Atlantic population, but Mediterranean turtles mature earlier at a smaller size. The heterogenous growth rates and ASM reported so far in the Mediterranean are probably due, at least in part, to different foraging areas

First ecotoxicological assessment of Caretta caretta (Linnaeus, 1758) in the Mediterranean Sea using an integrated nondestructive protocol

The loggerhead sea turtle (Caretta caretta) is a long-lived carnivorous reptile included in the IUCN Red List. The IUCN assessment for this species underlines the lack of information regarding pollution and pathogens and indicates as a priority effort to investigate and reduce the impacts of these threats. In this contest, the aim of our study was to conduct the first ecotoxicological assessment of this species in the Mediterranean Sea using a nondestructive integrated methodology. We set up and applied a monitoring protocol which includes endpoints, such as CYP1A, LPO, ENA assay, B esterases, never investigated before in this species. Seventy-five loggerhead turtles were sampled in a nondestructive way in Italian Sea Turtle Rescue Centers or free-ranging along the Spanish coasts. Blood, skin and carapace samples were used to test biomarker responses (CYP1A, VTG, LPO, Comet and ENA assay, BChE, GGT) and contaminant (OCs, PAHs, Pb, Cd, Hg) levels. Elaboration of experimental results was carried out taking also into consideration different age classes of the specimens. Among the main results obtained we should underline the statistically significant correlation between carcinogenic PAHs in blood and DNA fragmentation as well as between Cd in carapace and GGT in plasma. We also evidenced as youngest animals showed significantly higher DNA fragmentations, BChE inhibition and increase of GGT. Older specimens showed the highest levels of erythrocyte nuclear abnormalities which may indicate a long term toxicological stress. This study contributed to expand the knowledge about the ecotoxicology of C. caretta in the Mediterranean. The nondestructive protocol could also be applied to other marine ecosystems and other sea turtle species

First ecotoxicological assessment of Caretta caretta (Linnaeus, 1758) in the Mediterranean Sea using an integrated nondestructive protocol

The loggerhead sea turtle (Caretta caretta) is a long-lived carnivorous reptile included in the IUCN Red List. The IUCN assessment for this species underlines the lack of information regarding pollution and pathogens and indicates as a priority effort to investigate and reduce the impacts of these threats. In this contest, the aim of our study was to conduct the first ecotoxicological assessment of this species in the Mediterranean Sea using a nondestructive integrated methodology. We set up and applied a monitoring protocol which includes endpoints, such as CYP1A, LPO, ENA assay, B esterases, never investigated before in this species. Seventy-five loggerhead turtles were sampled in a nondestructive way in Italian Sea Turtle Rescue Centers or free-ranging along the Spanish coasts. Blood, skin and carapace samples were used to test biomarker responses (CYP1A, VTG, LPO, Comet and ENA assay, BChE, GGT) and contaminant (OCs, PAHs, Pb, Cd, Hg) levels. Elaboration of experimental results was carried out taking also into consideration different age classes of the specimens. Among the main results obtained we should underline the statistically significant correlation between carcinogenic PAHs in blood and DNA fragmentation as well as between Cd in carapace and GGT in plasma. We also evidenced as youngest animals showed significantly higher DNA fragmentations, BChE inhibition and increase of GGT. Older specimens showed the highest levels of erythrocyte nuclear abnormalities which may indicate a long term toxicological stress. This study contributed to expand the knowledge about the ecotoxicology of C. caretta in the Mediterranean. The nondestructive protocol could also be applied to other marine ecosystems and other sea turtle species

Behavioural plasticity in the use of a neritic foraging area by loggerhead sea turtles: insights from 37 years of capture–mark–recapture in the Adriatic Sea (Mediterranean Sea)

Assessing sea turtle movements and connectivity among different areas is pivotal to understanding their biology and implementing efficient conservation actions. In the Adriatic Sea, one of the most important sea turtle foraging areas in the Mediterranean, a total of 311 capture-mark-recapture (CMR) records (mostly bycatch) from 294 loggerhead turtles (Caretta caretta) in the period 1984-2021 were analysed. A general fidelity pattern to Adriatic subareas was indicated by a significantly shorter CMR distance than the potential dispersal distance and by the significantly higher proportion of re-encounters in the same area of release than expected. No seasonal pattern was detected between subareas, and shorter re-encounter distances were observed in turtles released and re-encountered in the same season, suggesting different winter and summer residential areas. Results suggest that turtles frequenting the Adriatic can go anywhere in the Mediterranean basin and may exhibit a wandering behaviour regardless of their size. A substantial connectivity with nesting sites in Greece was observed, confirming with empirical evidence that this is the most important breeding area for turtles foraging in the Adriatic Sea. This study highlights the value of cooperation among different groups and shows a main behavioural pattern of fidelity to neritic foraging grounds