Life after death? Exploring biochemical and molecular changes following organismal death in green turtles, Chelonia mydas (Linnaeus, 1758).

Green turtles, Chelonia mydas, have been included in biomonitoring efforts given its status as an endangered species. Many studies, however, rely on samples from stranded animals, raising the question of how death affects important biochemical and molecular biomarkers. The goal of this study was to investigate post mortem fluctuations in the antioxidant response and metabolism of carbohydrates in the liver of C. mydas. Liver samples were obtained from six green turtles which were submitted to rehabilitation and euthanized due to the impossibility of recovery. Samples were collected immediately after death (t = 0) and at various time intervals (1, 2, 3, 4, 5, 6, 12, 18 and 24 h post mortem), frozen in liquid nitrogen and stored at -80 °C. The activities of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) were analyzed, as were the levels of lipid peroxidation, glycogen concentration, RNA integrity (RNA IQ) and transcript levels of carbonic anhydrase and pyruvate carboxylase genes. Comparison between post mortem intervals showed a temporal stability for all the biomarkers evaluated, suggesting that changes in biochemical and molecular parameters following green turtle death are not immediate, and metabolism may remain somewhat unaltered up to 24 h after death. Such stability may be associated with the overall lower metabolism of turtles, especially under an oxygen deprivation scenario such as organismal death. Overall, this study supports the use of biomarkers in sea turtles sampled within a period of 24 h post mortem for biomonitoring purposes, though it is recommended that post mortem fluctuations of particular biomarkers be evaluated prior to their application, given that proteins may show varying degrees of susceptibility to proteolysis.

Cholinesterase inhibition and behavioral toxicity of carbofuran on Oreochromis niloticus early life stages.

Nile tilapia Oreochromis niloticus at 9 days post-hatch were exposed in semi-static experiments to the carbamate insecticide carbofuran, which is applied in agricultural systems in Brazil. Although the molecular mechanism of carbofuran toxicity is well known, a detailed understanding of the ecological mechanisms through which carbofuran effects can propagate towards higher levels of biological organization in fish is incomplete. Mortality rates were quantified for larvae exposed for 96 h to 8.3, 40.6, 69.9, 140, 297 and 397 µg/L carbofuran, and the LC(50) 96 h was 214.7 µg/L. In addition, the biochemical biomarker cholinesterase inhibition and behavioral biomarkers related to vision, swimming, prey capture and predator avoidance were quantified in individual larvae, as well as their growth in weight. The behavioral parameters were quantified by analysis of digitally recorded videos of individual larvae within appropriate experimental setups. The activity of the enzyme cholinesterase decreased after exposure to carbofuran with a lowest observed effects concentration (LOEC) of 69.9 µg/L. Visual acuity deficits were detected after carbofuran exposure with a LOEC of 40.6 µg/L. Swimming speed decreased with carbofuran exposure, with a LOEC of 397.6 µg/L. The number of attacks to prey (Daphnia magna nauplii) decreased in larvae exposed to carbofuran, with a LOEC of 397.6 µg/L. Growth in weight was significantly reduced in a dose dependent manner, and all carbofuran groups exhibited a statistically significant decrease in growth when compared to controls (p<0.05). The number of predator attacks necessary to capture larvae decreased after exposure to carbofuran, and the LOEC was 69.9 µg/L. These results show that exposure of sensitive early life stages of tilapia O. niloticus to sublethal concentrations of carbofuran can affect fundamental aspects of fish larval ecology that are relevant to recruitment of fish populations, and that can be better understood by the application of behavioral biomarkers.