Lipid metabolism in crocodilians: A field with promising applications in the field of ecotoxicology.

In the last years, lipid physiology has become an important research target for systems biology applied to the field of ecotoxicology. Lipids are not only essential components of biological membranes, but also participate in extra and intracellular signaling processes and as signal transducers and amplifiers of regulatory cascades. Particularly in sauropsids, lipids are the main source of energy for reproduction, growth, and embryonic development. In nature, organisms are exposed to different stressors, such as parasites, diseases and environmental contaminants, which interact with lipid signaling and metabolic pathways, disrupting lipid homeostasis. The system biology approach applied to ecotoxicological studies is crucial to evaluate metabolic regulation under environmental stress produced by xenobiotics. In this review, we cover information of molecular mechanisms that contribute to lipid metabolism homeostasis in sauropsids, specifically in crocodilian species. We focus on the role of lipid metabolism as a powerful source of energy and its importance during oocyte maturation, which has been increasingly recognized in many species, but information is still scarce in crocodiles. Finally, we highlight priorities for future research on the influence of environmental stressors on lipid metabolism, their potential effect on the reproductive system and thus on the offspring, and their implications on crocodilians conservation.

A comprehensive approach using multiple biomarkers to detect damage induced by pesticides in broad-snouted caiman (Caiman latirostris) under ex-situ conditions.

Caiman latirostris is one of the two species of the order Crocodylia that inhabit Argentina and is considered a species of vital ecological and economic importance in the north-east of Argentina. In this region, pesticides are the most common contaminants in natural environments and wild caiman populations are subject to this contamination constantly. The aim of this study was to evaluate the effects the main pesticides used in the region: glyphosate (GLY), cypermethrin (CYP) and chlorpyrifos (CPF) -based formulations, as well as the mixture of them, on C. latirostris juveniles under semi-controlled condition of exposure (ex-situ) during 75 days. One hundred yearling caimans (10-month-old) were equally distributed into five experimental groups (20 animals per group): a negative control (NC -tap water), GLY 2% (Roundup® Full II formulation -RU), CYP 0.12% (Atanor® formulation), CPF 0.8% (Lorsban® formulation), and a mixture of the three pesticides (Mx3: GLY 2% + CYP 0.12% + CPF 0.8%). We applied early warning biomarkers to detect damage induced by these chemicals in peripheral blood: activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), analysis of lipid peroxidation (LPO) by the thiobarbituric acid reactive substances (TBARS), DNA damage and specific base oxidation through the standard and modified comet assay (CA), chromosome damage by micronucleus (MN) test and other nuclear abnormalities (NAs), hematological and growth parameters. Results showed a statistically significant increase in MN and NAs frequency, DNA damage, with an important contribution of base oxidation for all exposed groups compared to the NC. Total white blood cells count (TWBCC), and growth parameters showed effects mainly at the Mx3. The principal component analysis (PCA) demonstrated more sensitivity for biomarkers associated to genetic damage, including base oxidation to DNA than LPO, antioxidant enzyme modulation, immunotoxicity or growth parameters, to detect pesticides effects, applied under conditions similar to that found in natural environments.

Micronuclei and other nuclear abnormalities on Caiman latirostris (Broad-snouted caiman) hatchlings after embryonic exposure to different pesticide formulations.

This study aimed to evaluate the embryotoxicity and genotoxicity of pesticide commercial formulations widely used in soybean crops through the Micronucleus (MN) test and other Nuclear Abnormalities (NAs) in erythrocytes of broad-snouted caiman (Caiman latirostris), exposed by topical application through the eggshell. Embryos were exposed (during all incubation: 70 days approximately) to sub-lethal concentrations of two glyphosate formulations PanzerGold® (PANZ) and Roundup® Full II (RU) (500, 750, 1000µg/egg); to the endosulfan (END) formulation Galgofan® and the cypermethrin (CYP) formulation Atanor® (1, 10, 100, and 1000µg/ egg). Blood samples were taken at the moment of hatching from the spinal vein for the application of the MN test and analysis of other NAs in erythrocytes, as markers of genotoxicity. Results indicated a significant increase in the frequency of MN for PANZ1000, END 10, CYP 1 and CYP 100 (p<0.05), and in the frequency of other NAs including Buds: END 100, 1000 and CYP 10 (p<0.05), eccentric nuclei: END 1, 10, 1000, CYP 10, 100, 1000 (p<0.01) and END 100 (p<0.05), notched nuclei: END 1, 10 (p<0.01) and END 1000, CYP 10, 100,1000 (p<0.05), and total nuclear abnormalities: END 1, 10, 100, 1000, CYP 10, 100 and 1000 (p<0.01), and the positive control (PC) (p<0.05), compared with the negative control. It was demonstrated a concentration dependent-effect in MN frequency only for PANZ (R2=0.98; p<0.01). Our study demonstrated that commercial formulations of pesticides induced genotoxic effects on C. latirostris, and NAs are a good indicator of genotoxicity in this specie.

In vitro evaluation of synergistic activity between ciprofloxacin and broad snouted caiman serum against Escherichia coli.

The in vitro synergistic activity between ciprofloxacin and serum of broad snouted caiman on Escherichia coli was studied. The estimated MIC value of ciprofloxacin was 0.0188 µg/ml, and two assays of kill curve during 5 hours were performed: the first one in a standard culture medium and the second one in the presence of caiman serum. Different concentrations of ciprofloxacin were tested. Ciprofloxacin showed higher values of bacterial elimination rate in the presence of caiman serum in all concentrations tested. The combined activity of sub-inhibitory concentrations of ciprofloxacin and the humoral immune factors present in caiman serum determined an increase in the bacterial elimination observed in this assay. We suggest that the antibacterial activity of complement and natural antibodies present in caiman serum, which can bind to both Gram-negative and Gram-positive bacteria and acting through the classical complement pathway, can inhibit bacterial growth of Escherichia coli by lysis.

DNA extraction from skins of wild (Hydrochoerus hydrochaeris and Pecari tajacu) and domestic (Sus scrofa domestica) species using a novel protocol.

Sometimes, commercial products obtained from wild animals are sold as if they were from domestic animals and vice versa. At this point of the productive chain, legal control of possible wildlife products is difficult. Common in the commerce of northern Argentina, skins of two wild species, the carpincho and the collared peccary, look very similar to each other and to those of the domestic pig; it is extremely difficult to differentiate them after they have been tanned. Because there was no an adequate methodology to discriminate between leather of these three species, we developed a new methodology of DNA extraction from skin and leather. This new method involves digesting a leather sample using proteinase K, followed by precipitation of proteins with 5 M NaCl, cleaning with absolute isopropanol and DNA precipitation with 70% ethanol. DNA is hydrated in Tris-EDTA buffer. This protocol provided good-quality DNA suitable for analysis with molecular markers. This new protocol has potential for use in identifying leather products of these species using molecular markers based on RAPDs.

Ultraviolet radiation-induced genotoxic effects in the broad-snouted caiman, Caiman latirostris.

Ultraviolet radiation (UVR) has many positive effects, but overexposure of organisms can generate alterations in DNA, either directly or indirectly, inducing mutagenic and cytotoxic lesions. The aim of this study was to evaluate the genotoxic effects of UVA-B/visible light in the broad-snouted caiman (Caiman latirostris), using the micronucleus (MN) test as a biomarker. Seventy two juvenile caimans, approximately 5 months old, were maintained for during 3 months under total darkness, or under 8 or 16h of daily exposure to artificial UV/visible light. MN test was applied before and after the experiment and the difference in MN frequencies was determined. Our results indicate significant increases in MN frequency with all treatments, compared to the basal (before experiment) values. Animals exposed to UV radiation showed a greater increase in MN frequency, compared to the animals exposed to total darkness (TD) treatment. These results provide information about the possible harmful effects generated by sub-chronic exposure to UVR in zoos, reptile hobbyist and breeding programs, as well as the deleterious consequences of increased UV environmental impact on wild species such as the broad-snouted caiman.