Pablo A. Siroski

Genetic, enzymatic and developmental alterations observed in Caiman latirostris exposed in ovo to pesticide formulations and mixtures in an experiment simulating environmental exposure

In South America, economic interests in last years have produced a constant increase in transgenic soybean cropping, with the corresponding rise in pesticide formulated products. The aim of this study was to determine the effects of pesticides formulations and mixtures on a South American caiman, Caiman latirostris, after in ovo exposure. We conducted a field-like experiment which simulates the environmental exposure that a caiman nest can receive in neighbouring croplands habitats. Experimental groups were Control group, Treatment 1: sprayed with a glyphosate herbicide formulation, and Treatment 2: sprayed with a pesticide mixture of glyphosate, endosulfan and cypermethrin formulations. Results demonstrated genotoxicity, enzymatic and metabolic alterations, as well as growth delay in caimans exposed in ovo to Treatments 1 and 2, showing a higher toxicity for the mixture. Integral evaluation through biomarkers of different biological meaning is highly informative as early indicators of contamination with pesticides and mixtures in this wildlife species.

Allometry of Reproduction in Wild Broad-Snouted Caimans (Caiman latirostris)

Abstract We studied 20 nesting female Caiman latirostris and their clutches in Santa Fe Province, Argentina. We regressed clutch and egg parameters and hatchling size with several measurements of female size (ln-transformed) to evaluate the allometry of reproduction in the wild. Larger females produced relatively smaller clutches. Larger females produced larger eggs and hatchlings. Egg width, not length, limits egg size relative to female body size. Future studies should address the relationship between female fecundity and hatchling fitness.

Embryological development of Caiman latirostris (Crocodylia: Alligatoridae).

A standard development embryological series is the primary basis to organize information of any embryological study and is also used to determine the age of eggs and embryos in field conditions. In this article, we calibrate developmental series of the broad‐snouted caiman, Caiman latirostris, against an established series for Alligator mississippiensis. Morphometric measures and extend of the opaque‐shell banding were also related to embryo age. In earlier stages, external morphological features alone can account for embryo age, but we suggest that morphometric measurements should be introduced later in the development. Unlike morphologic and morphometric attributes, the opaque patch was not a useful age predictor. As expected, a close correlation between embryonic development of C. latirostris and A. mississippiensis was observed. genesis 46:401–417, 2008.

The temperature-sensitive period (TSP) during incubation of broad-snouted caiman (Caiman latirostris) eggs

All crocodiles studied to date exhibit temperature-dependent sex determination. During the many weeks from egg laying to hatch there is a period of 10 to 15 d in the middle third of incubation (in the American alligator) during which the sex of the embryo is irreversibly fixed, referred to as the temperature-sensitive period or TSP. In this work we investigated the TSP in Caiman latirostris eggs incubated at female-inducing and male-inducing temperatures (29 ◦ C and 33 ◦ C respectively) by switching eggs from 29 ◦ Ct o 33 ◦ C and vice versa at timed interval throughout incubation. Compared to Alligator mississippiensis the duration of TSP was longer, and the onset of TSP was at an earlier stage of incubation.

Genotoxicity induced by Roundup® (Glyphosate) in tegu lizard (Salvator merianae) embryos

Environmental contaminants produce multiple adverse consequences at individual, population and ecosystem levels. High volumes of agrochemicals applied to great variety of crops, together with agricultural expansion, generate great concerns due to the impact for the environment and large risk implicated for wildlife. The lack of data on these threats is striking. The tegu lizard (Salvator merianae) is one of the species that live in environments under contaminant effects. Several characteristics allow proposing this species as a potential sentinel organism for the monitoring of pesticides in their habitat. The present study is the first report about genotoxicity in tegu lizard neonates after embryonic exposure to Roundup® (glyphosate 66.2%). The micronucleus test (MN), nuclear abnormalities (NAs) assay and comet assay (CA) were used as biomarkers of genotoxic effects induced in erythrocytes by topical exposure of the eggs to the glyphosate commercial formulation Roundup® (RU), in laboratory controlled conditions. A total of 96 eggs were distributed in six groups exposed to RU (50, 100, 200, 400, 800, 1600μg/egg), one positive control (PC; 200μg cyclophosphamide/egg) and one negative control (NC; distilled water). No teratogenic effects were observed in any of the exposed or control neonates. A significant increase in DNA damage was observed in all concentrations higher than 100μg/egg with respect to NC (p<0.05). However, no statistical differences were found in the frequencies of MN and NAs in any group exposed to RU compared to the NC. No statistically significant differences were found in the size of the lizards at birth or after six months post-exposure (p>0.05). Our results provide new information about the undesirable effects of the glyphosate-based herbicide formulations RU on this lizard species that inhabits areas permanently exposed to several pesticide formulations. We consider of utmost necessity a strict regulation of the agrochemical application conditions in those environments near to places where wild populations of terrestrial and aquatic species live, in order to minimize the adverse effects on ecosystems.

Effects of in vivo exposure to Roundup® on immune system of Caiman latirostris

Abstract The present study aimed to evaluate the effect of Roundup® (RU, glyphosate-based formulation) on some parameters of the immune system and growth of Caiman latirostris. Seventy-two caimans (20-day-old) from Proyecto Yacaré (Gob. Santa Fe/MUPCN) were used. Two groups were exposed for 2 months to different concentrations of RU (11 or 21 mg/L; taking into account the concentration recommended for its application in the field), while one group was maintained as control. The RU concentration was progressively decreased through the exposure period to simulate glyphosate degradation in water. Animals were measured and weighed at the beginning and end of the experiment, and blood samples taken after exposure to determine total and differential white blood cell (WBC) counts as well as total protein concentration (TPC), and for performing protein electrophoresis. The results showed that, compared against control hosts, there was a decrease in WBC counts, a higher percentage of heterophils, a higher TPC (with a low percentage of F2 protein fraction), and a negative effect on growth in the young caimans exposed to RU. These results demonstrate that in vivo exposure to RU induced alterations in the selected immune parameters, plasma proteins, and growth of caimans, thereby providing relevant information about the effects of this type of pesticide in this important species in the Argentinian wetlands.

Cranial sexual discrimination in hatchling broad-snouted caiman (Caiman latirostris)

Broad-snouted caiman (Caiman latirostris) hatchlings present a consistent sexual dimorphism in their cranium shape and size. Male hatchlings have smaller crania than females. Using multivariate statistical analyses it is possible to discriminate sex in broad- snouted caiman hatchlings by their cranial shape with a reasonable efficiency. The understanding of sexual dimorphism of crocod ilian hatchlings might be possibly improved by experimental approach considering, genetic and phenotypic variables such as incubation temperature and clutch of origin.

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.

Spontaneous genetic damage in the tegu lizard (Tupinambis merianae): The effect of age

Several studies indicate that certain factors such as age, sex or nutritional status among others, may affect the level of DNA damage, both induced and spontaneous, so it is very important to consider them for a more accurate interpretation of the findings. The aim of this study was to analyze the influence of age, sex, and nest of origin on spontaneous genetic damage of Tupinambis merianae determined by the comet assay (CA) and the micronucleus (MN) test, in order to improve reference data for future in vivo studies of xenobiotics exposure in this species. Sixty-five tegu lizards of three different ages: newborns (NB), juveniles (JUV) and adults (AD), both sexes and from different nests of origin were used. Blood samples were collected from the caudal vein of all animals and the MN test and CA were applied on peripheral blood erythrocytes to determine basal frequency of MN (BFMN) and basal damage index (BDI). The comparison between age groups showed statistically significant differences in the BFMN and BDI (p<0.05). NB animals showed significantly higher BDI values in relation to JUV and AD (p<0.016), but no statistically differences were found between the latter two. NB showed lower BFMN respect to other age groups, being statistically significant only when compared to AD (p<0.016). BFMN or BDI showed no statistically significant differences between sexes or nests of origin (p>0.05). A weak negative relationship was found only between BFMN and weight of NB tegu lizard (p=0.014; R(2)=0.245). Basal values of genetic damage obtained with both biomarkers in the tegu lizard evidenced that age is an intrinsic factor that should be taken into account to avoid misunderstanding of the results in future biomonitoring studies.

Detection and Characterization of Phospholipase A2 (PLA2) in Caiman latirostris and Caiman yacare Plasma

Reptiles have proven to have a versatile and efficient nonspecific immune system adapted to the environments in which they commonly live. Phospholipase A2 (PLA2) is important hydrolytic enzyme involved in the regulation of specific types of messengers, with significant roles in the innate immune response. A number of agents that exert effects on cellular receptors emit a series of signals leading to the increased activity of PLA2. Phospholipase A2 has been identified and characterized in temperature, plasma concentration, and kinetic dependence in two species of caiman. The results of these studies suggest that the high PLA2 activities observed in caiman plasma may be an important component of a well-developed innate immunity. Based on the knowledge of their properties, this powerful immunologic component should be evaluated as a possible application in the veterinary or even human therapeutic industry. Additionally, this is another reason to consider these animals excellent models for the study of immune phylogenetic mechanisms.