Pablo R. Grenat

Variation in erythrocyte size between juveniles and adults of Odontophrynus americanus

Differences in erythrocyte size between tadpoles and adults have been found in numerous anuran species but there are no references about erythrocyte size of juveniles. We measured the size of red blood cells and nuclei of juvenile and adult individuals of Odontophrynus americanus. Erythrocyte and nuclear size of juvenile were significantly smaller than the corresponding sizes of adults. The erythrocytes of juvenile O. americanus were comparable in size to the erythrocytes of adult O. cordobae. We discuss about the possibility of correctly distinguishing juvenile O. americanus (tetraploid) from adult O. cordobae (cryptic diploid counterpart) specimens using erythrocytes. We also discuss possible causes of variation.

Intra-specific variation in advertisement call of Odontophrynus cordobae (Anura, Cycloramphidae): a multilevel and multifactor analysis

Advertisement call is a complex signal that can vary at different scales and the characterization of this variation is crucial to understand its adaptive function. In our study, we examined different factors that could influence intra and inter-population call variation in Odontophrynus cordobae. We analyzed calls of individuals from different breeding sites across the species distribution, including a syntopic site with their related species O. americanus. Dominant frequency, pulse rate, call duration and pulses/call were the acoustic parameters that showed the lowest intra-individual variation (CV ≤ 3%). The discriminant function analysis (DFA) assigned 92% of calls to the correct individual, indicating that the advertisement calls of O. cordobae are individually distinctive. Univariate and multivariate statistical analysis showed a significant contribution of pulse rate in differentiation of populations. Interpulse interval and pulse rate showed a moderate clinal variation, even when syntopic populations were excluded from the analysis. We found no association between acoustic and linear geographical or altitudinal distances and observed an evident divergence between the syntopic population and the other populations analyzed. This result is reinforced by DFA, which showed a differentiation among syntopic and allopatric populations of O. cordobae. We discuss our results from the hypothesis of a possible character displacement influencing call properties in this species in syntopy with their related species O. americanus. However, new syntopic areas and tests of female preferences would be needed to strengthen our results.

Assessment in situ of genotoxicity in tadpoles and adults of frog Hypsiboas cordobae (Barrio 1965) inhabiting aquatic ecosystems associated to fluorite mine.

Non-lethal biological techniques such as blood biomarkers have gained attention due to their value as early signals of anthropic effects of contamination representing significant tools to evaluate ecosystems health. We evaluate and characterize in situ genotoxicity of water samples collected from aquatic ecosystems around a fluorite mine using amphibian frogs Hypsiboas cordobae as bioindicator species complemented with 16 physicochemical parameters. Four stations associated with fluorite mine sampling were sampled: a stream running on granitic rock with natural high fluorite content; two streams both running on metamorphic rock with low fluorite content; and an artificial decantation pond containing sediments produced by fluorite flotation process with high variation in physicochemical parameters. We analyses the blood of tadpoles and adults of H. Cordobae, calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, immature and enucleated erythrocytes. Individuals were measured and weighed and body condition was calculated. The results of this study indicate that individuals of decantation pond are exposed to compounds or mixtures which are causing cell damage when compared to those that were collected of stream. Larval stage was more vulnerable than the adult phase and it could be related mainly to the higher exposure time to xenobiotics, which can penetrate easily by skin, mouth and gills; additionally this site offers a reduced availability of food than other sites. Therefore, chronic exposure to pollutants could derive in degenerative and neoplastic diseases in target organs. Moreover these individuals may experience reproductive and behavioral disturbances which could lead to population decline in the long term.

Evaluation in situ of genotoxicity and stress in South American common toad Rhinella arenarum in environments related to fluorite mine

Little attention has been paid to the impact of wastewater generated by mining activities on fluoride. In this study, we evaluated the hematology responses of common South American toad Rhinella arenarum inhabiting natural and artificial environments associated with a fluorite mine from central Argentina. We analyzed three sampling stations associated with the fluorite mine: (I) Los Cerros Negros stream (CN), which runs on granitic rock with a high fluorite content; (II) Los Vallecitos stream (LV), which runs on metamorphic rock with low fluorite content; and (III) artificial decantation ponds (DP) containing sediments produced by fluorite flotation process. We calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, and immature erythrocytes. In addition, we performed a differential leukocyte count and determined neutrophils/lymphocyte ratio as a stress response estimator. We found high micronucleus (MN) and erythrocyte nuclear abnormality (ENA) frequencies in DP and CN but low frequencies in LV. The neutrophil/lymphocyte ratio was different among sites, with a significant increase in individuals from DP. Values registered in DP could be caused by exposure to mixture of compounds registered in dams that hold wastewater, while high values registered in CN stream might be due to natural concentrations of fluoride. Our results suggest that blood is an effective and non-destructive sensitive indicator for monitoring genotoxic agents in freshwater ecosystems.

Geographic variation in the advertisement call of Hypsiboas cordobae (Anura, Hylidae)

Geographic variation in the advertisement call of anuran is commonly observed among conspecific populations. We analyzed the geographic variation of advertisement calls in six populations of the Argentinean treefrog species Hypsiboas cordobae throughout its geographic distribution. The advertisement calls of this species consisted of three, four or five tonal notes. Spectral call variables showed significant differences among populations, as in the first and second inter-note intervals. Discriminant function analysis demonstrated significant multivariate differences among populations. All spectral variables exhibited clinal variation, with frequencies increasing significantly from north to south. These same variables were negatively correlated with the altitudes of the six populations. Mantel tests showed no significant correlation between geographic distances and bioacoustic distances or between altitudinal distances and bioacoustic distances, attributable to that call variations are due to different levels of latitudinal and altitudinal populations studied, and not to the distances between pairs of localities. Altitudinal variation in spectral properties may be attributable to an inverse relation with body size, which varies clinally along the same geographical axis.

External changes in embryonic and larval development of Odontophrynus cordobae Martino et Sinsch, 2002 (Anura: Cycloramphidae)

We provide the first description of development for a species belonging to Odontophrynus genus by describing all external changes of embryonic and larval stages for Odontophrynus cordobae. External morphological changes through development were analyzed on specimens bred in captivity. Embryonic and larval development, from fertilization to metamorphosis, was completed in 62 days and 46 stages were defined. We split the staging series into ten developmental groups: fertilization (stages 1 and 2); segmentation (stages 3–9); gastrulation (stages 10–12); neurulation (stages 13–16); elongation (stages 17–19); external gill larva (stages 20–24); internal gill larva (stage 25); pre-metamorphosis (stages 26–41); pro-metamorphosis (stage 42); metamorphic climax (stages 43–46). Marked increases in total length were evidenced during elongation and during stage 25, when the tadpole begins to feed. Stage 25 was the longest one (8 days) and it was related to organs rearrangement, morphological progression and body mass increment typical of free life larval form. Similar studies on related species are needed to compare different developmental stages at different taxonomic levels.

Morphology and size of blood cells of Rhinella arenarum (Hensel, 1867) as environmental health assessment in disturbed aquatic ecosystem from central Argentina

Four populations of Rhinella arenarum from aquatic environments with different degrees of disturbance in central Argentina were compared to assess the ability of cytomorphology and cytomorphometry of blood cells as a hematological biomarker. A total of 93 specimens of R. arenarum (adults sexually mature) were captured during the spring. From the analysis of cell, no variations were found in terms of morphology, whereas in nuclear and cell areas and Price-Jones curves, we observed a smaller size in erythrocytes of individuals inhabiting the site most altered, “Villa Dalcar,” as well as for leukocytes, lymphocytes, neutrophils, and eosinophils for the same site. This could be caused by presence of different pollutants in the lake. Furthermore, this was confirmed by the high levels of environmental variables (conductivity, total dissolved solids, and salinity) show that Villa Dalcar is the site most affected by human activities.

Reproductive behaviour and development dynamics of Odontophrynus cordobae (Anura, Cycloramphidae)

We provide detailed information on breeding sites, release call, satellite behaviour, courtship, and embryonic and larval development of Odontophrynus cordobae. Field observations were carried out during two breeding seasons (2004–2005 and 2008–2009). Clutches were raised in captivity (artificial ponds) to analyse the development of embryos and tadpoles. Breeding sites were represented by streams and their backwaters, as well as temporary and permanent ponds of varying sizes. Operational sex ratio was male-biased varying between 0.154 and 0.25. Acoustic activity started about 8–10 pm and ended between 2 and 4 am the next day. Distance among calling males was approximately three metres. Male satellite behaviour was observed. Amplexus attempts among males were common. Release calls were structurally similar although shorter than advertisement calls. Courtship and formation of amplexus were documented.