Nancy E. Salas

Reproductive Ecology of the Common South American Toad Rhinella arenarum (Anura: Bufonidae): Reproductive Effort, Clutch Size, Fecundity, and Mate Selection

Abstract Understanding the causes of population declines is difficult when the ecology of the organisms themselves is insufficiently known. The common South American toad, Rhinella arenarum, inhabits a wide variety of habitats. Their populations are abundant; however, life-history characteristics of the adult, including reproductive traits, are poorly known. We studied the reproductive ecology of R. arenarum for several populations in central Argentina during two breeding seasons (2007–08). Breeding activity was compared for the two seasons with respect to variation in habitat variables. Females deposited a mean of 23,226.67 eggs per clutch (N  =  10), and snout–vent length did not show any significant effect on the number of eggs. Spawning females lost 27% of their mass when spawning occurred, and the heavier females had greater reproductive output. We found a significantly positive relationship between amplexed male and female snout–vent lengths, indicating size-assortative mating. Females in good body and somatic condition produced larger clutches independent of snout–vent length. Because clutch hydration in ponds occurs, reproductive effort as a function of body loss mass of spawning females is a more appropriate estimate of reproductive output for R. arenarum. Trends in the number of egg strings over several breeding periods can provide information about changes in population size. Long-term studies designed to monitor populations are needed to determine the impacts and consequences of environmental changes.

Health status of tadpoles and metamorphs of Rhinella arenarum (Anura, Bufonidae) that inhabit agroecosystems and its implications for land use

Perturbations of water bodies near agricultural and livestock systems can affect embryonic and larval stages of anurans and negatively impact adult populations and structure of amphibian communities. This study is focused on early development of Rhinella arenarum, for which body growth, abnormalities in the oral disc and genetic damage on erythrocytes were analyzed to establish the impact of agroecosystems on local populations of amphibians. Tadpoles and metamorphs of R. arenarum were collected in three agroecosystems (namely, C1, C2, and C3) and in a site without agricultural and livestock activities (SM) from central Argentina. Egg masses of C1 were extracted for breeding tadpoles under laboratory conditions (Lab). Tadpoles were in small size and lighter in weight in C1 and C2. Metamorphs were shorter and lighter in weight in C1 and C3. In SM and Lab samples, no tadpoles with abnormal LTRF (labial tooth row formula) or without labial teeth were observed. In C1, the highest frequency of abnormal LTRF was recorded and was the only site in which tadpoles without labial teeth were found. In C1 and C2 the tadpoles had highest micronucleus frequencies and nuclear abnormalities. C1 can be considered as the site with the highest anthropogenic perturbation and with less healthy tadpoles. Livestock practices such as alternating cattle between parcel and keeping a buffer between crops and water bodies, would allow a better development of the first aquatic stages that are essential for the conservation of the anuran populations.

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.

Adverse effect of agroecosystem pond water on biological endpoints of common toad (Rhinella arenarum) tadpoles

Chemical prroducts used in farming and wastes from livestock can contaminate pond water in agroecosystems due to runoff. Amphibians using these ponds for breeding are probably exposed to pollutants, and serious consequences might be observed afterward at the population level. Assessment biological endpoints of anuran to water quality give a realistic estimate of the probability of occurrence of adverse effects and provide an early warning signal. In this study, the ecotoxicity of agroecosystem ponds from the south of Córdoba province, Argentina, was investigated. Ponds in four sites with different degrees of human disturbance were selected: three agroecosystems (A1, A2, A3) and a site without crops or livestock (SM). The effect of pond water quality on the biological endpoint of Rhinella arenarum tadpoles was examined using microcosms with pond water from sites. Biological endpoints assessed were as follows: mortality, growth, development, morphological abnormalities (in body shape, gut, and labial tooth row formula), behavior, and blood cell parameters (micronucleus and nuclear abnormalities). Results indicated that water from agroecosystems has adverse effect on early life stage of R. arenarum. High mortality and fewer metamorphs were recorded in the A1 and A3 treatments. Tadpoles and metamorphs from A1 and A2 treatments had lower body condition. Tadpoles from A1 and A3 showed the highest prevalence of morphological abnormalities. The lowest amount of tadpoles feeding and the highest percentage of tadpoles swimming on the surface were observed in treatments with agroecosystem pond water. The higher frequencies of micronuclei and nuclear abnormalities were recorded in tadpoles from A1, A2, and A3 treatments. We check the sensitivity of the biological endpoints of R. arenarum tadpoles like early warning indicators of water quality. We found that the poor water quality of agroecosystem ponds has impact on the health of the tadpoles, and this could affect the persistence of populations. We recommend implementation of management actions before the harmful effects of agroecosystem pond water on early life stage of anuran become evident in higher ecological levels.

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.

Redescription of the karyotype of five species of the family Bufonidae (Amphibia: Anura) from central area of Argentina

In this study karyotypic features of the five species of the family Bufonidae from the central area of Argentina are described. The species are Rhinella achalensis, Rhinella arenarum, Rhinella fernandezae, Rhinella schneideri and Melanophryniscus stelzneri. The metaphases were obtained from intestinal and testis cells, using conventional techniques. Twenty metaphasic figures per individual were analyzed and the total length of each chromosome and the length of the four arms were measured. The obtained measurements were processed using Excel 2000 to obtain the average length of the arms p and q, the arm ratio, the centromeric index, the relative chromosome length and the relative arm length. All species showed karyotype 2n = 22, and karyotype formula of 6: 5. Pairs one to six were large, with a relative chromosome length between 18.64–7.59%; pairs seven to eleven were small, with a relative chromosome length between 7.18–2.42%. In all species the chromosome morphology was metacentric or submetacentric. Karyotype and ideograms were made for all species, based on morphometric parameters of the chromosome complement. Finally, discriminant analysis was used to separate the five species analyzed, with a highly significant classification rate of 80% and P < 0.0001. These results agree, in general, with those presented by other authors, however, in M. stelzneri detailed karyological studied have not been made so far, thus this work represents a significant contribution to the karyotypic decryption features of this species and the Rhinellla species from central area of Argentina.

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.