Hemoparasites Do Not Affect Life-History Traits and Cellular Immune Response in Treefrog Hosts Boana cordobae.

We provide the first evidence for hemoparasites in the endemic Cordoba treefrog Boana cordobae. We collected 37 adult frogs at 1200 m a.s.l. in the Comechingones Mountains in the Córdoba province (Argentina). Each individual was sexed, then snout-vent length and body mass were recorded, a toe was collected for skeletochronological age determination, and a slide with a blood smear was prepared for hemoparasite screening, before releasing the frogs in situ. A total of 81% (n = 30) of the frogs were infected by hemogregarines and trypanosomes with a high intensity of infections. Dactylosoma was found for the first time in Argentina. Hemoparasites had no significant effect on the leukocyte profile, which we assessed from the May-Grünwald-Giemsa-stained blood smears. The neutrophils/lymphocytes ratio, indicative of stress, was insignificantly higher (0.06) in parasitized frogs than in parasite-free individuals (0.04). Infected frogs were larger than the controls, but this effect vanished when correcting size data for age. Young frogs (first-breeders) dominated the age distribution of parasite-free individuals, suggesting that infection of frogs takes usually place after sexual maturation. Vectors transmitting hemoparasites to B. cordobae remain to be identified. We demonstrate that moderate to high intensities of hemoparasites do not significantly affect the cellular immune response of B. cordobae, or any of the life-history traits studied, nor did they show any external sign of disease.

Effect on growth and development of common toad (Rhinella arenarum) tadpoles in environment related to fluorite mine.

Fluorite mining activities have been scarcely evaluated so far and the potential effect of this activity on larval stages is poorly known. Thus, studies addressing the effect of contaminants present in water bodies on the health of amphibian larvae are crucial information for their conservation and constitute a warning sign of environmental modification. This study aims to evaluate the effects of natural and artificial surface waters associated with a fluorite mine on the early life stages of Rhinella arenarum. Using microcosms, we assessed four sites with different degrees of disturbance by the fluorine mine: Vallecitos stream (undisturbed); Cerros Negros upstream of mining camp (low disturbance); Cerros Negro downstream of mining camp (medium disturbance); Decantation Ponds (high disturbance). For 65 days we measured different endpoints at different periods of tadpole development. The highest mortality of tadpoles was observed in the Decantation Ponds. In the same way, larval body condition was lower at Decantation Ponds throughout the study, due mainly to the lower weight. A significant decrease in growth and development was observed in Cerros Negro downstream of the mining camp and Decantation Ponds. No significant differences in growth, development and mortality were observed for the other two sites. At the metamorphic climax we observe a lower body condition and a reduced recruitment of individuals in Decantation Ponds. This study allowed us to assess the effect on larvae of R. arenarum of fluorite mining wastewater compared with other near-natural sites. Given the potentially negative synergic effects of mixed water pollutants on tadpoles, this study suggests that chronic exposure to fluorite mining water may significantly impact the adult amphibian population structure, thus altering population viability. Therefore, we propose to monitor the correct functioning of the mine and especially of Decantation Ponds to avoid discharges into the natural streams.

Asymmetric Male Mating Success in Lek-Breeding Rhinella arenarum.

Mate choice is the attempt of an individual to gain higher reproductive fitness by preferring to mate with some individuals and not with others. We studied the role of mate choice in the mating system of the neotropical toad Rhinella arenarum by assessing male reproductive tactics for mate acquisition and the contribution of female choice for pair formation. In a shallow pond in central Argentina, we estimated male mating success and the corresponding reproductive tactics by focal observation. The variation of phenotypic and genotypic traits (size and shape, longevity, vocalization features, heterozygosity) was related to the observed mating success in 110 males. The phonotactic response of 21 reproductive females to conspecific advertisement call features was tested in arena experiments. Mating success was limited to 32 males, pair formation was size-assortative. The dominant reproductive tactics were advertising from call positions near suitable breeding sites and pre-mating fights with intruding males, whereas the interception of amplectant pairs and the displacement of mated males were never observed. Female phonotaxis was directed to conspecific choruses but complex and simple call structures were not distinguished. We conclude that the mating system is a lek combining pre-mating fights among males and female choice of slightly smaller males. Fights interfere with female choice, undermining size-assortative mating. This is a unique system in the R. marina species group, in which interception behavior dominates reproduction.

Body size, age and growth in males populations of Boana pulchella (Anura, Hylidae).

Age determination in amphibians is crucial to the investigation of life-history traits. In this context, we studied, for the first time, the life-history traits of Boana pulchella from a sample (63 adult males) of three populations (Las Acequias, Río Cuarto, Alejandro Roca) in central Argentina using the skeletochronological method. All adults of B. pulchella studied showed recognizable bone structures that allowed age determination. The average snout-vent length of sexually mature males was 43.39 mm. The maximum observed longevity was 5 years (5 Las Acequias, 4 Río Cuarto, 3 Alejandro Roca) and minimum age at sexual maturity was 2 years (same in the three populations), with mean of 2.96 years. Body size and age were positively correlated (except in Alejandro Roca populations). The growth patterns, estimated by the von Bertalanffy growth equation, do not show differences between populations, and the growth rate decrease after the attainment of sexual maturity. In conclusion, the determination of the individual age of the different populations of B. pulchella allowed us for the first time to establish the characteristics of the life history of the species, important for future comparisons whit others populations and future conservation biology studies.

Anuran tadpoles inhabiting a fluoride-rich stream: diets and morphological indicators.

We in situ assessed the influence of natural fluoride concentrations in lotic freshwater ecosystems on diet and morphology of Boana cordobae tadpoles. Two streams were sampled in Argentina: Los Vallecitos stream (LF-LV) and Los Cerros Negros stream (HF-CN) with low and high fluoride levels, respectively. We captured tadpoles of B. cordobae in each stream using nets. Body weight (BW), total length (TL) and body condition (BC) of tadpoles was registered. Food items were identified to genus level and assigned to functional traits. Tadpoles showed significant differences in TL between streams, with smaller individuals in HF-CN, while did not show differences in BW and BC. The diet of tadpoles consisted mostly of microalgae. In tadpoles from HF-CN stream the proportion of cyanobacteria was lower than tadpoles from LF-LV. In relation to functional traits, small algae, high profile and colonial algae were more abundant in HF-CN. Algae attached by pads showed a higher proportion in HF-CN diets and stalked algae were more abundant in LF-LV. The differences in TL and diet of tadpoles can be attributed to differences in algal community composition, with genera that are affected by high concentrations of natural fluoride; for example cyanobacteria. The low algal richness registered in HF-CN stream does not affect the physiological state of the tadpoles, possibly because of a higher algal density in HF-CN diets. However, in further studies it would be important to evaluate the population status of B. cordobae from the HF-CN, because a small body length of tadpoles could have consequences at the population level.

Evaluation in situ of genotoxic and cytotoxic response in the diploid/polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems.

Polyploidization has been documented across a wide range of vertebrates. Gene duplication could promote better adaptation to environmental changes and to chronic injury or stress. We investigated if genotoxic and cytotoxic responses to agricultural impact are affected by ploidy. We evaluate syntopic populations of the cryptic diploid/polyploid complex Odontophrynus cordobae/O. americanus breeding in an agroecosystem from Central Argentina. The blood of 72 adult anurans was analysed. We used erythrometry to distinguish Odontophrynus individuals with different ploidy levels. We calculated micronucleus frequencies (Mn) and erythrocytic nuclear abnormalities (ENAs) as genotoxic effects and enucleated, mitotic, pyknotic and immature erythrocytes as cytotoxic endpoints (CYT). Mn, ENAs and CYT frequencies were significantly different between diploid and polyploid organisms. The higher frequencies of Mn and CYT were recorded in polyploid organisms, and the higher frequency of ENAs was recorded in diploids. These results indicate that stress response, as indicated by most genotoxic and cytotoxic endpoints, was higher in polyploids respect to diploids. Polyploidy could provide greater genetic flexibility increasing buffering against exogenous DNA-damaging agents and thus confer an advantage over diploids under certain environmental conditions.

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.

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.

Erythrocyte nuclear size as a better diagnostic character than cell size in the identification of live cryptic polyploid species.

It is well documented in anurans the cryptic condition of many species complexes involving polyploids. In these complexes the character that clearly differentiates them is the number of chromosome complements. The blood cells of amphibians conserve their nucleus, and so the erythrocyte size is correlated with the DNA content. We analyzed two cryptic-polyploid complexes occurring in the center of Argentina: Odontophrynus cordobae (2n)/O. americanus (4n) and Pleurodema kriegi (4n)/P. cordobae (8n). Our aim was evaluate the efficiency in the utilization of nuclear area with respect to cellular area of the erythrocytes to define the limits values for the identification of cryptic-polyploid species. We studied 110 individuals of Pleurodema and 116 individuals of Odontoprhynus. For each individual, we measured the cellular and nuclear length (L) and width (A) of 40 erythrocytes (Area = L*A*π/4) and boundary values were calculated using distribution curves. In both complexes studied, the erythrometric parameters showed significant differences between related species. Moreover, in both complexes the nuclear area was more efficient for identifying the species (Pleurodema: 34.39 µm2 (probability = 99.96%) and Odontophrynus: 24.02 µm2 (99.075%)) than the cell area (Pleurodema: 273.08 µm2 (97.55%) y Odontophrynus: 197.69 µm2 (97.94%)). Greater efficiency found using nuclear area is novel and significant because most studies use only the cell area to differentiate polyploid complexes.