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.

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.

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.