Changes in call properties of Boana pulchella (Anura, Hylidae) in response to different noise conditions.

The increasing expansion of urban areas leads to the emergence of new noisy environments that can affect animal communication. Calls play a crucial role in the mating displays of anurans, and the negative impact of anthropogenic noise-induced auditory masking has been reported in several species. We investigated the acoustic variation in 96 males (n = 971 calls) of the treefrog Boana pulchella across acoustically undisturbed sites and different noise conditions, associated with urban areas (URBAN) and roads (ROAD), in Central Argentina. We analyzed the effect of anthropogenic noise conditions on 6 temporal (call duration [CD], intercall interval, first and second note duration, internote interval, and call rate) and 3 spectral (dominant frequency of first and second note, frequency difference between them) call properties. The effects of temperature and size on acoustical variables were controlled. We observed differences in all call attributes among the noise conditions, except for intercall interval. Males exposed to URBAN and ROAD exhibited significant changes in CD, dominant frequency of the second note, and frequency difference between them. URBAN males had longest internote interval, while ROAD individuals displayed increased first and second note duration and call rates. Interestingly, ROAD males exhibited immediate changes in call rate in direct response to passing heavy vehicles. Our study emphasizes the impact of anthropogenic noise on the acoustic characteristics of B. pulchella calls. Understanding how animals adapt to noisy environments is crucial in mitigating the adverse effects of urbanization on their communication systems. Future investigations should explore whether the observed call adjustments are effective in avoiding or mitigating the negative consequences of anthropogenic noise on reproductive success.

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.

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.

Morphological abnormalities in natural populations of the common South American toad Rhinella arenarum inhabiting fluoride-rich environments.

Morphological abnormalities in amphibians may be attributed to contaminants, ultraviolet radiation and trematode parasites, or a synergistic effect between them. In the present study, morphological abnormalities in Rhinella arenarum adults from natural and artificial fluoride-rich environments were identified and evaluated. Three sites were sampled in central Argentina: Los Vallecitos stream (LF-LV), Los Cerros Negros stream (MF-CN), and Decantation ponds (HF-DP), with low (0.33 mg/L), middle (2.03 mg/L) and high (14.0 mg/L) fluoride levels respectively; the latter site is associated with a fluoride mine. Abnormal individuals were photographed and then standard radiographs were taken. Abnormality frequencies and relative percentage of abnormal individuals were calculated for each site. In addition, skeletochronology was used to estimate toad's age. Five abnormality types were identified: syndactyly, ectrodactyly, polydactyly, microphthalmia and ectromelia. Percentages of abnormal individuals per site were: LF-LV = 4%, MF-CN = 21.2% and HF-DP = 6.4%. The MF-CN and HF-DP populations had morphological abnormality frequencies that exceeded the reference value (5%) reported in the literature. The average age did not differ between sites. The results of this study indicate that there is an association between frequency of morphological abnormalities and high fluoride levels.

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.

Common toad Rhinella arenarum (Hensel, 1867) and its importance in assessing environmental health: test of micronuclei and nuclear abnormalities in erythrocytes.

Anthropogenic activities may generate significant changes in the integrity of aquatic ecosystems, so long-term monitoring of populations that inhabit them is crucial. Counting micronucleated erythrocytes (MN) and erythrocytic nuclear abnormalities (ENA) in peripheral blood is a widely used method for detecting chromosomal damage due to chemical agents in the water. We analyzed MN and ENA frequency in blood obtained from the common toad Rhinella arenarum populations in sites with different degrees of environmental degradation. The results of this study indicate that there is an association between the frequency of micronuclei and nuclear abnormalities and the degree of environmental alteration recorded for the sites studied.

Determinación de la frecuencia de micronúcleos en eritrocitos de Bufo arenarum que habitan ambientes urbanizados / Determination of micronuclei frequency in erythrocytes of Bufo arenarum inhabiting urbanized environments

Los anfibios son constituyentes integrales de ecosistemas naturales y agrícolas, y debido a su sensibilidad pueden ser utilizados como bioindicadores. La frecuencia de micronúcleos se emplea como un biomarcador que evalúa la respuesta a agentes genotóxicos. El objetivo del trabajo fue determinar y comparar la frecuencia de micronúcleos en eritrocitos de Bufo arenarum que habitan ambientes antrópicos de la ciudad de Río Cuarto. Se escogieron tres sitios modificados: un lago urbano Lago Villa Dálcar (33º06´S-64º22´W) y dos sitios de cultivo denominados Cultivo1 (33º05'S-64º26'W) y Cultivo 2 (33º05'S-64º25'W). Como control se escogió un área ubicada en las sierras de Comechingones, en el Campo Experimental Las Guindas (32º35'S- 64º42'W), pertenecientes a la UNRC. En cada sitio se recolectaron ocho individuos utilizando trampas de caída y relevamientos por encuentros visuales. A cada individuo se le extrajo sangre de la vena angularis inmediatamente después de la captura. Se realizaron dos extendidos por cada individuo, se dejaron secar al aire por 24 hs, luego se fijaron con metanol por 20 minutos y se colorearon con Giemsa al 5% durante 10 minutos. Posteriormente los frotis fueron analizados con microscopio (1000X) y se tomaron fotografías con cámara digital. La frecuencia de micronúcleos fue contabilizada para 4000 células por individuo. Se realizaron comparaciones utilizando el test de Kruskal-Wallis. Las frecuencias de micronúcleos encontradas son: Las Guindas: 0,17 ± 0,23; Cultivo1: 0,78 ± 0,28; Cultivo 2: 0,61 ± 0,45 y Villa Dálcar: 0,76 ± 0,47. Existen diferencias significativas entre el control y los demás sitios (p< 0,01). Los resultados obtenidos demuestran la relevancia en la aplicación del test de micronúcleos in vivo en anfibios anuros para comparar distintos ambientes. Estos resultados indicarían la necesidad de nuevas investigaciones y de un monitoreo más prolongado en el tiempo para estos ambientes, incluyendo por ejemplo otros tipos de biomarcadores, así como otras posibles especies representativas de la región.

Amphibians are integral constituents of natural and agricultural ecosystems, and because of their sensitivity can be used as bioindicators. Micronuclei test is used as a biomarker for determining the response to genotoxic agents. The purpose of this study was to determine and compare the frequency of micronuclei in peripheral blood erythrocytes of Bufo arenarum toads that inhabiting anthropic environments for the city of Rio Cuarto. Organisms used in the MN test were collected from three sites modified: an urban lakeVilla Dalcar (33º06´S-64º22´W) and two cultivation sites called Crop 1 (33º05'S-64º26'W) and Crop 2 (33º05'S-64º25'W). As a control site, an area in the mountains of Comechingones, Las Guindas (32º35'S-64º42'W), belonging to the UNRC was sampled. At each site, eight individuals were collected using pitfall traps and by visual encounter surveys. To each individual is bled from the vein angularis immediately after capture. We performed two extended for each individual, air-dried for 24 hours, then fixed with methanol for 20 minutes and stained with 5% Giemsa for 10 minutes. Then the smears were analyzed with microscope (1000X) and took pictures with digital camera. For each individual the frequency of micronuclei for 4000 cells was recorded. Comparisons were made using the Kruskal-Wallis test. Micronuclei frequencies found are: Las Guindas: 0.17± 0.23; Crop 1: 0.78 ± 0.28, Crop 2: 0.61 ± 0.45 and Villa Dalcar: 0.76 ± 0.47. There are significant differences between the control and the other sites (p< 0.01). These findings show the relevance in the application of micronucleus test in vivo for anuran amphibians to compare different environments. These results suggest the need for further research and monitoring longer time for these environments, including such other biomarkers, as well as other possible species representative of the region.

Estudio comparativo de la frecuencia de micronúcleos y anormalidades nucleares en eritrocitos de tres especies ícticas / Comparative studies of the frequency of micronuclei and nuclear abnormalities in erythrocytes of three species fishes

Los ecosistemas acuáticos cercanos a las zonas urbanas sufren un deterioro constante ocasionado por diversos factores físico-químicos relacionados con la actividad antrópica, representando un riesgo para los organismos que los habitan, en especial los peces. Estos efectos pueden ser biomonitoreados mediante técnicas de amplia utilización, como el Test de Micronúcleos (Mn) y el de Anormalidades nucleares (AN), entre otros. El objetivo de este trabajo fue verifcar y comparar la frecuencia de micronúcleos y anormalidades nucleares en tres especies ícticas colectadas en un lago urbano, Cyprinus carpio, Astyanax eigenmanniorum y Cheirodon interruptus. Luego de la captura y anestesia de los peces, se extrajo de cada individuo una muestra de sangre y se registró el peso y la longitud total. Las muestras fueron fjadas y luego teñidas con Giemsa al 10%, posteriormente se las analizó con microscopio (1000X) y se les tomaron fotografías con cámara digital. Se calculó, además, el índice de condición corporal (K) para cada uno de los ejemplares capturados. Se aplicó un análisis de correlación de rangos de Spearman (rs) entre frecuencia de Mn y AN vs K para cada especie. La prueba de Kruskal-Wallis se aplicó para evaluar diferencias de Mn y AN entre las especies. Los resultados no mostraron una correlación estadísticamente signifcativa (P> 0,05) entre las frecuencias de Mn y AN vs K. Las frecuencias de Mn entre las diferentes especies no arrojaron diferencias signifcativas (P> 0,05), mientras que las AN sí (P< 0,05). Astyanax eigenmanniorum fue la especie que mayor sensibilidad presentó. Las diferencias preliminares de AN entre las especies utilizadas, convierten a A. eigenmanniorum en una especie "centinela" y posible biomonitor de agentes xenobióticos.

Aquatic ecosystems near urban areas suffer a steady deterioration caused by physical and chemical factors related to human activity, representing a danger to organisms that inhabit them, especially the fsh. These effects may be biomonitored by widely used techniques, such as the Micronucleus Test (Mn) and Nuclear Abnormalities (NA), among others. The aim of this study was to verify and compare the frequency of micronuclei and nuclear abnormalities in three fsh species collected in an urban lake, Cyprinus carpio, Astyanax eigenmanniorum and Cheirodon interruptus. After the capture and anesthesia of the fsh, weight and length were registered, and a blood sample was obtained from each individual. The samples were fxed and stained with 10% Giemsa, subsequently they were analyzed with the microscope (1000X) and photographed with a digital camera. The body con-dition index (K) was calculated for each fsh caught. A correlation analysis Spearman rank (rs) between frequency of Mn and NA vs. K was performed for each species. The Kruskal-Wallis test was applied to assess differences in Mn and NA between species. The results showed no statistically signifcant correlation (P> 0.05) between the frequencies of Mn and NA vs K. The frequencies of Mn between different species yielded no signifcant differences (P> 0.05), whereas NA (P <0.05) did. A. eigenmanniorum was the most sensitive species. NA preliminary differences between the species used, convert A. eigenmanniorum in a sort of "sentinel specie" and a possible biomonitor of xenobiotic agents.