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.

Effect to age on the geographic variation in morphometric traits among populations of Boana cordobae (Anura: Hylidae) / Efecto de la edad en la variación geográfica de características morfométricas entre poblaciones de Boana cordobae (Anura: Hylidae)

Abstract The morphometric variation of body size is an important topic of the natural history of the species which has been received particular interest. In this study, we estimated differences in body size and age structure of six populations of Boana cordobae living at different altitudes, 808-2 310 m.a.s.l. in Córdoba and San Luis provinces (Argentina). We measured 15 morphometric variables and used skeletochronology to age determination of 79 individuals. Morphometric variables showed significant differences between sexes, being females larger than males, even when the effect of age was taken into account. We found a significant relationship between age and most of the morphometric variables. When removing the effects of age, we found significant inter-population differences in body size. Males from the high-elevation populations were larger than individuals from low-elevation populations. These results suggest that a difference in age structure between populations is a main factor for the geographic variation in body size of this species.(AU)

Resumen La variación morfométrica del tamaño corporal es un aspecto importante de la historia natural de las especies, que ha sido de particular interés. En este estudio se estimaron diferencias en el tamaño corporal y la estructura de edad de seis poblaciones de Boana cordobae que viven a diferentes altitudes, 808-2 310 m.a.s.l. en las provincias de Córdoba y San Luis (Argentina). La técnica de esqueletocronología se utilizó para la determinación de la edad, mediante la medición de 15 variables morfométricas en 79 individuos. Las variables morfométricas mostraron diferencias significativas entre sexos, siendo las hembras de mayor tamaño que los machos, incluso cuando el efecto de la edad se tuvo en cuenta; y se encontró una relación significativa entre la edad y la mayoría de las variables morfométricas. Cuando el efecto de la edad fue removido, se encontraron diferencias significativas en el tamaño del cuerpo entre las poblaciones estudiadas. Machos de poblaciones de mayor altitud son más grandes que los de altitudes menores. Estos resultados sugieren que las diferencias en la estructura de edad entre las poblaciones es un factor de importancia a tener en cuenta para analizar la variación en el tamaño corporal de esta especie según el área geográfica.(AU)

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.

Biomarcadores hematológicos del sapo común Bufo (Rhinella) arenarum en ecosistemas alterados de la provincia de Córdoba / Hematologic biomarkers of the common toad Bufo arenarum in altered ecosystem of Córdoba province

El uso de biomarcadores hematológicos es importante para poder evaluar riesgos ambientales y la exposición a sustancias químicas potencialmente tóxicas y así poder desarrollar medidas que sirvan como señales de alarma temprana en ambientes contaminados. El objetivo de este trabajo fue determinar biomarcadores hematológicos en el sapo común Bufo (Rhinella) arenarum que proporcionen información para el diagnóstico de la salud de distintos ecosistemas de la provincia de Córdoba. Un total de 93 individuos adultos de Bufo (Rhinella) arenarum fueron capturados en cuatro sitios de la provincia de Córdoba y distintas variables ambientales del agua fueron medidas en cada sitio. Las muestras de sangre se obtuvieron mediante la punción de la vena angularis. Luego fueron teñidas con la tinción diferencial May Grünwald-Giemsa y fueron observadas mediante microscopio Zeiss Primo Star iLED para realizar el conteo de glóbulos blancos. Las variables ambientales del agua mostraron diferencias estadísticamente significativas entre las poblaciones, se registraron valores menores en “Alpa Corral”, mientras que “Villa Dálcar” presentó los mayores valores para dichas variables. No se encontraron diferencias estadísticamente significativas entre localidades en el recuento de glóbulos blancos (cada 1000 eritrocitos); sin embargo, en “Villa Dálcar” se registraron los ejemplares con mayor cantidad de leucocitos en el conteo de glóbulos blancos (425,48 ± 481,34). Mientras que para la fórmula leucocitaria, los linfocitos fueron las células más abundantes para los ejemplares de todos los sitios; encontrándose diferencias estadísticamente significativas entre cada localidad y “Alpa Corral”. Los ejemplares de “Villa Dálcar” presentaron diferencias estadísticamente significativas en relación a los monocitos y basófilos, registrándose altos valores para ambas células en los ejemplares de este sitio; mientras que en los ejemplares de “Cultivo” las diferencias se encontraron en eosinófilos y la relación neutrófilos/linfocitos. Estos resultados revelaron que de las cuatro localidades analizadas, “Villa Dálcar” representa la más afectada por las actividades antrópicas. Estas variaciones registradas en los parámetros hematológicos podrían considerarse evidencia de la posible presencia de agentes contaminantes en el lago “Villa Dálcar”.

Hematologic biomarkers are important to assess the environmental and health risks of exposure to potentially toxic chemicals, and for developing measures that serve as early warning signals in polluted areas. The aim of this study was to determine hematologic biomarkers of common toad Bufo (Rhinella) arenarum that provide information for the diagnosis of the health of ecosystems of Córdoba province. A total of 93 adult individuals of Bufo (Rhinella) arenarum were collected from four sites of Córdoba Province and hydric environmental variables were measured on each site. Blood samples were obtained by angularis vein puncture, smears of fresh blood were stained with May Grünwald-Giemsa and observed by using a microscope Carl Zeiss Primo Star (Pack 5), for white blood cell count. Water environmental variables showed statistically significant differences among localities, with the lowest values in “Alpa Corral” and highest values in “Villa Dálcar”. There were no statistically significant differences among individuals of different sites for white blood cells count. However, individuals from "Villa Dálcar" showed the highest number of white cells (425.48 ± 481.34). In the leukocyte formula, lymphocytes were most abundant white blood cells in individuals from all study sites. The blood parameters of toads from all sites showed statistically significant differences with individuals from “Alpa Corral”. "Villa Dálcar" Individuals exhibited significant differences in relation to monocytes and basophils with the highest values for both cell types. “Cultivo” locality showed significant differences for eosinophils and for the relationship neutrophils/ lymphocytes. These results revealed that “Villa Dálcar” represent the site most affected by human activities. In particular, changes recorded in hematological parameters could be giving evidence of the possible presence of contaminants in Lake “Villa Dálcar”.

Biomarcadores hematológicos del sapo común Bufo (Rhinella) arenarum en ecosistemas alterados de la provincia de Córdoba / Hematologic biomarkers of the common toad Bufo arenarum in altered ecosystem of Córdoba province

El uso de biomarcadores hematológicos es importante para poder evaluar riesgos ambientales y la exposición a sustancias químicas potencialmente tóxicas y así poder desarrollar medidas que sirvan como señales de alarma temprana en ambientes contaminados. El objetivo de este trabajo fue determinar biomarcadores hematológicos en el sapo común Bufo (Rhinella) arenarum que proporcionen información para el diagnóstico de la salud de distintos ecosistemas de la provincia de Córdoba. Un total de 93 individuos adultos de Bufo (Rhinella) arenarum fueron capturados en cuatro sitios de la provincia de Córdoba y distintas variables ambientales del agua fueron medidas en cada sitio. Las muestras de sangre se obtuvieron mediante la punción de la vena angularis. Luego fueron teñidas con la tinción diferencial May Gr³nwald-Giemsa y fueron observadas mediante microscopio Zeiss Primo Star iLED para realizar el conteo de glóbulos blancos. Las variables ambientales del agua mostraron diferencias estadísticamente significativas entre las poblaciones, se registraron valores menores en “Alpa Corral”, mientras que “Villa Dálcar” presentó los mayores valores para dichas variables. No se encontraron diferencias estadísticamente significativas entre localidades en el recuento de glóbulos blancos (cada 1000 eritrocitos); sin embargo, en “Villa Dálcar” se registraron los ejemplares con mayor cantidad de leucocitos en el conteo de glóbulos blancos (425,48 ± 481,34). Mientras que para la fórmula leucocitaria, los linfocitos fueron las células más abundantes para los ejemplares de todos los sitios; encontrándose diferencias estadísticamente significativas entre cada localidad y “Alpa Corral”. Los ejemplares de “Villa Dálcar” presentaron diferencias estadísticamente significativas en relación a los monocitos y basófilos, registrándose altos valores para ambas células en los ejemplares de este sitio; mientras que en los ejemplares de “Cultivo” las diferencias se encontraron en eosinófilos y la relación neutrófilos/linfocitos. Estos resultados revelaron que de las cuatro localidades analizadas, “Villa Dálcar” representa la más afectada por las actividades antrópicas. Estas variaciones registradas en los parámetros hematológicos podrían considerarse evidencia de la posible presencia de agentes contaminantes en el lago “Villa Dálcar”.(AU)

Hematologic biomarkers are important to assess the environmental and health risks of exposure to potentially toxic chemicals, and for developing measures that serve as early warning signals in polluted areas. The aim of this study was to determine hematologic biomarkers of common toad Bufo (Rhinella) arenarum that provide information for the diagnosis of the health of ecosystems of Córdoba province. A total of 93 adult individuals of Bufo (Rhinella) arenarum were collected from four sites of Córdoba Province and hydric environmental variables were measured on each site. Blood samples were obtained by angularis vein puncture, smears of fresh blood were stained with May Gr³nwald-Giemsa and observed by using a microscope Carl Zeiss Primo Star (Pack 5), for white blood cell count. Water environmental variables showed statistically significant differences among localities, with the lowest values in “Alpa Corral” and highest values in “Villa Dálcar”. There were no statistically significant differences among individuals of different sites for white blood cells count. However, individuals from "Villa Dálcar" showed the highest number of white cells (425.48 ± 481.34). In the leukocyte formula, lymphocytes were most abundant white blood cells in individuals from all study sites. The blood parameters of toads from all sites showed statistically significant differences with individuals from “Alpa Corral”. "Villa Dálcar" Individuals exhibited significant differences in relation to monocytes and basophils with the highest values for both cell types. “Cultivo” locality showed significant differences for eosinophils and for the relationship neutrophils/ lymphocytes. These results revealed that “Villa Dálcar” represent the site most affected by human activities. In particular, changes recorded in hematological parameters could be giving evidence of the possible presence of contaminants in Lake “Villa Dálcar”.(AU)

Reproductive biology of Pleurodema guayapae (Anura: Leptodactylidae: Leiuperinae).

Pleurodema guayapae is a species that inhabits saline environments and semidesert zones from central Argentina. To date, the knowledge about the reproductive biology of this species is very poor, and our aim is to contribute to its knowledge with the description of some important reproductive aspects. For this, field work was undertaken in an area near to Patquia, La Rioja province. Sampling was undertaken during three summer periods (2006-2007; 2007-2008; 2008-2009) in Chamical-Patquia area, where we could find reproductively active populations. We observed and described breeding sites, type of clutch, process of foam nest construction, clutch and egg number and sizes, and hatching time and stage. Behaviour observations were performed from the time that males began to call until the pairs ended up the foam nests building, and layed the eggs. Additionally, one amplected pair was observed and filmed in the process of foam nest construction, and four amplectant pairs were collected and separatelly placed in plastic containers, for nests observations in the laboratory. Hatching time was based on three different foam nests of known age. We found that P. guayapae populations were acoustically active only after a rainfall. Its breeding sites were represented by ephemeral ponds of fresh water, product of rains. The males emitted their calls inside or outside these ponds. A detailed description of the foam nest construction process by both females and males was made. The clutches were in dome-shaped foam nest type of 6-9cm in diameter and 1-3cm in height, some of which were in communal nests. The nests had an average of 1 137 pigmented eggs. This species showed a short hatching time. Our results allow us to conclude that this species should be considered an extreme explosive breeder. Our results are discussed with others obtained for related species.

Reproductive biology of Pleurodema guayapae (Anura: Leptodactylidae: Leiuperinae) / Biología reproductiva de Pleurodema guayapae (Anura: Leptodactylidae: Leiuperinae)

Pleurodema guayapae is a species that inhabits saline environments and semidesert zones from central Argentina. To date, the knowledge about the reproductive biology of this species is very poor, and our aim is to contribute to its knowledge with the description of some important reproductive aspects. For this, field work was undertaken in an area near to Patquía, La Rioja province. Sampling was undertaken during three summer periods (2006-2007; 2007-2008; 2008-2009) in Chamical-Patquía area, where we could find reproductively active populations. We observed and described breeding sites, type of clutch, process of foam nest construction, clutch and egg number and sizes, and hatching time and stage. Behaviour observations were performed from the time that males began to call until the pairs ended up the foam nests building, and layed the eggs. Additionally, one amplected pair was observed and filmed in the process of foam nest construction, and four amplectant pairs were collected and separatelly placed in plastic containers, for nests observations in the laboratory. Hatching time was based on three different foam nests of known age. We found that P. guayapae populations were acoustically active only after a rainfall. Its breeding sites were represented by ephemeral ponds of fresh water, product of rains. The males emitted their calls inside or outside these ponds. A detailed description of the foam nest construction process by both females and males was made. The clutches were in dome-shaped foam nest type of 6-9cm in diameter and 1-3cm in height, some of which were in communal nests. The nests had an average of 1 137 pigmented eggs. This species showed a short hatching time. Our results allow us to conclude that this species should be considered an extreme explosive breeder. Our results are discussed with others obtained for related species.

Pleurodema guayapae es una especie que habita en zonas semidesérticas y salinas del área central de Argentina. Los conocimientos actuales sobre la biología reproductiva de esta especie son muy pobres, por lo que nuestro objetivo fue contribuir a su conocimiento con la descripción de algunos aspectos reproductivos importantes. Para ello se realizaron tres campañas de muestreo durante los veranos (2006-2007, 2007-2008 y 2008-2009) en el área de Chamical-Patquía, provincia de La Rioja, donde se encontraron poblaciones reproductivamente activas. Se observaron y describieron los sitios reproductivos, tipo de puesta, proceso de construcción del nido de espuma, tamaño de la puesta y de los huevos, número de huevos por puesta y tiempo y estado de eclosión de la larva. Las observaciones de comportamiento se realizaron desde el momento en que los machos comenzaron a emitir los cantos hasta que la pareja en amplexo terminó de construir el nido de espuma con los huevos en su interior. Además, se observó y filmó una pareja en amplexo, en el proceso de construcción del nido de espuma y se recolectaron cuatro parejas en amplexo y se colocaron separadamente en recipientes plásticos para que sus puestas pudieran ser observadas en el laboratorio. El tiempo de eclosión se determinó en tres nidos de espuma de edad conocida. Se encontró que las poblaciones de P. guayapae solo estuvieron acústicamente activas luego de una lluvia torrencial. Los sitios de reproducción fueron charcas efímeras de agua de lluvia. Los machos emitieron sus cantos de advertencia desde adentro o fuera de esas charcas. Se realizó una descripción detallada del proceso de construcción del nido de espuma por parte de la hembra y el macho de P. guayapae. El tipo de la puesta en esta especie es un nido de espuma flotante con forma de domo de 6 a 9cm de diámetro y de 1 a 3cm de alto, algunos de los cuales estaban unidos entre sí formando puestas comunales. Los nidos de espuma poseen en promedio 1 137 huevos pigmentados. Esta especie mostró un tiempo de eclosión corto. Nuestros resultados nos permiten concluir que P. guayapae debe ser considerada como reproductor explosivo extremo. Nuestros resultados son discutidos con los obtenidos en otras especies afines.