RESUMEN

Degradation of the environment by agriculture affects the persistence and health of the amphibian populations. Characteristics related to reproduction of anuran can be used to evaluate the status of populations and as endpoints in environmental health assessment. In this in situ study the aspects related to the amplexus and ovipositions of the bioindicator species Rhinella arenarum that inhabits agroecosystems were analyzed. The hypothesis of this study is that perturbations of agroecosystems have a negative impact on the size of reproductive adults, on the size of ovipositions and eggs, and on the survival of eggs and embryos. Study area is located in the rural landscape of central Argentina. Four sampling sites were selected: C1, C2 and C3 are ponds on agroecosystems; and SM is a reference site that is not affected by agriculture or livestock. Abundance of amplexus pairs, oviposition and tadpoles per site was recorded. Individuals´ snout-vent length (SVL) in amplexus was measured. The fecundity was calculated like number of eggs per oviposition. The eggs' Gosner stage, the diameter eggs and the frequency of dead and abnormal eggs were recorded by oviposition. Killing-power between egg-embryo and egg-tadpole was calculated. The higher phosphate concentration was detected in all agroecosystems and nitrate was detected in C1 and C2. Conductivity, salinity and SDT were higher in C1 site Male SVL from the SM site was lower than the other sites while the largest SVL was of female from the C3 site. The higher frequencies of sprouted eggs and of dead eggs were recorded in the C2 site. Egg diameter was associated with SM and correlated negatively to SVL of the male and female. No correlation between female SVL and oviposition size was recorded. Killing-power in the passage from egg to tadpole classes was higher in the three agroecosystems. The hypothesis of this study was corroborated in part. Reproductive adults in agroecosystems did not have smaller body size. However, in the agroecosystem ponds, the eggs with smaller diameter were registered, the oviposition had higher frequency of abnormal eggs and the higher mortality was registered. This confirms the high sensitivity of the early stages to environmental disturbances and sustains their use as endpoints for the environmental health assessment.

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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.

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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.

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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”.

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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)

RESUMEN

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.

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The advertisement call plays an important role in the life history of anuran amphibians, mainly during the breeding season. Call features represent an important character to discriminate species, and sound emissions are very effective to assure or reinforce genetic incompatibility, especially in the case of sibling species. Since frogs are ectotherms, acoustic properties of their calls will vary with temperature. In this study, we described the advertisement call of C. cranwelli, quantifying the temperature effect on its components. The acoustic emissions were recorded during 2007 using a DAT record Sony TCD-100 with stereo microphone ECM-MS907 Sony and tape TDK DAT-RGX 60. As males emit their calls floating in temporary ponds, water temperatures were registered after recording the advertisement calls with a digital thermometer TES 1300+/-0.1 degreeC. Altogether, 54 calls from 18 males were analyzed. The temporal variables of each advertisement call were measured using oscillograms and sonograms and the analyses of dominant frequency were performed using a spectrogram. Multiple correlation analysis was used to identify the temperature-dependent acoustic variables and the temperature effect on these variables was quantified using linear regression models. The advertisement call of C. cranwelli consists of a single pulse group. Call duration, Pulse duration and Pulse interval decreased with the temperature, whereas the Pulse rate increased with temperature. The temperature-dependent variables were standardized at 25 degreeC according to the linear regression model obtained. The acoustic variables that were correlated with the temperature are the variables which emissions depend on laryngeal muscles and the temperature constraints the contractile properties of muscles. Our results indicated that temperature explains an important fraction of the variability in some acoustic variables (79% in the Pulse rate), and demonstrated the importance of considering the effect of temperature in acoustic components. The results suggest that acoustic variables show geographic variation to compare data with previous works.

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The advertisement call plays an important role in the life history of anuran amphibians, mainly during the breeding season. Call features represent an important character to discriminate species, and sound emissions are very effective to assure or reinforce genetic incompatibility, especially in the case of sibling species. Since frogs are ectotherms, acoustic properties of their calls will vary with temperature. In this study, we described the advertisement call of C. cranwelli, quantifying the temperature effect on its components. The acoustic emissions were recorded during 2007 using a DAT record Sony™ TCD-100 with stereo microphone ECM-MS907 Sony™ and tape TDK™ DAT-RGX 60. As males emit their calls floating in temporary ponds, water temperatures were registered after recording the advertisement calls with a digital thermometer TES 1 300±0.1°C. Altogether, 54 calls from 18 males were analyzed. The temporal variables of each advertisement call were measured using oscillograms and sonograms and the analyses of dominant frequency were performed using a spectrogram. Multiple correlation analysis was used to identify the temperature-dependent acoustic variables and the temperature effect on these variables was quantified using linear regression models. The advertisement call of C. cranwelli consists of a single pulse group. Call duration, Pulse duration and Pulse interval decreased with the temperature, whereas the Pulse rate increased with temperature. The temperature-dependent variables were standardized at 25°C according to the linear regression model obtained. The acoustic variables that were correlated with the temperature are the variables which emissions depend on laryngeal muscles and the temperature constraints the contractile properties of muscles. Our results indicated that temperature explains an important fraction of the variability in some acoustic variables (79% in the Pulse rate), and demonstrated the importance of considering the effect of temperature in acoustic components. The results suggest that acoustic variables show geographic variation to compare data with previous works.

El canto de advertencia en anuros es especie-específico, pero como los anfibios son organismos ectotermos, las variables acústicas pueden estar afectadas por la temperatura. Se describe el canto de advertencia de Ceratophrys cranwelli cuantificando el efecto de la temperatura sobre sus componentes. Los cantos se grabaron in situ y se registró la temperatura del sitio de canto. Un total de 54 cantos de 18 individuos fueron digitalizados y analizados. Las variables temporales de cada canto de advertencia fueron medidas a partir de los oscilogramas y sonogramas, mientras que el análisis de frecuencia se realizó a través del espectrograma. Mediante análisis de correlación múltiple se identificaron las variables acústicas temperatura-dependientes y el efecto de la temperatura fue cuantificado mediante los modelos de regresión lineal obtenidos. C. cranwelli posee un canto de advertencia simple pulsado. Las variables Duración del canto, Duración del pulso e Intervalo entre pulso decrecen con la temperatura, en tanto que la Tasa de pulsos está directamente correlacionada con la temperatura. Las variables temperatura-dependientes fueron estandarizadas a 25ºC según el modelo de regresión lineal obtenido para ser comparadas con datos publicados. Se demuestra la importante variabilidad que produce la temperatura en algunas propiedades del canto de C. cranwelli.

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Morphometric analyses are particularly important, and for many years they have supported evolutionary and ecological phenomena, and have been useful for the classification of new species, mainly to the lowest taxonomic levels. In order to assess the degree of sexual dimorphism, the intra-specific morphometric variation and the inter-specific morphological differences, we performed morphometric analyses of two morphologically cryptic species, Odontophrynus cordobae (diploid) and O. americanus (tetraploid). For this, we measured 15 morphometric variables on 211 individuals from 18 localities of Córdoba province. We found sexual dimorphism in six and three parameters in O. cordobae and O. americanus, respectively. Diploid and tetraploid males significantly differed in six morphometric variables. Discriminant Function Analysis (DFA) including all populations correctly classified a 76.37% of individuals within their respective species. DFA based on four groups (1- allopatric O. cordobae, 2-syntopic O. cordobae; 3- allopatric O. americanus; 4- syntopic O. americanus) accurately classified a 73.45% of individuals within their respective groups. Allopatric individuals of each species and, allopatric and syntopic individuals within each species were clearly segregated. Syntopic individuals of O. cordobae and O. americanus showed the lowest percentages of correct classification. Morphometric differences between sympatric diploids and tetraploids were not greater than those observed in allopatry. These findings deviate from the expected results under the hypothesis of character displacement, and they suggest that external morphological characters would not have a major influence on the recognition and choice of conspecific males by females.

El análisis morfométrico ha servido como apoyo para explicar fenómenos ecológicos y evolutivos y para la clasificación de nuevas especies. Nosotros realizamos el análisis morfométrico de dos especies morfológicamente crípticas con el objeto de evaluar el grado de diferenciación intra e interespecífica. Se midieron 15 variables sobre 211 individuos pertenecientes a 18 localidades del centro de Argentina. Se encontró dimorfismo sexual en seis variables morfométricas en Odontophrynus cordobae y en tres variables en O. americanus. Se encontraron diferencias significativas entre especies en seis variables. El análisis discriminante incluyendo los individuos de todas las poblaciones relevadas para O. cordobae y O. americanus, mostró una clasificación positiva de los individuos dentro de su respectiva especie del 76.37%. En el análisis discriminante realizado con base en cuatro grupos (O. cordobae alopátrico y sintópico y O. americanus alopátrico y sintópico) se obtuvo una reclasificación de los individuos dentro de cada grupo del 73.45%. Las diferencias morfométricas entre especies fueron mayores en sintopía. Los resultados se alejan de los resultados esperados bajo la hipótesis del desplazamiento de caracteres y podrían sugerir que los caracteres morfológicos externos no tendrían una influencia importante en el reconocimiento y elección de machos coespecíficos por parte de las hembras.

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Intra- and inter-specific morphometric variation between Odontophrynus populations (Anura: Cycloramphidae) of central Argentina. Morphometric analyses are particularly important, and for many years they have supported evolutionary and ecological phenomena, and have been useful for the classification of new species, mainly to the lowest taxonomic levels. In order to assess the degree of sexual dimorphism, the intra-specific morphometric variation and the inter-specific morphological differences, we performed morphometric analyses of two morphologically cryptic species, Odontophrynus cordobae (diploid) and O. americanus (tetraploid). For this, we measured 15 morphometric variables on 211 individuals from 18 localities of Córdoba province. We found sexual dimorphism in six and three parameters in O. cordobae and O. americanus, respectively. Diploid and tetraploid males significantly differed in six morphometric variables. Discriminant Function Analysis (DFA) including all populations correctly classified a 76.37% of individuals within their respective species. DFA based on four groups (1- allopatric O. cordobae, 2-syntopic O. cordobae; 3- allopatric O. americanus; 4- syntopic O. americanus) accurately classified a 73.45% of individuals within their respective groups. Allopatric individuals of each species and, allopatric and syntopic individuals within each species were clearly segregated. Syntopic individuals of O. cordobae and O. americanus showed the lowest percentages of correct classification. Morphometric differences between sympatric diploids and tetraploids were not greater than those observed in allopatry. These findings deviate from the expected results under the hypothesis of character displacement, and they suggest that external morphological characters would not have a major influence on the recognition and choice of conspecific males by females.

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