Bufo Arenarum Research Articles

Validation of Electrochemiluminiscence Immunoassay for Ovarian Steroid Determination in Rhinella arenarum.

In this work, we describe the validation of an electrochemiluminescence immunoassay (ECLIA) that allowed us for the first time to determine the levels of progesterone (P4 ) and testosterone (T) secreted by Rhinella arenarum follicles during the preovulatory (POP) and reproductive (RP) periods. We also verified the relation between P4 and T levels and oocyte maturation. Moreover, we demonstrated that the extraction protocol developed for the determinations of P4 and T by ECLIA proved to be efficient and reproducible since the efficacy of the extraction was above 95% in all assays conducted. The results indicate that in the validation process the variation coefficient (CV) between assays is compatible with the analytical procedures based on automated immunoassays (CV < 8%) and that the adaptation proposed for the samples allows the determination of T and P4 with the Cobas e-411 analyzer. Our results indicate that in basal conditions the levels of T released by R. arenarum follicles were higher than those of P4 during POP and RP. In these conditions, steroid secretion failed to induce germinal vesicle break down (GVBD) in the follicles. Under gonadotropin stimulation, steroidogenesis showed a remarkable increase in both periods, especially during POP. This increase was correlated with a high maturation percentage in the follicles incubated in vitro (GVBD = 72 ± 16%) during POP. During RP, human Chorionic Gonadotropin (hCG) induced 81.75 ± 9.1% GVBD. This study is the first report of the seasonal steroidogenic activity in the ovary of R. arenarum in situ using an ECLIA-modified protocol developed in our laboratory.

Combined endosulfan and cypermethrin-induced toxicity to embryo–larval development of Rhinella arenarum

ABSTRACTThe combined effects of two widely used pesticides, endosulfan and cypermethrin, on survival of embryo–larval development of the South American toad (Rhinella arenarum) were examined. The toxicity bioassays were performed according to the AMPHITOX test. Embryos and larvae were exposed to mixtures of these pesticides at equitoxic ratios from acute or chronic exposure to evaluate interaction effects. The results were analyzed using both Marking’s additive index and combination index (CI)–isobologram methods. Acute (96-h) and intermediate (168-h) toxicity of endosulfan–cypermethrin mixtures remained almost constant for larvae and embryos, but when exposure duration was increased, there was a significant elevation in toxicity, obtaining chronic (240-h) no-observed-effect concentrations (NOEC) values of 0.045 and 0.16 mg/L for embryos and larvae, respectively. These are environmentally relevant concentrations that reflect a realistic risk of this pesticide mixture to this native amphibian species. The toxicity increment with the exposure duration was coincident with the central nervous system development on embryos reaching the larval period, the main target organ of these pesticides. The interactions of the pesticide mixtures at acute and chronic exposure were antagonistic for embryo development (CI > 1), and additive (CI = 1) for larvae, while chronic exposure interactions were synergistic (CI < 1) for both developmental periods. Data indicated that endosulfan–cypermethrin mixtures resulted in different interaction types depending on duration and developmental stage exposed. As a general pattern and considering conditions of overall developmental period and chronic exposure, this pesticide mixture usually applied in Argentine crop fields is synergistic with respect to toxicity for this native amphibian species.

Overcrowding-mediated stress alters cell proliferation in key neuroendocrine areas during larval development inRhinella arenarum

Exposure to adverse environmental conditions can elicit a stress response, which results in an increase in endogenous corticosterone levels. In early life stages, it has been thoroughly demonstrated that amphibian larval growth and development is altered as a consequence of chronic stress by interfering with the metamorphic process, however, the underlying mechanisms involved have only been partially disentangled. We examined the effect of intraspecific competition on corticosterone levels during larval development of the toad Rhinella arenarum and its ultimate effects on cell proliferation in particular brain areas as well as the pituitary gland. While overcrowding altered the number of proliferating cells in the pituitary gland, hypothalamus, and third ventricle of the brain, no differences were observed in areas which are less associated with neuroendocrine processes, such as the first ventricle of the brain. Apoptosis was increased in hypothalamic regions but not in the pituitary. With regards to pituitary cell populations, thyrotrophs but not somatoatrophs and corticotrophs showed a decrease in the cell number in overcrowded larvae. Our study shows that alterations in growth and development, produced by stress, results from an imbalance in the neuroendocrine systems implicated in orchestrating the timing of metamorphosis.

Apoptosis, proliferation and presence of estradiol receptors in the testes and Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura).

The dynamic equilibrium between spermatogonial proliferation and testicular apoptosis determines the progression of spermatogenesis in amphibians. Estrogens and their receptors play a central role in regulating spermatogenesis in vertebrates, and in some species of anurans, estradiol (E2 ) is involved in the regulation of spermatogonial proliferation and apoptosis of germ cells. Bidder's organ (BO) is a structure characteristic of Bufonidae that has historically been compared to an undeveloped ovary. In adult Rhinella arenarum males, BO is one of the main sources of plasma E2 . The aim of this study was 1) to describe the seasonal variations in testicular apoptosis, spermatogonial proliferation, and cellular proliferation in BO; and 2) to analyze the presence and localization of estrogen receptor β (ERβ) in the testes and BO of R. arenarum. Testicular fragments and BOs from animals collected during the year were labeled with 5-bromo-2'-deoxyuridine (BrdU) and BrdU incorporation was determined using immunohistochemistry. Apoptosis in testicular sections was detected using the TUNEL method, and ERβ localization was assessed using immunohistochemistry in testes and BOs. The results indicate that spermatogonial proliferation is highest during the reproductive season and that cysts of spermatocytes and spermatids undergo apoptosis during the postreproductive season. Furthermore, the proliferation of follicular cells is highest during the reproductive and postreproductive seasons. ERβ was primarily detected by immunolocalization in Sertoli cells, follicular cells, and oocytes. Taken together, these results suggest that cysts that do not form spermatozoa are removed from testes by apoptosis and that estrogens regulate both spermatogenesis and oogenesis in adult males of R. arenarum.

Harmful Effects of the Dermal Intake of Commercial Formulations Containing Chlorpyrifos, 2,4-D, and Glyphosate on the Common Toad Rhinella arenarum (Anura: Bufonidae)

Amphibians have complex life cycles with aquatic and terrestrial life and uncovered skins; therefore, they are exposed to chemical contamination, where dermal exposure is a significant route for pesticide uptake in both habitats. In this study, measurements in blood samples such as levels of butyrylcholinesterase (BChE), carboxylesterase (CbE), glutathione S-transferases (GST), thiobarbituric acid reactive substances (TBARS), modified alkaline comet assay (ACA) for detection of oxidized bases (FPG and Endo III sites), as well as the ratio of heterophils and lymphocytes (H/L), were evaluated as non-destructive biomarkers to monitor dermal pesticide exposure in male toads of Rhinella arenarum. Toads were exposed to a solution containing a nominal concentration of commercial formulations of the insecticide chlorpyrifos (CPF, 10 mg/L), and herbicides 2,4-D and glyphosate (GLY) (20 mg/L, respectively). After 48 h of exposure, the levels of plasma B-sterases (BChE and CbE) were inhibited (55 and 43 %, respectively) in toads exposed to CPF. Also, the activity of GST was inducted for dermal exposure to 2,4-D, as well as the levels of TBARS due to CPF exposure. Besides this, CPF and 2,4-D exposure induced oxidative DNA damage, and the H/L ratio decreased for the both herbicide exposures. Our results showed that exposure via dermal uptake to CPF, 2,4-D, and GLY in the common toad R. arenarum induced neurotoxicity, oxidative stress, and immunological depression. Thus, some blood biomarkers employed in our study (B-esterases, GST, levels of TBARS, ACA, and H/L ratio) might be used as predictors in health and ecological risk assessment of amphibian populations exposed to OP insecticides and herbicides.

Abatement of toxicity of effluents containing Cr(VI) by heterogeneous photocatalysis. Toxicity assessment by AMPHITOX assay

Toxicity of a Cr(VI) solution before and after treatment by TiO2 heterogeneous photocatalysis (HP) was performed with AMPHITOX bioassay. Changes in toxicity on Rhinella arenarum larvae for 10-d were monitored after exposure to an untreated Cr(VI) solution and to the same solution after HP treatment. The HP treatment of a 41.60mgL−1 Cr(VI) solution reduced to 37.5% the concentration of the metal ion. A 10-fold reduction in toxicity at acute exposure (72h) and 150-fold reduction in toxicity after 240h was found. Further, the LOEC value increased from 0.001% for the untreated solution to 0.153% after HP treatment. Moreover, the safe concentration in untreated solution corresponded to 0.0001% sample, and it was 0.01% after the treatment, i.e., 100 times higher. A saving of water of about 100,000L per L of effluent would be possible through dilution to allow safer concentrations for discharge; the saving would reach the highest value (1,000,000L per L) at 240h. Sub-lethal effects were completely absent in larvae exposed to the treated solution. The AMPHITOX test allowed to detect chronic effects at low Cr concentrations, i.e. at environmentally relevant levels.

Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes.

Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

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.

Age structure, growth and longevity in the common toad, Rhinella arenarum, from Argentina

Age structure, growth and longevity was determined in the common toad, Rhinella arenarum, from a sub - urban pond located in the Pampa plains, central Argentina during two breeding seasons, in 2000 and 2008 by using skeletochronology, which relies on the analysis of the annual lines of arrested growth (LAGs) in bones. Both females and males were captured in 2008, while only males were recorded in 2000. Females were significantly larger than males. Mean population age was 2.4 ± 0.9 years in 2000. In 2008, the difference in age was not significant between the sexes (Males: 3.0 ± 0.7, n = 21; Females: 2.6 ± 0.9, n = 12), neither between males in 2000 and 2008. The longev - ity in males of 2000 was 6 LAGs and exceeded that of males (5 LAGs) and females (4 LAGs) in 2008. Von Bertalanffy curves showed that the growth coefficient in the males of 2000 (K = 2.97 ± 0.47) was almost double that of females (K = 1.21 ± 0.10) and males (K = 1.01 ± 0.14) of 2008. Males and females Rhinella arenarum show different morpho - logical and life history traits and the year of sampling can significantly influence the estimation of the studied param -

Living organisms influence on environmental conditions: pH modulation by amphibian embryos versus aluminum toxicity

The LC10, 50 and 90/24h of aluminum for Rhinella arenarum embryos at complete operculum stage were 0.55, 0.75 and 1mgAl3+/L respectively. Those values did not change significantly by expanding the exposure period till 168h. The aluminum toxicity was evaluated in different pH conditions by means of a citrate buffer resulting for instance, 1mgAl3+/L at pH 4, 4.1, 5 and 6 in 100%, 70%, 35% and 0% of lethality respectively. As an outstanding feature, the embryos changed the pH of the maintaining media both in the case of Al3+ or citrate buffer treatments toward neutral. 10 embryos in 40mL of AMPHITOX solution were able to increase the pH from 4.2 to 7.05, a fact related with a metabolic shift resulting in an increase in nitrogen loss as ammonia. Our study point out the natural selection of the most resistant amphibian embryos both for pH or aluminum as well as the capacity of living organisms (as a population) to alter their chemical environment toward optimal conditions for their survival. As these facts occur at early life stages, it expand the concept that living organisms at ontogenic stages are biomarker of environmental signatures of the evolutionary process (Herkovits, 2006) to a global Onto-Evo concept which imply also the feedback mechanisms from living organisms to shape environmental conditions in a way that benefits them.

Differential uptake of endosulfan in the South American toad under sublethal exposure.

Agroecosystems are usually polluted with a wide variety of contaminants with pesticides being very frequently detected. Endosulfan, an organochlorine pesticide, has been shown to cause both lethal and sublethal effects on aquatic organisms such as amphibians and especially on its early developmental stages. In this context, the aim of this study was to evaluate the uptake of environmentally relevant concentrations of endosulfan and its correlation with differential sensitivity in the early development stages of the common South American toad, Rhinella arenarum. Embryos and larvae were exposed to sublethal concentrations of endosulfan for several periods of exposures. According to the developmental stage at which they were exposed, the uptake rate was different. Bioconcentration factors (BCFs) for embryos significantly decreased with exposure time and concentration (p < 0.05) reaching a BCF of ≤1679 for embryos at 96 h of exposure to 0.001 mg endosulfan L(-1). BCFs for larvae significantly increased with exposure time (p < 0.05) obtaining a maximum of 40 at 504 h. In our previous study, we reported that embryos were less sensitive to the effects of endosulfan than larvae, which is in line with the main tendency of embryos to bioconcentrate endosulfan as observed in this study.

Health status of tadpoles and metamorphs of Rhinella arenarum (Anura, Bufonidae) that inhabit agroecosystems and its implications for land use

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.

Acute toxicity of arsenic and oxidative stress responses in the embryonic development of the common South American toad Rhinella arenarum.

Arsenic (As), a natural element of ecological relevance, is found in natural water sources throughout Argentina in concentrations between 0.01 mg/L and 15 mg/L. The autochthonous toad Rhinella arenarum was selected to study the acute toxicity of As and the biochemical responses elicited by the exposure to As in water during its embryonic development. The median lethal concentration (LC50) value averaged 24.3 mg/L As and remained constant along the embryonic development. However, As toxicity drastically decreased when embryos were exposed from heartbeat-stage on day 4 of development, suggesting the onset of detoxification mechanisms. Given the environmental concentrations of As in Argentina, there is a probability of exceeding lethal levels at 1% of sites. Arsenic at sublethal concentrations caused a significant decrease in the total antioxidant potential but generated an increase in endogenous glutathione (GSH) content and glutathione S-transferase (GST) activity. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with GSH for its excretion. The authors conclude that toad embryos are more sensitive to As during early developmental stages and that relatively high concentrations of this toxic element are required to elicit mortality, but oxidative stress may be an adverse effect at sublethal concentrations.

Evaluation and Validation of a Semiautomatic Image Analysis Method: Quantification of Nares Cell Proliferation of Rhinella arenarum (Anura) Detected with the Avidin-Biotin Complex/Peroxidase, DAB

The current report presents the development and application of a novel methodological approach for computer-based methods of processing and analysis of proliferative tissues labeled by ABC-peroxidase method using 3, 3′-diaminobenzidine (DAB) as chromogen. This semiautomatic method is proposed to replace the classical manual approach, widely accepted as gold standard. Our method is based on a visual analysis of the microscopy image features from which a computational model is built to generate synthetic images which are used to evaluate and validate the methods of image processing and analysis. The evaluation allows knowing whether the computational methods applied are affected by the change of the image characteristics. Validation allows determining the method’s reliability and analyzing the concordance between the proposed method and a gold standard one. Additional strongness of this new approach is that it may be a framework adaptable to other studies made on any kind of microscopy.

Biochemical biomarkers of sublethal effects in Rhinella arenarum late gastrula exposed to the organophosphate chlorpyrifos

We determined the biochemical and molecular effects of the organophosphate insecticide chlorpyrifos (CPF) in the late gastrula embryonic stage of the South American toad Rhinella arenarum continuously exposed from fertilization (24 h). Our objective was to evaluate these responses as potential biomarkers at low, sublethal levels of the toxicant. We first established the EC50 for embryo arrest in 21.3 mg/L, with a LOEC of 16 mg/L. At 4 mg/L CPF, some embryos were unable to complete the dorsal lip of the blastopore and the yolk plug became blur, probably because of abnormal cell migration. Acetylcholinesterase activity, the specific biomarker for organophosphates, was unaffected by any of the tested concentrations of CPF (2–14 mg/L). In turn, 2 mg/L CPF increased the reduced glutathione levels and inhibited glutathione-S-transferase activity, suggesting an oxidative stress and antioxidant response. Catalase was induced by CPF exposure at higher concentrations (8 and 14 mg/L). We also studied transcription factor c-Fos as a signaling event related to development in early embryogenesis. Analysis of nuclear c-Fos protein showed two bands, both enhanced in embryos exposed to 2 and 8 mg/L CPF. While nuclear Erk protein was practically unaffected, Mek protein levels were induced by the OP. Transcription factor c-Fos may be then linking oxidative stress with developmental alterations observed due to CPF exposure. These molecular and biochemical responses observed in R. arenarum gastrula at sublethal CPF exposures may replace non-responsive AChE as very early biomarkers in toad gastrula.

Assessment of polyaniline nanoparticles toxicity and teratogenicity in aquatic environment using Rhinella arenarum model

With the rapid growth of nanotechnology and the applications of nanoparticles, environmental exposure to these particles is increasing. However, their impact in human and environmental health is not well studied. Anurans, with life stage comprising embryos, tadpoles and adults, have an extremely permeable skin which makes them excellent indicators of environmental health. This study evaluated the acute toxicity effects of polyaniline nanoparticles (PANI-Np) in different dispersant on embryos and larvae of Rhinella arenarum. The results showed that LC50 of PANI-Np dispersed in polyvinylpyrrolidone (PVP) were 1500mg/L, while LC50 by PANI-Np dispersed in PVP+PNIPAM (polyN-isopropylacrilamide) showed a highest toxicity (1170mg/L). The embryo teratogenicity increased with increasing exposure concentration in both kinds of PANI-Np although in PANI-Np1, there is an increased teratogenic effect associated with the polymer stabilizer PVP.

Effect of estradiol on apoptosis, proliferation and steroidogenic enzymes in the testes of the toad Rhinella arenarum (Amphibia, Anura)

Estrogens inhibit androgen production and this negative action on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. Estrogens are also involved in the regulation of amphibian spermatogenesis by controlling testicular apoptosis and spermatogonial proliferation. The Bidder’s organ (BO) is a structure characteristic from the Bufonidae family and in adult males of Rhinella arenarum it is one of the main sources of plasma estradiol (E2). The purpose of this study is to analyze the effect of E2 on testicular steroidogenic enzymes, apoptosis and proliferation in the toad R. arenarum. For this purpose, testicular fragments were treated during 24h with or without 2 or 20nM of E2. After treatments, the activities of cytochrome P450 17α-hydroxylase-C17–20 lyase (CypP450c17) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/I) were measured by the transformation of radioactive substrates into products, and CypP450c17 expression was determined by Western blot analysis. Apoptosis in testicular sections was detected with a commercial fluorescent kit based on TUNEL method, and proliferation was evaluated by BrdU incorporation. Results indicate that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3β-HSD/I activity during the post reproductive season. Furthermore, although E2 has no effect on apoptosis during the pre and the post reproductive seasons, it stimulates testicular apoptosis during the reproductive season, mostly in spermatocytes. Finally, E2 has no effect on testicular proliferation all year long. Taken together, these results suggest that E2 is involved in the regulation of testicular steroidogenesis and spermatogenesis.

Toxicity of the insecticide chlorpyrifos to the South American toad Rhinella arenarum at larval developmental stage

Chlorpyrifos (CPF) is an insecticide widely used for pest control in the fruit-productive region of North Patagonia, Argentina, where it is found in superficial waters. The aim of this study was to establish the toxic effects of CPF in Rhinella arenarum toad larvae as a potentially exposed species. We determined the 96h-LC50 (1.46±0.27mg/L), the LOEC (0.81mg/L, LC10) and NOEC (0.43mg/L, LC1) for CPF lethality as endpoint. We also analyzed biochemical biomarkers in larvae exposed to sublethal CPF concentrations. The IC50 for cholinesterase was 0.113±0.026mg/L, one order of magnitude lower than the LC50. Carboxylesterase activity was inhibited, buffering OP toxicity on cholinesterase. Reduced glutathione increased after 24h as an antioxidant response, and decreased at 96h together with catalase activity, due to oxidative stress. These biochemical effects suggest that environmentally relevant CPF concentrations pose a threat to R. arenarum larvae progression.

Use of local visual cues for spatial orientation in terrestrial toads (Rhinella arenarum): The role of distance to a goal.

The use of environmental visual cues for navigation is an ability present in many groups of animals. The effect of spatial proximity between a visual cue and a goal on reorientation in an environment has been studied in several vertebrate groups, but never previously in amphibians. In this study, we tested the use of local visual cues (beacons) to orient in an open field in the terrestrial toad (Rhinella arenarum). Experiment 1 showed that toads could orient in space using 2 cues located near the rewarded container. Experiment 2 used only 1 cue placed at different distances to the goal and revealed that learning speed was affected by the proximity to the goal (the closer the cue was to the goal, the faster toads learned its location). Experiment 3 showed that the position of a cue results in a different predictive value. Toads preferred cues located closer to the goal more than those located farther away as a reference for orientation. Present results revealed, for the first time, that (a) toads can learn to orient in an open space using visual cues, and that (b) the effect of spatial proximity between a cue and a goal, a learning phenomenon previously observed in other groups of animals such as mammals, birds, fish, and invertebrates, also affects orientation in amphibians. Thus, our results suggest that toads are able to employ spatial strategies that closely parallel those described in other vertebrate groups, supporting an early evolutionary origin for these spatial orientation skills.

Trophic ecology of syntopic anuran larvae, Rhinella arenarum (Anura: Bufonidae) and Hypsiboas cordobae (Anura: Hylidae): its relation to the structure of periphyton

The grazing of amphibian tadpoles has a significant effect on structure and function of freshwater algal communities. The aim of this study was to evaluate the composition of the diet, the amplitude and degree of overlap of the trophic niche of tadpoles of Rhinella arenarum and Hypsiboas cordobae. We captured tadpoles using nets in pond. Food items were identified to genus level and assigned to morphological guilds. The diet of both species consisted of microalgae, with a predominance of diatoms, but representatives of Cyanobacteria, Chlorophyta, Euglenozoa, Dinozoa and Ciliophora, were also recorded. A greater abundance was observed in H. cordobae diet, with diversity and richness showed significant differences between species. The proportion of individuals of each guild was different in the two species. As regards the size of the taxa consumed, both species consumed a higher proportion of small organisms. The differences in the diet of these species could be explained by different behaviors for food acquisition in the water column, as H. cordobae tends to be nektonic and R. arenarum benthic. The analysis of diets suggest that the quantity of food available to the tadpole anurans is very low; this could be due to water turbidity, depth, substrate type and strong variations in conductivity in a short period of time.