Individual and mixture toxicity of chromium and copper in development, oxidative stress, lipid metabolism and apoptosis of Bufo gargarizans embryos.

In natural ecosystems, living organisms are always subjected to a mixture of multiple heavy metals exposure, yet it is more common to study the effect of individual, rather than combined exposure. This study assessed the impacts of single or combined exposure to Cr and Cu on embryonic development, oxidative stress, lipid metabolism and apoptosis in the early development of Bufo gargarizans embryos. The total length, development stage and malformations of embryos were measured, and the mRNA expression of genes related to oxidative stress, lipid metabolism and apoptosis at Gs 18 and Gs 22 were determined by RT-qPCR. The results showed that all treatments significantly reduced the total length of embryos, delayed the stage of embryonic development and increased the proportion of malformed embryos. The Cr-Cu mixture treatment showed the greatest suppression of embryonic development and induced the highest rate of embryo malformation, compared to individual Cr and Cu treatments. In addition, the expression levels of oxidative stress genes (HSP90, SOD and GPx) and fatty acid ß-oxidation-related genes (ACOXL, ECHS1 and SCP) showed an up-regulated trend in treatments compared to control groups. Conversely, the lipid synthesis-related mRNA gene expressions (KAR, TECR, ACSL3 and ACSL4) were down-regulated. Among them, the Cr-Cu mixture had the greatest impact on lipid metabolism gene expression. The treatments showed significant effects on the expression of apoptosis genes (Bcl-1 and Bax), with Bcl-1 mRNA expression increasing and Bax mRNA expression decreasing. These results indicated that exposure to individual Cr, Cu and a Cr-Cu mixture can lead to oxidative stress, disrupt lipid metabolism and promote apoptosis, and the Cr-Cu mixture could cause more serious negative effects on B. gargarizans embryos than Cr or Cu individually.

One of the most significant discoveries in Developmental Biology from an Indian laboratory - Iqbal A. Niazi and the role of retinoids in limb regeneration.

In this short commentary, we reflect upon the fascinating paper by I.A. Niazi entitled "Background to work on retinoids and amphibian limb regeneration: Studies on anuran tadpoles - a retrospect." originally published in J. Biosciences (1996), and herein reproduced with the kind permission of the author and the Journal of Biosciences. It is fitting that this landmark publication is included in this India-related Special Issue of the Int. J. Dev. Biol., because it sketches the background to what is arguably one of the two most significant discoveries in Developmental Biology to come from an Indian laboratory. Besides being of intrinsic interest, it spawned an entire area of research, one that deals with the role of retinoids in morphogenesis and development generally.

Response Patterns of Biomarkers as Tools to Identify Toxic Effects of Cadmium and Lead on Bufo gargarizans Embryo.

Molecular biomarkers play an increasing crucial role in evaluating and predicting toxicity of metals. Expressions patterns of genes related to oxidative stress, apoptosis, immune and inflammation response in the Bufo gargarizans embryo exhibited a development dependent manner. The genes related to oxidative stress (HSP, GPx and SOD) are the first response in the development of embryo, followed by the apoptosis (Bax, BCLAF1 and TRAIL) and inflammation and immune response (SOCS3, IL-27 and IL-17D), respectively. Then, we have verified the HSP, Bax and SOCS3 IL-27 (expressed highest in their respective processes) exhibited the most significant changes in Cd-Pb mixed group compared with control. In addition, we found exposure of Cd-Pb mixed metals causes greater adverse effects than Cd, Pb alone on development and morphology of embryo. Overall, our results provide a useful tool to use the sensitive molecular biomarkers as indicators of developmental toxicity in amphibian embryo.

Effects of nitrate on development and thyroid hormone signaling pathway during Bufo gargarizans embryogenesis.

Nitrate is known to disrupt the thyroid hormone, which is essential for the metamorphosis of amphibians. However, few studies are focused on the effects of nitrate on the maternal thyroid hormone in early amphibian embryos. We aimed to determine the impact of nitrate on maternal thyroid hormone signaling pathway in Bufo gargarizans embryos. B. gargarizans embryos were exposed to different concentrations of nitrate-nitrogen (NO3-N) for 7 days. High concentration of NO3-N (50, 100, and 200 mg/L) could induce embryonic malformation and influence the development of embryos. In addition, maternal T4 and components of the thyroid hormone (TH) signaling pathway were detected by ELISA and RNA-seq, respectively. The expression levels of mRNA related to thyroid hormone and oxidative stress were affected in the early developing embryos in all NO3-N treatment groups. However, the T4 levels and the spatial expression patterns of type II iodothyronine deiodinase (D2), type III iodothyronine deiodinase (D3), thyroid hormone receptor α (TRα), and thyroid hormone receptor ß (TRß) mRNA were not changed by nitrate. In conclusion, the results of our study highlight the crucial role of the maternal thyroid hormone signaling pathway in normal embryonic development, and the adverse effects of nitrate on the expression levels of mRNA related to thyroid hormone signaling pathway and oxidative stress in B. gargarizans embryos.

Effect-based environmental monitoring for thyroid disruption in Swedish amphibian tadpoles.

It is well-known that the metamorphosis process in amphibians is dependent on thyroid hormones. Laboratory studies have shown that several environmental contaminants can affect the function of thyroid hormones leading to alterations in the amphibian metamorphosis. The basic idea of the present study was to elucidate if the amphibian metamorphosis might be a useful tool as biomarker for effect-based environmental monitoring, examining wild tadpoles for potential thyroid hormone disruption. A laboratory test was performed to identify the responses from exposure to 6-propylthiouracil (PTU), which has a well-known mechanism on the thyroid system, on Swedish tadpoles from the Rana genus. This was followed by an environmental monitoring study where tadpoles of Rana arvalis, R. temporaria, and Bufo bufo were sampled from various sites in Sweden. Morphological data such as body weight, histopathological measurements of the thyroid glands, and environmental parameters were recorded. The results revealed that Rana tadpoles respond similar as other amphibians to PTU exposure, with interrupted development and increased size relative to the developmental stage. Data on some wild tadpoles showed similar features as the PTU exposed, such as high body weight, thus suggesting potential thyroid disrupting effects. However, histological evaluation of thyroid glands and pesticide analyses of the water revealed no clear evidence of chemical interactions. To a minor degree, the changes in body weight may be explained by natural circumstances such as pH, forest cover, and temperature. The present study cannot fully explain whether the high body weights recorded in some tadpoles have natural or chemical explanations. However, the study reveals that it is clearly achievable to catch tadpoles in suitable stages for the use in this type of biomonitoring and that the use of these biomarkers for assessment of thyroid disruption seems to be highly relevant.

Endocrine disruption, oxidative stress and lipometabolic disturbance of Bufo gargarizans embryos exposed to hexavalent chromium.

The aim of the current study was to determine the potential developmental and metabolic abnormalities caused by Cr (VI) exposure on Bufo gargarizans (B. gargarizans) embryos. B. gargarizans embryos were treated with different concentrations of Cr (VI) (13, 52, 104, 208, and 416 µg Cr6+ L-1) for 6 days. Morphological abnormalities, total length, weight and developmental stage were monitored. Malformations of embryos were also examined using scanning electron microscopy (SEM). In addition, the transcript levels of several genes associated with lipid metabolism, oxidative stress, and thyroid hormones signaling pathways were also determined. Our results showed a time-dependent inhibitory effect of Cr (VI) on the growth and development of B. gargarizans embryos. On day 4, total length, weight, and developmental stage were significantly lower at 416 µg Cr6+ L-1 relative to control embryos. On day 6, significant reductions in total length, weight, and developmental stage were observed at 104, 208, and 416 µg Cr6+ L-1. Malformed embryos were found in all Cr (VI) treatments, which were characterized by axial flexures, yolk sac edema and rupture, surface tissue hyperplasia, stunted growth, wavy fin and fin flexure. RT-qPCR results showed that exposure to Cr (VI) down-regulated TRß and Dio2 mRNA expression and up-regulated Dio3 mRNA level at 416 µg Cr6+ L-1. The transcript levels of SOD and GPx were upregulated at 52, 208, and 416 µg Cr6+ L-1, while the transcript level of HSP90 was downregulated at 52, 208, and 416 µg Cr6+ L-1. Also, mRNA expression of lipid synthesis-related genes (FAE and ACC) were significantly downregulated in embryos treated with 208 and 416 µg Cr6+ L-1, but mRNA expression of fatty acid ß-oxidation-related genes (ACOX, CPT, and SCP) was significantly upregulated at 416 µg Cr6+ L-1. Therefore, our results suggested that Cr (VI) could disrupt thyroid endocrine pathways and lipid synthesis, leading to the inhibition of growth and development in B. gargarizans embryos. Furthermore, the decreased ability of scavenging ROS induced by Cr (VI) might be responsible for the teratogenic effects of Cr (VI).

Toxic effects of NH4+-N on embryonic development of Bufo gargarizans and Rana chensinensis.

Although nitrogen fertilizer is commonly used worldwide, little information is currently available about NH4+-N toxicity on amphibians. This study determined the acute and chronic toxic effects of NH4+-N on two native Chinese amphibian species (Bufo gargarizans and Rana chensinensis), and compared the negative sensitivity of different embryos to NH4+-N. Static renewal aqueous exposures were performed using B. gargarizans and R. chensinensis embryos at Gosner stage 2 over 96 h. In terms of 96 h-LC50, B. gargarizans and R. chensinensis embryos had significantly different responses to NH4+-N, and the latter was more sensitive to NH4+-N than the former. In the chronic toxicity test, exposure to 10 mg L-1NH4+-N or higher significantly decreased the hatching rate of embryos in both species. Significant increases in the abnormality rate of embryos at 50 mg L-1NH4+-N or higher were observed and morphological abnormalities were characterized by axial flexures, yolk sac edema, and hyperplasia in both species. Additionally, the total length of embryos decreased in a dose-dependent manner after exposure to NH4+-N. The results indicate that NH4+-N exposure can increase abnormality and inhibit the hatching and development of embryos in B. gargarizans and R. chensinensis.

Post-metamorphic development of skin glands in a true toad: Parotoids versus dorsal skin.

Chemical defenses in amphibians are a common antipredatory and antimicrobial strategy related to the presence of dermal glands that synthesize and store toxic or unpalatable substances. Glands are either distributed throughout the skin or aggregated in multiglandular structures, being the parotoids the most ubiquitous macrogland in toads of Bufonidae. Even though dermal glands begin to develop during late-larval stages, many species, including Rhinella arenarum, have immature glands by the end of metamorphosis, and their post-metamorphic growth is unknown. Herein, we compared the post-metamorphic development of parotoids and dorsal glands by histological and allometric studies in a size series of R. arenarum. Histological and histochemical studies to detect proteins, acidic glycoconjugates, and catecholamines, showed that both, parotoids and dorsal glands, acquire characteristics of adults in individuals larger than 50 mm; that is, a moment in which the cryptic coloration disappears. Parotoid height increased allometrically as a function of body size, whereas the size of small dorsal glands decreased with body size. The number of glands in the dorsum was not linearly related to body size, appearing to be an individual characteristic. Only adult specimens had intraepithelial granular glands in the duct of the largest glands of the parotoids. Since toxic secretions accumulate in the central glands of parotoids, allometric growth of parotoids may translate into greater protection from predators in the largest animals. Conversely, large glands in the dorsum, which produce a proteinaceous secretion of unknown function, grow isometrically to body size. Some characteristics, like intraepithelial glands in the ducts and basophilic glands in the dorsum, are limited to adults.

Effects of a fungicide formulation on embryo-larval development, metamorphosis, and gonadogenesis of the South American toad Rhinella arenarum.

Sublethal toxicity of the formulated fungicide Maxim(®) XL on embryonic, larval and juvenile development of Rhinella arenarum was evaluated by means of standardized bioassays. Maxim(®) XL, one of the most used fungicides in Argentina, is based on a mixture of two active ingredients: Fludioxonil and Metalaxyl-M. Maxim(®) XL exposure induced severe sublethal effects on the embryos, expressed as general underdevelopment, axial flexures, microcephaly, cellular dissociation, abnormal pigmentation, underdeveloped gills, marked edema and wavy tail. As the embryo development advanced, alterations in behavior as spasmodic contractions, general weakness and inanition were observed. Maxim(®) XL did not affect neither the time required to complete metamorphosis nor sex proportions, but gonadal development and differentiation were impaired. Gross gonadal analysis revealed a significant proportion of exposed individuals with underdevelopment of one or both gonads. Histological analysis confirmed that 18% and 10% of the individuals exposed to 0.25 and 2mg/L Maxim(®) XL, respectively, exhibited undifferentiated gonads characterized by a reduced number (or absence) of germ cells. Taking into account the risk evaluation performed by means of Hazard Quotients, this fungicide could be a threat to R. arenarum populations under chronic exposure. This study represents the first evidence of toxic effects exerted by Maxim(®) XL on amphibians. Finally, our findings highlight the properties of this fungicide that might jeopardize non-target living species exposed to it in agricultural environments.

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.

Role of sediments in modifying the toxicity of two Roundup formulations to six species of larval anurans.

The role of sediment in modifying the toxicity of the original formulation of Roundup® and Roundup WeatherMAX® was examined in aqueous laboratory tests. Six species of anurans (Bufo fowleri, Hyla chrysoscelis, Rana catesbeiana, Rana clamitans, Rana sphenocephala, and Rana pipiens) were exposed at Gosner stage 25 to concentrations of the 2 herbicide formulations in 96-h, static, nonrenewal experiments in the presence and absence of sediment. All species tested had lower median lethal concentration values in water-only exposures of both formulations compared with exposures with sediment. Sediment significantly altered the potency slopes in all tests with the exceptions of H. chrysoscelis and R. clamitans when exposed to the original formulation of Roundup and H. chrysoscelis and R. sphenocephala when exposed to Roundup WeatherMAX. Thresholds were significantly different in all tests, including those in which potency slopes did not differ. Based on water-sediment exposures of the original formulation of Roundup, all 6 species tested had a margin of safety when compared with the predicted environmental concentration of the highest label application rate. Of the 6 species, 5 had a margin of safety when exposed to Roundup WeatherMAX. During incidental exposures in the field, sediments and organic matter present in aquatic systems provide significant sources of environmental ligands. If used according to label instructions, both herbicides should pose minimal risk to anuran amphibians in actual field applications. Environ Toxicol Chem 2014;33:2616-2620. © 2014 SETAC.

Differential toxicity and uptake of Diazinon on embryo-larval development of Rhinella arenarum.

Diazinon, an anti-cholinesterase organophosphate, is an extensively used pesticide. The main objective of this work was to assess the lethal and sublethal effects of Diazinon and its comparison with the uptake by embryos and larvae of the common South American toad Rhinella arenarum by means of standardized bioassays during acute (96 h), short-term chronic (168 h) and chronic (504 h) exposures. Toxicity resulted time- and stage-dependent, thus the lethal concentration 50 for 96 h, 168 h and 504 h were 27.2; 20.1 and 6.8 mg Diazinon L(-1) for embryos and 8, 6.7 and 1.9 mg Diazinon L(-1) for larvae. It is noteworthy the remarkable differences found in the concentration which caused lethality with those causing adverse effects on development such as malformations (teratogenic effects). Therefore, the teratogenic index from 144 h was greater than two; the main adverse effects were axial flexures, irregular borders, wavy tail, microcephaly, malformed mouth and adhesive structures, gut miscoiling, underdeveloped gills, cloacal edema, desquamation and severe hydropsy. Moreover, the characteristic sublethal effect of Diazinon on larvae was abnormal behavior related to neurotoxicity with a NOEC-168 h of 4.5 mg Diazinon L(-1). Diazinon contents in R. arenarum were time-dependent and significantly related to exposure concentration for both embryos and larvae. Diazinon contents were also stage-dependent, as it was up to 27 times higher for organisms exposed from blastula stage onwards than early larvae. These facts and the Hazard Quotients, a numerical expression of ecological risk, of 2.73, which is above USEPA's Level of Concern, showed the threat that Diazinon represents for R. arenarum populations.

Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla.

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.

Chronic toxicity of copper on embryo development in Chinese toad, Bufo gargarizans.

This study examined the effects of copper exposure on embryonic development of Chinese toad, Bufo gargarizans. Firstly, the LC(50) values from 24 to 96 h of exposure were 3.61×10(-6) M, by means of a 4 d toxicity test with B. gargarizans embryos. Secondly, Chinese toad embryos were exposed to 10(-9)-10(-6) M copper from mid gastrula stage to operculum completion stage. Measurements included mortality, tadpole weight, tadpole total length, growth retardation, duration of different embryo stages and malformation. Embryonic survival was not affected by copper. Relative to control tadpoles, significantly decreased weight and total length were found at 10(-9)-10(-6) M reduced percentage of the embryos in right operculum stage after 10 d exposure to copper and reduced percentage of embryos in operculum completion stage after 12 d exposure to copper were also observed. Moreover, the duration of embryonic development increased at neural, circulation and operculum development stage in copper-treated groups. For the scanning microscope and histological observation, the abnormalities were malformation of wavy dorsal fin, flexural tail, curvature body axis, yolk sac oedema and reduced pigmentation in the yolk sac. Histopathological changes in olfactory, retinal epithelium and skin were also observed. DNA strand breaks exposed to the copper were analyzed by DNA ladder. In conclusion, copper induced toxic effects on B. gargarizans embryos. The present study indicated chronic toxicity tests may provide more accurate way in formulating the "safe levels" of heavy metals to amphibian.

[Apoptosis and differentiation in presumptive neural retina and presumptive retinal pigmented epithelium during early eye development in toad, Bufo raddei Strauch].

Apoptosis and differentiation in presumptive neural retina (PNR) and presumptive retinal pigmented epithelium (PRPE) wert investigated during early retina development of toad, Bufo raddei Strauch. TUNEL staining was used to evaluate apoptotic cells and the immunohistochemistry was used to assess the expression levels ofglial fibrillary acidic protein (GFAP), RT97 and tyrosinase (Tyr) during early eye development respectively. The density of apoptotic cells and protein expression were quantitated with Image-Pro Plus 6.0. Apoptosis was found in both PNR and PRPE and the density of apoptotic profiles in PRPE was higher than that in PNR (most P<0.01) at the same stage during early eye development. The expression levels of GFAP and RT97 changed from low to high in PNR, but from high to low in PRPE, whereas the expression level of Tyr, was contrary to those of GFAP and RT97 in both PNR and PRPE. The point of intersection of these, increase and decrease respectively was found at 5-6 h after formation of optic vesicle (FOV). PRPE becomes thinner than PNR, one of the reasons might be due to higher density of apoptosis in PRPE than that in PNR during early eye development. Molecular differentiation, however, occurred after the contact of the optic vesicle outer wall with the overlying ectoderm which promotes the expression of specific molecules and inhibits the expression of non-specific molecules in PNR and PRPE respectively.

Differences in susceptibility to Saprolegnia infections among embryonic stages of two anuran species.

Many amphibians are known to suffer embryonic die-offs as a consequence of Saprolegnia infections; however, little is known about the action mechanisms of Saprolegnia and the host-pathogen relationships. In this study, we have isolated and characterized the species of Saprolegnia responsible for infections of embryos of natterjack toad (Bufo calamita) and Western spadefoot toad (Pelobates cultripes) in mountainous areas of Central Spain. We also assessed the influence of the developmental stage within the embryonic period on the susceptibility to the Saprolegnia species identified. Only one strain of Saprolegnia was isolated from B. calamita and identified as S. diclina. For P. cultripes, both S. diclina and S. ferax were identified. Healthy embryos of both amphibian species suffered increased mortality rates when exposed to the Saprolegnia strains isolated from individuals of the same population. Embryonic developmental stage was crucial in determining the sensitivity of embryos to Saprolegnia infection. The mortalities of P. cultripes and B. calamita embryos exposed at Gosner stages 15 (rotation) and 19 (heart beating) were almost total 72 h after challenge with Saprolegnia, while those exposed at stage 12 (late gastrula) showed no significant effects at that time. This is the first study to demonstrate the role of embryonic development on the sensitivity of amphibians to Saprolegnia.

Stage-dependent toxicity of 2,4-dichlorophenoxyacetic on the embryonic development of a South American toad, Rhinella arenarum.

The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenoxyacetic acid (2,4-D) and a commercial formulation (CF) were evaluated on Rhinella (= Bufo) arenarum embryos at different developmental stages. Adverse effects were analyzed by means of the isotoxicity curves for lethality, malformations, stage-dependent susceptibility, and ultrastructural features. For all experimental conditions, the CF was more toxic, up to 10 times, than the active ingredient, being the open mouth stage (S.21) the most susceptible to the herbicide. For continuous treatment conditions, the early embryonic development was the most susceptible to 2,4-D and the LC50s for 96 and 168 h were 9.06 and 7.76 mg L(-1) respectively. In addition, both the active ingredient and the CF were highly teratogenic, resulting in reduced body size, delayed development, microcephaly, agenesis of gills, and abnormal cellular proliferation processes as the main adverse effects. According to US EPA, 2,4-D in agricultural scenarios may be up to three times higher than the NOEC values for teratogenic effects reported in this study. Therefore, they might represent a risk for amphibians. This study also points out the relevance of reporting the susceptibility of embryos at different developmental stages to both the active ingredient and the CF of agrochemicals in order to protect nontarget organisms.

Época reproductiva y distribución geográfica del sapo Incilius aucoinae (Bufonidae) en Golfito, Costa Rica / Reproductive period and geographic distribution of the toad Incilius aucoinae in Golfito, Costa Rica

Reproductive period and geographic distribution of the toad Incilius aucoinae in Golfito, Costa Rica. The biology of neotropical amphibians is not well known. Some toad species of the Bufonidae family are common, thus allowing the study of their populations. We studied a population in río Cañaza, Golfito, Costa Rica, in a sector 360 m upstream of Barrio Ureña, divided in 36 sectors of 10 m. The study was carried out for five years, but taking samples of adults in January 1995, 1997, 1998 (except March), and 1999. We also studied tadpoles during four days in 1997. In total, 443 males and 7 females were marked, with 315 males recaptured and no females. Females are bigger (91.1 mm) than males (61.3 mm). Most recaptured individuals occurred in the same or adjacent sectors, with a maximum movement of 28 sectors. One individual was recaptured repeatedly in all the samples. This indicates that they survived at least five reproductive cycles. The reproductive cycle takes place during the dry season, between December and April. We found more individuals during the reproductive cycle of 1997 and less during 1995. Sector 6 had the most number of males and sector number 4 had the least. Sectors with semi-open vegetation had more males compared to sectors with more forest cover. February is the month with the highest abundance of males. There are intermediate values in December and January, and fewer individuals in March and April. In every month, except February, the abundance of individuals was greater where there were beaches. Tadpoles were found both during the day and night in the 31 river sectors, but it was less likely to find tadpoles in the river section during the night. During the day all tadpoles were found scattered around the river, at night they congregated near river margins. The reproductive cycle of this toad occurs during the dry season and females are present only in the reproductive season, resulting in an almost only male sex ratio (only seven females were found). Males can reproduce at least in 5 reproductive cycles, and -unexpectedly- day and night distribution of tadpoles varies. Rev. Biol. Trop. 57 (Suppl. 1): 301-311. Epub 2009 November 30.

Se estudian algunos aspectos de la biología reproductiva del sapo Incilius aucoinae en una población en el río Cañaza, Golfito, Costa Rica; en una sección de 360 m aguas arriba dividida en 36 sectores de 10 m. La duración del estudio con adultos fue de cuatro años, con muestreos en enero de 1995, 1997, 1998 (excepto marzo) y enero de 1999; los renacuajos se estudiaron en cuatro días de 1997. Se marcaron 443 machos (con 315 reobservaciones) y 7 hembras (no fueron recapturadas). Un individuo fue recapturado en todos los periodos de estudio, lo que indica que por los menos sobreviven a 5 épocas reproductivas. La época reproductiva corresponde a los meses secos, de diciembre a abril. En la época reproductiva de 1997 se encontró más individuos y menos en 1995; el sector 6 es el que tiene más machos y el 4 menos; la mayor cantidad de machos se encontró en sectores con vegetación semiabierta, los sectores con mayor cobertura vegetal tienen menos; febrero es el mes con mayor abundancia de machos, con valores intermedios en diciembre y enero, y con muy pocos individuos en marzo y abril. En todos los meses, excepto febrero, la abundancia fue mayor donde había playones. Este sapo se reproduce en la época seca, y en el sitio reproductivo las hembras apenas entran a reproducirse. Se da una relación de sexos de casi solo machos, pues solo hubo 7 hembras observadas, y sorprende los cambios de distribución entre el día y la noche en los renacuajos.

Desarrollo embrionario y larva del sapo Incilius aucoinae (Bufonidae) en Golfito, Costa Rica / Embryonic development and larva of the toad Incilius aucoinae (Bufonidae) in Golfito, Costa Rica

Embryonic development and larva of the toad Incilius aucoinae (Bufonidae) in Golfito, Costa Rica. The larva and developmental stages of Incilius aucoinae are described using specimens from Río Cañaza, Golfito, Costa Rica. We collected the individuals in January 1997, and January and February 1998. The total sample was 696 individuals, 317 eggs in 1-12 stages, 176 eggs with embryos in stages 15-16, and 204 tadpoles in stages 21-43. The average number of eggs per nest was 8 940 (n = 4). These eggs were laid in strings, attached to the river strata. Eggs in stages 1-12 had an average diameter of 1.75 (s = 0.38) mm and eggs with embryos in stages 15 -16 measured 1.33 (0.34) mm wide and 3.06 (1.46) mm long. The description of the tadpole based on UCR 19982 is as follows: dorsal, ventral surfaces, and tail are brown with dark brown spots. Length of tail 9.43 mm, body length 6.18 mm (total length 15.62). Translucent light brown small fins, dorsal fin with scattered spots, ventral fin without dark pigments. Dorsal-ventral flattened oval body with anteroventral snout. A few individuals had varied colors, from black (3.4%) to uniform (0.5-1.5%). Rev. Biol. Trop. 57 (Suppl. 1): 291-299. Epub 2009 November 30.

Se describe la larva y los estadios del desarrollo del huevo y larva de Incilius aucoinae utilizando especímenes del río Cañaza, Golfito, Costa Rica, recolectados en enero de 1997 y enero-febrero de 1998. La muestra fue de 696 individuos, 317 huevos en estadios 1-12, 176 huevos con embriones en estadios 15-16, y 204 renacuajos en estadios 21-43. El promedio del número de huevos por nidada fue de 8 940 (n = 4), los cuales fueron colocados en una hilera, sujetos al sustrato del río. Los huevos en estadios 1-12 tienen un promedio de diámetro de 1.75 (s = 0.38) mm y los huevos con embriones en estadios 15-16 miden 1.33 (0.34) de ancho y 3.06 (1.46) mm de largo. La descripción del renacuajo basada en el espécimen UCR 19982, es como sigue: el dorso, el vientre y la cola son de color café y punteado de café oscuro; la cola mide 9.43 mm y el cuerpo 6.18, para un tamaño total de 15.62; las aletas son pequeñas café claro translúcidas, la dorsal con puntos dispersos y la ventral sin ningún tipo de pigmento oscuro. También es el único renacuajo ovoide con una tendencia a ser aplastado dorso-ventralmente, y con la boca antero-ventral. Dentro de la variación observada, algunos individuos son de color negro (3.4 %) y pocos son uniformes (0.5- 1.5 %).