Gonadal sex differentiation, development up to sexual maturity and steroidogenesis in the skipper frog, Euphlyctis cyanophlyctis.

Gonadal sex differentiation, development up to sexual maturity and steroidogenesis were studied in the Indian skipper frog, Euphlyctis cyanophlyctis. In stage 25 tadpoles, gonads contained a few yolk laden germ cells and somatic cells. Ovarian differentiation occurred at stage 27 with the initiation of meiosis. Interestingly, meiosis preceded the formation of a central lumen that was discernible at stage 28. Folliculogenesis in the developing ovary was observed at stage 29. Vitellogenesis was observed in the 3 months old frogs and the females attained sexual maturity around 4 months. Testicular differentiation occurred indirectly through an ovarian phase. In some animals, from stage 37 onwards, oocyte degeneration was observed that was completed around metamorphic climax. Concurrently, large numbers of mesonephric cells were invading the gonads. Around metamorphosis, reorganization of the germ and somatic cells into testicular cords was observed. Following metamorphosis, the formation of seminiferous tubules was observed in the 2 weeks old males. Meiosis in the developing testes was observed in 1.5 months old males. In 3 months old males, the testes contained all stages spermatogenesis including spermatozoa. Steroidogenesis in the developing gonads was studied by immunohistochemical localization of 3ß-HSD enzyme. At stage 26, a few immunoreactive cells were seen in the kidneys (interrenal cells). However, during and after differentiation, gonads failed to show positive immunoreaction. In the developing ovary at stage 37, follicular cells surrounding the oocytes were positive for 3ß-HSD immunoreactivity. In the ovaries of 3 months old females, follicular cells surrounding the vitellogenic oocytes and stromal cells were positive for 3ß-HSD immunoreaction. E. cyanophlyctis exhibits undifferentiated type of gonadal differentiation, in which gonadal differentiation precedes steroidogenesis.

First report on microplastics contamination in a meteorite impact Crater Lake from India.

Microplastic pollution is a worldwide concern affecting all environmental matrices, including pristine lakes. Lentic lakes operate as a sink for microplastics (MPs), which interfere with a biogeochemical cycle and, therefore, deserve immediate attention. We present a comprehensive assessment of MPs contamination in the sediment and surface water of a geo-heritage site, Lonar lake (India). It is the third largest natural saltwater lake and only basaltic crater in the world formed by meteoric impact around 52,000 years ago. Mean MPs abundance in lakeshore sediment and surface water was 14.44 particles/kg and 2.66 particles/L, respectively. Small-sized MPs dominate the hypersaline region of the lake. Transparent and green fragments and filaments morphotypes were abundant. Most of the MPs in Lonar lake were secondary in origin. FTIR-ATR analysis revealed 16 types of polymers in the lake, of which polypropylene, polyvinyl chloride, polyethylene, high-density polyethylene, low-density polyethylene, polystyrene, and polyester were the most common. The overall pollution load index (PLI) for Lonar lake sediment and water was 1.39 and 2.58, respectively. Although all sampling stations had significant MPs pollution (PLI > 1), there was noticeable station-specific variability, which could be linked to anthropogenic activities. Irresponsible tourist behavior and religious activities, coupled with poor waste management are the leading causes of MPs contamination in the lake. The current work fills a gap in the investigation of MP pollution in a crater lake formed by a meteorite impact by being the first to provide a precise estimate of the MPs contamination in the Lonar lake.

Effect of ethylenethiourea on metamorphosis and ovary development: A comparative study of three larval frogs.

Amphibian endocrine systems interact with each other during normal development. Interference with one of the endocrine systems may influence others. We studied the effect of a thyroid inhibitor (ethylenethiourea [ETU]) on metamorphosis and ovary development of three species, Sphaerotheca pashchima, Indosylvirana caesari, and Euphlyctis cyanophlyctis with different larval durations. We treated the tadpoles of these species with 50, 100, and 200 mg/L concentrations of ETU and studied their larval duration, size at metamorphosis, and ovary development. The results revealed that ETU affects metamorphosis, depending on the species and concentration. ETU delayed metamorphosis of E. cyanophlyctis tadpoles and did not affect metamorphosis in S. pashchima tadpoles. Lower concentrations of ETU stimulated metamorphosis in I. caesari tadpoles while high concentration delayed metamorphosis. In the tadpoles (E. cyanophlyctis) treated with higher concentrations of ETU, ovary development was advanced with an increased size of the diplotene oocytes. Oocyte size was smaller in the tadpoles (of I. caesari) treated with lower concentrations of ETU. These results demonstrated that the tadpoles of these species show different responses to the thyroid inhibitor, possibly due to the differences in the larval duration and sensitivity. Inhibition or acceleration of metamorphosis did not interfere in the ovary development of E. cyanophlyctis and I. caesari. These results will be useful in understanding the impact of endocrine disruptors on the interaction between thyroid and sex steroid hormones.

Importance of genetic data in resolving cryptic species: A century old problem of understanding the distribution of Minervarya syhadrensis Annandale 1919, (Anura: Dicroglossidae).

Frogs of the genus Minervarya are cryptic and widely distributed in South Asia. However, many of them lack information about the precise type locality, genetic data, and distribution range. The present study aimed to examine the genetic affinities of a widely distributed species Minervarya syhadrensis around its type locality in the northern Western Ghats (Pune, Maharashtra). We studied the type specimen of M. syhadrensis and collected similar sized Minervarya frogs from Pune district. In the field, we observed two different calls from morphologically similar (M. syhadrensis like) males suggesting the sympatric occurrence of two cryptic species (that we initially named Minervarya species A and Minervarya species B). We analyzed morphology, call pattern, and mitochondrial 16S rRNA gene sequence of both species. Minervarya species A has a long call with a low pulse repetition rate and higher dominant frequency compared to that of the Minervarya species B. These species cannot be differentiated based on morphometric data. However, they can be sorted out using morphological characters such as the presence of longitudinal skin folds on the dorsal side (Minervarya species A) and differences in foot webbing. DNA sequences of Minervarya species A and Minervarya species B are matching with those of M. caperata and M. agricola respectively. After studying the type specimens of M. syhadrensis and M. caperata, we found morphological similarities (longitudinal skin folds) with the samples of Minervarya species A collected during the present study. Based on the results of our study (morphology and genetic) and available literature, we propose to redefine M. syhadrensis as applying to the lineage initially named Minervarya species A, and to treat the species M. caperata as a junior synonym of M. syhadrensis. Our study will be helpful in further taxonomic revision of the genus, and provides natural history information for M. syhadrensis and M. agricola.

A new species of Fejervarya Bolkay, 1915 (Anura: Dicroglossidae) from the northern Western Ghats parts of Maharashtra, India.

In the recent past the systematic position and taxonomy of genus Fejervarya, Bolkay is undergoing changes in its systematic position due to fairly good amount of phylogenetic resolution, cryptic morphological characters and lack of systematic sampling for phylogenetic studies across the range of distribution. In our sampling in the northern Western Ghats, we encountered a new lineage sister to the phylogenetic cluster which comprises the 'Rufescens complex'. This new lineage is described here as new species Fejervarya marathi sp. nov. based on distinctness in a combination of morphological characters, genetic distance and geography. The problems in morphological groupings for the Fejervarya frogs of the Western Ghats in the recent studies are discussed with the sub-clade composition based on geography in the phylogenetic tree.

An integrative taxonomic review of the South Asian microhylid genus Uperodon.

Based on a recent molecular phylogenetic study, the South Asian microhylid genus Uperodon (subfamily Microhylinae) currently comprises of 12 valid species that are largely restricted to India and Sri Lanka. Considering the revised generic-level status of its various members, here we review the taxonomy of all known species in this genus and clarify their nomenclatural status and geographical distribution, by integrating evidence from genetics, adult and tadpole morphology, breeding ecology, and bioacoustics. Our molecular analyses of a mitochondrial 16S rRNA gene fragment combined with external and internal morphological studies also revealed a distinct new species in the genus. This species, formally described as Uperodon rohani sp. nov., is endemic to Sri Lanka and widely distributed at lower elevations in the island. For nomenclatural stability of various previously known members, the following actions are also undertaken: (1) redescription of the poorly-defined species Ramanella anamalaiensis Rao (= Uperodon anamalaiensis) and Hylaedactylus montanus Jerdon (= Uperodon montanus); (2) neotype designation for Ramanella anamalaiensis Rao (= Uperodon anamalaiensis), Ramanella minor Rao, Ramanella mormorata Rao (= Uperodon mormorata), and Ramanella triangularis rufeventris Rao; (3) lectotype designation for Callula variegata Stoliczka (= Uperodon variegatus); and (4) synonymization of Ramanella minor Rao with Uperodon anamalaiensis.

High temperatures influence sexual development differentially in male and female tadpoles of the Indian skipper frog, Euphlyctis cyanophlyctis.

Although sex determination in amphibians is believed to be a genetic process, environmental factors such as temperature are known to influence the sex differentiation and development. Extremely low and high temperatures influence gonadal development and sex ratio in amphibians but the mechanism of action is not known. In the present study, effect of different temperatures on gonadal development, sex ratio and metamorphosis was studied in the Indian skipper frog, Euphlyctis cyanophlyctis. The embryos of Gosner stage 7 were exposed to 20, 22, 24, 26, 28, 30 and 32 degrees C up to tadpole stage 42. The embryos (stage 7) were also exposed to 20 and 32 degrees C up to tadpole stage 25 (non-feeding stages). Tadpoles of stage 25 were reared at 20 and 32 degrees C up to stage 42 (feeding stages). The results show that exposure to higher temperatures (28, 30 and 32 degrees C) during stages 7-42 produced male-biased sex ratio. Rearing of tadpoles at 32 degrees C during stages 25-42 produced malebiased sex ratio, while exposure during stages 7-25 did not affect sex ratio. Embryos and tadpoles exposed to lower temperatures (20 and 22 degrees C) died during the early stages. High temperatures stimulated testis development, and disturbed ovary development. Exposure to high temperatures resulted in the early metamorphosis of tadpoles with reduced body size. These results demonstrated that high temperatures influence gonadal development differently in male and female tadpoles, leading to male-biased sex ratio. These results suggest that high temperature probably acts through stress hormones and favours the small-sized sex.