Euphlyctis Cyanophlyctis Schneider, 1799 (Amphibia: Dicroglossidae) in district Lower Dir, Pakistan / Euphlyctis Cyanophlyctis (Schneider, 1799) Amphibia: Dicroglossidae no distrito de Lower Dir, Paquistão

Euphlyctis cyanophlyctis (the skittering frog) is one of the most widespread species in Pakistan. Present study was aimed to know the presence of Euphlyctis cyanophlyctis in urban and rural areas of Lower Dir, the North-western Pakistan. A total of 33 frogs were collected, including 15 from rural and 18 from urban areas. The frogs were caught by hands covered with gloves instead of using nets. The collection was managed from August to October 2016 and from April to May 2018. Morphometric analysis, coloration as well as photographs of the frogs have been provided in detail. Skittering frogs were seen frequent in swampy areas near the water bodies. These frogs were mostly seen after sunset.

Euphlyctis cyanophlyctis (a rã que desliza) é uma das espécies mais comuns no Paquistão. O presente estudo teve como objetivo conhecer a presença de Euphlyctis cyanophlyctis em áreas urbanas e rurais de Lower Dir, noroeste do Paquistão. Um total de 33 sapos foram coletados, incluindo 15 de áreas rurais e 18 de áreas urbanas. As rãs foram apanhadas com as mãos cobertas com luvas em vez de redes. A coleta foi gerenciada de agosto a outubro de 2016 e de abril a maio de 2018. Análises morfométricas, coloração e também fotografias das rãs foram fornecidas em detalhes. Rãs saltitantes foram vistas freqüentemente em áreas pantanosas próximas aos corpos d’água. Essas rãs eram vistas principalmente após o pôr do sol.

Altered temperature affect body condition and endochondral ossification in Bufo gargarizans tadpoles.

Bufo gargarizans is one kind of economic animals with higher medicinal value in China. In this study, B. gargarizans (Bufo gargarizans) tadpoles were reared at three different water temperature (15, 22 and 29 °C) from Gosner stages 28-46. We investigated the effects of temperature on growth, development, survival, metamorphic duration, size and skeletal ossification at Gosner stage 40, 42, and 46, as well as thyroid tissue reached metamorphic climax (Gs42). Besides, we examined the transcription levels of endochondral ossification-related genes in hind limb at metamorphic climax (Gs42). Our results showed that the growth and development of tadpoles conform to the temperature-size rule (TSR). While warm temperature resulted in the decrease in body size and hind limb length, and shorten larval period, cold temperature led to increase in body size and hind limb length but prolonged larval period. Histological examinations revealed that warm and cold temperatures caused damage to thyroid tissue. Also, warm and cold temperatures inhibited the degree of ossification with the double staining methodology. Additionally, the real-time PCR results suggested that warm and cold temperatures significantly up-regulated Runx2, VEGF and VEGFR mRNA levels, and down-regulated TRß, MMP9, MMP13 and Runx3 mRNA levels. The up-regulation of Dio2 level and down-regulation of Dio3 level were observed in warm temperature. TRα mRNA level was significantly increased in warm temperature, but decreased in cold temperature. Collectively, these observations demonstrated that warm and cold temperatures affected endochondral ossification in B. gargarizans tadpoles, which might influence their capacity to terrestrial locomotion.

Effects of temperature on growth, development and the leptin signaling pathway of Bufo gargarizans.

Climate change is one of the most important causes of the decline in amphibians. Changes in temperature have an important effect on the growth and development and energy metabolism of amphibians. The aim of this study is to unravel the effects of temperature on the leptin signaling pathway of Bufo gargarizans and its molecular mechanisms. Our results showed that high temperature accelerated the development rate of tadpoles, but reduced body size and mass, while low temperature deferred the development of tadpoles, but increased size and mass. Both high temperature and low temperature exposure caused pathological damage of the liver in B. gargarizans. The results of RT-qPCR revealed that the high temperature treatment significantly upregulated the transcript levels of genes related to thyroid hormone (DIO2 (D2), Thyroid Hormone Receptor-α (TRα)) and the leptin signaling pathway (Leptin Receptor (LepR), Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Tyrosine kinase 2 (TYK2), Signal Transducer And Activator Of Transcription 3 (STAT3), Signal Transducer And Activator Of Transcription 3.1 (STAT3.1), and Signal Transducer And Activator Of Transcription 6 (STAT6)), while there was a decrease of mRNA expression of these genes (TRα, Thyroid Hormone Receptor-Beta (TRß), LepR, JAK1, and TYK2) in the liver of tadpoles exposed to high temperature compared with the intermediate temperature treatment. Therefore, our results suggested that temperature extremes might interfere with the thyroid and leptin signaling pathways and affect the growth and development of B. gargarizans. Furthermore, tissue injury of the liver could occur due to exposure to temperature extremes. This work promotes public awareness of environmental protection and species conservation needs, also provides valuable experimental data and a theoretical basis for the protection of amphibians.

Spindly leg syndrome in Atelopus varius is linked to environmental calcium and phosphate availability.

Spindly leg syndrome (SLS) is a relatively common musculoskeletal abnormality associated with captive-rearing of amphibians with aquatic larvae. We conducted an experiment to investigate the role of environmental calcium and phosphate in causing SLS in tadpoles. Our 600-tadpole experiment used a fully-factorial design, rearing Atelopus varius tadpoles in water with either high (80mg/l CaCO3), medium (50mg/l CaCO3), or low calcium hardness (20mg/l CaCO3), each was combined with high (1.74 mg/l PO4) or low (0.36 mg/l PO4) phosphate levels. We found that calcium supplementation significantly improved tadpole survival from 19% to 49% and that low calcium treatments had 60% SLS that was reduced to about 15% at the medium and high calcium treatments. Phosphate supplementation significantly reduced SLS prevalence in low calcium treatments. This experimental research clearly links SLS to the calcium: phosphate homeostatic system, but we were unable to completely eliminate the issue, suggesting an interactive role of other unidentified factors.

Exposure to artificial light at night during the larval stage has delayed effects on juvenile corticosterone concentration in American toads, Anaxyrus americanus.

Artificial Light At Night (ALAN) is an environmental stressor that can disrupt individual physiology and ecological interactions. Hormones such as corticosterone are often responsible for mediating an organism's response to environmental stressors. We investigated whether ALAN was associated with a corticosterone response and whether it exacerbated the effects of another common stressor, predation. We tested for consumptive, non-consumptive, and physiological effects of ALAN and predator presence (dragonfly larvae) on a widespread amphibian, the American toad (Anaxyrus americanus). We found predators had consumptive (decreased survival) and non-consumptive (decreased growth) effects on larval toads. ALAN did not affect larval toads nor did it interact with the predator treatment to increase larval toad predation. Despite the consumptive and non-consumptive effects of predators, neither predators nor ALAN affected corticosterone concentration in the larval and metamorph life-stages. In contrast to studies in other organisms, we did not find any evidence that suggested ALAN alters predator-prey interactions between dragonfly larvae and toads. However, there was an inverse relationship between corticosterone and survival that was exacerbated by exposure to ALAN when predators were absent. Additionally, larval-stage exposure to ALAN increased corticosterone concentration in juvenile toads. Our results suggest the physiological effects of ALAN may not be demonstrated until later life-stages.

Two New Ansonia from Mountains of Borneo (Anura, Bufonidae).

Two lineages of stream toads in the genus Ansonia from Malaysian Borneo have long been suspected to be specifically distinct on the basis of molecular data. We assessed the taxonomic status of these lineages using morphological and additional genetic data. In mtDNA phylogeny, each lineage-one from Bario, Kelabit Highlands of Sarawak, the other from Mt. Mulu of Sarawak and the Crocker Range of Sabah-is separated from other congeners by large genetic distances, comparable with those observed between heterospecific species in the genus. These lineages are also morphologically distinguishable from other species, and are considered to represent valid, independently evolving species. We therefore describe them as A. kelabitensis sp. nov. and A. kanak sp. nov.

Induction of hyperpigmentation and heat shock protein 70 response to the toxicity of methomyl insecticide during the organ development of the Arabian toad, Bufo arabicus (Heyden,1827).

Methomyl (MET) is a carbamate insecticide which is used as a substitute for organophosphorus compounds to protect crops against insects. The present study aims to evaluate the cytoprotection response of pigment cells and heat shock protein 70 (HSP70) after exposure to MET during the tadpole developmental stages of the Arabian toad, Bufo arabicus. Three developmental larval stages of the toad were selected and divided into two groups; Control and MET-exposed (MET-EX) tadpoles (10ppm). MET-EX tadpoles showed an increased number of pigment cells in the liver, kidney, anterior eye chamber, and skin tissues as compared to the control. The glycogen content in the developing liver and muscles (myotomes) of MET-EX tadpoles was decreased as compared to the control. In the MET-EX tadpoles, immunohistochemical staining showed an increase of HSP70 expression in the liver hepatocytes, the nucleated red blood cells (nRBC) in kidney glomeruli, the iridocorneal angle of anterior eye chamber, and the skin as compared to the control. The current study concluded that pigment cells and HSP70 represented a cytoprotecting response against MET insecticide during the organ development of B. arabicas tadpoles. Therefore, MET use should be regularly monitored in the environment to protect animals and human from exposure to this insecticide.

Naproxen and Its Phototransformation Products: Persistence and Ecotoxicity to Toad Tadpoles (Anaxyrus terrestris), Individually and in Mixtures.

Although pharmaceutical pollution is a global environmental concern, much remains unknown about the transformation of pharmaceuticals in the wild and their effects on wildlife. In the environment, pharmaceuticals typically transform to some extent into different, structurally related compounds. Pharmaceutical transformation products resulting from exposure to sunlight (i.e., ultraviolet radiation) in surface waters are of particular concern; these products can be more hydrophobic, persistent, and toxic than their parent compounds. In the present study, naproxen, a widely used nonsteroidal anti-inflammatory drug, and its phototransformation products were studied to assess the overall persistence and photochemical fate of naproxen. Southern toad (Anaxyrus terrestris) larvae were used as model aquatic vertebrates to evaluate the acute toxicity of naproxen and its phototransformation products singly and in mixtures. The phototransformation products were observed to be more persistent and more toxic than naproxen itself. The slower phototransformation of the phototransformation products relative to naproxen suggests a greater potential to accumulate in the environment, particularly when naproxen is continually released. Mixtures of naproxen and its phototransformation products, in ratios observed during phototransformation, were more toxic than naproxen alone, as predicted by the model of concentration addition and the greater toxicity of the phototransformation products. Together, these results indicate that the ecological risk of naproxen may be underestimated by considering environmental levels of naproxen alone. Environ Toxicol Chem 2019;38:2008-2019. © 2019 SETAC.

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.

Developmental Stage Affects the Consequences of Transient Salinity Exposure in Toad Tadpoles.

Development can play a critical role in how organisms respond to changes in the environment. Tolerance to environmental challenges can vary during ontogeny, with individual- and population-level impacts that are associated with the timing of exposure relative to the timing of vulnerability. In addition, the life history consequences of different stressors can vary with the timing of exposure to stress. Salinization of freshwater ecosystems is an emerging environmental concern, and habitat salinity can change rapidly due, for example, to storm surge, runoff of road deicing salts, and rainfall. Elevated salinity can increase the demands of osmoregulation in freshwater organisms, and amphibians are particularly at risk due to their permeable skin and, in many species, semi-aquatic life cycle. In three experiments, we manipulated timing and duration of exposure to elevated salinity during larval development of southern toad (Anaxyrus terrestris) tadpoles and examined effects on survival, larval growth, and timing of and size at metamorphosis. Survival was reduced only for tadpoles exposed to elevated salinity early in development, suggesting an increase in tolerance as development proceeds; however, we found no evidence of acclimation to elevated salinity. Two forms of developmental plasticity may help to ameliorate costs of transient salinity exposure. With early salinity exposure, the return to freshwater was accompanied by a period of rapid compensatory growth, and metamorphosis ultimately occurred at a similar age and size as freshwater controls. By contrast, salinity exposure later in development led to earlier metamorphosis at reduced size, indicating an acceleration of metamorphosis as a mechanism to escape salinity stress. Thus, the consequences of transient salinity exposure were complex and were mediated by developmental state. Salinity stress experienced early in development resulted in acute costs but little long-lasting effect on survivors, while exposures later in development resulted in sublethal effects that could influence success in subsequent life stages. Overall, our results suggest that elevated salinity is more likely to affect southern toad larvae when experienced early during larval development, but even brief sublethal exposure later in development can alter life history in ways that may impact fitness.

Transport infrastructure severely impacts amphibian dispersal regardless of life stage.

Transport infrastructure such as roads has been reported to negatively affect dispersal. Their effects on dispersal are thought to be complex, depending on the characteristics of the structure and the intensity of the traffic using it. In addition, individual factors, such as age, may strongly affect dispersal decisions and success when individuals are confronted with transport infrastructure. Despite the importance of this topic for wildlife conservation, few studies have investigated the effect of transport infrastructure on individuals' dispersal decisions before and after sexual maturity. We examined the effects on two kinds of infrastructure, gravel tracks and paved roads, on the dispersal of an endangered amphibian, the yellow-bellied toad (Bombina variegata). We used capture-recapture data collected during a five-year period on a large, spatially structured population of B. variegata. Our study revealed that emigration rates increased with an individual's age, while dispersal distance decreased. It also showed that both tracks and roads had negative effects on immigration. The negative effect of roads was stronger than that of tracks. We additionally found that the effect of tracks on dispersal slightly decreased with a toad's age. In contrast, the negative effect of roads was severe and relatively similar across age classes.

Exposure to cadmium induced gut histopathological damages and microbiota alterations of Chinese toad (Bufo gargarizans) larvae.

Cadmium (Cd) is highly hazardous to both terrestrial and aquatic life and it also has multiple negative impacts on amphibian tadpoles and frogs. However, its effects on gut health of amphibian tadpoles are still poorly understood. We used Chinese toad (Bufo gargarizans) tadpoles to examine the effects of chronic cadmium exposure on gut histology and intestinal microbiota by using regular histology analysis and high-throughput sequencing techniques. Tadpoles were exposed to cadmium concentrations at 0, 5, 100 and 200 µg L-1 from Gosner stage 26 to 38. Our results showed 100 and 200 µg L-1 cadmium exposure caused severe gut histopathological alterations while 5 µg L-1 cadmium exposure induced subtle intestine damage. Moreover, species diversity, taxonomic composition and community structure of gut microbiota were influenced by cadmium exposure. Species diversity and richness decreased gradually with the increase of cadmium concentration. Microbial communities of tadpoles in 100 and 200 µg L-1 cadmium exposure groups were remarkably different from those in control group. Furthermore, the relative abundances of prevalent phyla such as Proteobacteria, Bacteroidetes and Firmicutes and dominant genera such as Klebsiella and Aeromonas were also affected by cadmium exposure. We concluded that cadmium could be harmful to tadpole health by inducing intestinal damages and gut microbiota changes.

Exposure to nitrate alters the histopathology and gene expression in the liver of Bufo gargarizans tadpoles.

Nitrate is a ubiquitous component in aquatic environment and the concentrations of anthropogenic nitrate-nitrogen (NO3N) can exceed 25 mg/L in surface waters and 100 mg/L in ground waters. The exceed nitrate has adverse effects on survival, development, and metamorphosis of amphibian. Liver is the hub of many biological processes, including lipid metabolism and bile salts secretion. However, there is little information about the effects of nitrate on the liver in amphibians during metamorphosis. In this study, B. gargarizans was exposed to different concentrations of nitrate from embryo to metamorphosis climax to investigate the effects of nitrate on the liver. The survival rate, metamorphosis percent, body mass, total length, and hind-limb length were measured. The histopathological changes and transcriptome responses in the liver of B. gargarizans to nitrate were examined. Results indicated exposure to 50 and 100 mg/L NO3N delayed the metamorphosis and decreased the metamorphosis percent of B. gargarizans. The body size of B. gargarizans at 10 and 50 mg/L NO3N groups were decreased while it was increased at 100 mg/L NO3N group. In addition, exposure to 100 mg/L NO3N caused severe histopathological changes, including cellular atrophy, increased intercellular areas, degraded lipid droplets, hepatic fibrosis, bile canaliculus contraction and degraded mitochondria in liver. The results of RNA-seq and qRT-PCR interpreted the molecular responses, which might be the factors to induce histopathological changes in the liver of B. gargarizans under the pressure of nitrate exposure.

Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC®) exposure in Rhinella schineideri (Anura: Bufonidae) Neotropical tadpoles.

Brazil is an important consumer of herbicides. In sugarcane cultivation-the country's most extensive agricultural crop-atrazine-based formulations are the principal form of weed control. Several studies have investigated adverse effects of atrazine or their formulations on anurans, but not specifically on Brazilian species. Our aim was therefore to investigate the lethal and sublethal effects of an atrazine-based herbicide in Rhinella schneideri tadpoles and, in particular, effects on the pigmentation system as a new endpoint in ecotoxicological studies. Rhinella schneideri tadpoles at the Gosner-30 stage were exposed to the atrazine-based herbicide formulation, SIPTRAN 500 SC®, in acute bioassays at concentrations of 1.5-25 mg/L. The lethal and sublethal effects induced were analysed at different ecotoxicological levels: organismal level (alterations in behaviour, growth, development, and body mass; morphologic abnormalities), histological level (liver histopathology), the pigmentation system (melanomacrophages and dermal-melanophores), and cellular level (erythrocyte micronucleus formation and other nuclear-abnormalities). This herbicide induced sublethal effects at the organismal level with alterations in swimming and growth and morphologic abnormalities. These results demonstrated that, in anuran tadpoles, the atrazine-based agrochemical increased the frequency of micronucleus formation and other nuclear-abnormalities in erythrocytes and caused liver damage. In addition, we demonstrated for the first time effects of an atrazine-based formulation on the pigmentation system of anuran tadpoles, specifically an increase in the number of melanomacrophages and dermal melanophores. This study is the first to use several widely differing endpoints at different ecotoxicological levels in a comprehensive manner for assessment of the effects of environmental stressors in order to determine the health status of Neotropical anuran species. In doing so, this study establishes a foundation for future ecological assessments.

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).

The relationship between spindly leg syndrome incidence and water composition, overfeeding, and diet in newly metamorphosed harlequin frogs (Atelopus spp.).

Spindly Leg Syndrome (SLS) is a persistent animal welfare issue associated with the rearing of amphibians in captivity. We conducted two experiments to investigate the effects of diet, water composition and overfeeding on prevalence of SLS in newly metamorphosed harlequin frogs (Atelopus spp.). In our first experiment, we offered 400 full-sibling tadpoles of Atelopus certus isocaloric diets in treatments of 31%, 37%, 42% and 48% crude protein respectively. Tadpoles fed higher protein diets metamorphosed faster, but the incidence of SLS exceeded 80% in all treatments leading to the conclusion that variation in dietary protein was not responsible for causing SLS. We used 720 full-sibling Atelopus glyphus tadpoles in a second experiment to examine the effects of diet type, water composition and diet ration on SLS. We found that an overall incidence of 58% spindly leg in tadpoles reared in tap water, but reduced to about 10% in water treated by reverse osmosis and then reconstituted. It is possible that the reverse osmosis treatment removed some factor that caused the SLS, or that the reconstitution may have added a mineral lacking in the original tap water. Within tap water treatments, overfeeding tadpoles in tanks increased the incidence of SLS. We recommend further experimental research into this condition to identify the causative factors in the water. Additional research into the nutritional composition of food available to wild tadpoles would be useful in formulating captive diets, that have to date been solely based on surrogate species.

Endochondral ossification in hindlimbs during bufo gargarizans metamorphosis: A model of studying skeletal development in vertebrates.

Background Endochondral ossification, the process by which most of the skeleton is formed, is accurately regulated by many specific groups of molecules and extracellular matrix components. The molecular mechanisms of endochondral ossification have been extensively investigated in mammals. However, there are few studies about it in amphibians. Results Ossification of femur and tibiofibula was observed at Gs 40, and tarsals and metatarsals were ossified at Gs 42. Most of the skeletons in hindlimbs were completely ossified except for articular cartilages at Gs 46. Thirty-two genes related to endochondral ossification were found in the differentially expressed genes (DEGs) library of hindlimbs, and nine of these genes were validated by qRT-PCR during metamorphosis. Sox9 was expressed in the columnar, prehypertrophic, and upper hypertrophic zones, and the expression of Ihh was observed in prehypertrophic chondrocyte zone in hindlimbs of B. gargarizans. Conclusions The ossification of hindlimbs increased gradually, and the ossification sequence was from proximal to distal in B. gargarizans during metamorphosis. Thirty-two genes found in the DEGs library were related to the regulation of endochondral ossification of hindlimbs in amphibians. The present study will provide a valuable genomic resource for the future study of endochondral ossification in amphibian. Developmental Dynamics 247:1121-1134, 2018. © 2018 Wiley Periodicals, Inc.

Prolonged middle ear development in Rhinella horribilis.

Despite the benefit of the tympanic middle ear to airborne hearing sensitivity, anurans range in how soon they develop functional middle ears after transitioning to life on land. Previous evidence suggested that bufonids had particularly slow middle ear developmental rates, but precise timelines have not yet been published for this family. Here, we provide the first age-verified middle ear development timeline for a true toad species (family Bufonidae). We find that although middle ear development begins during metamorphosis in Rhinella horribilis, the middle ear remains incomplete 15 weeks after the transition from aquatic tadpole to land-dwelling toadlet. Using this new middle ear timeline, we discuss commonalities and differences in middle ear development among bufonids, as well as among Anura.

Transcriptome analyses reveal molecular mechanisms that regulate endochondral ossification in amphibian Bufo gargarizans during metamorphosis.

BACKGROUND: A developmental transition from aquatic to terrestrial existence is one of the most important events in the evolution of terrestrial vertebrates. Amphibian metamorphosis is a classic model to study this transition. The development of the vertebrate skeleton can reflect its evolutionary history. Endochondral ossification serves a vital role in skeletal development. Thus, we sought to unravel molecular mechanisms that regulate endochondral ossification during Bufo gargarizans metamorphosis. METHODS: The alizarin red-alcian blue double staining method was used to visualize the skeletal development of B. gargarizans during metamorphosis. RNA sequencing (RNA-seq) was used to explore the transcriptome of B. gargarizans in four key developmental stages during metamorphosis. Real-time quantitative PCR (RT-qPCR) was used to validate the expression patterns of endochondral ossification related genes. RESULTS: Endochondral ossification increased gradually in skeletal system of B. gargarizans during metamorphosis. A total of 137,264 unigenes were assembled and 44,035 unigenes were annotated. 10,352 differentially expressed genes (DEGs) were further extracted among four key developmental stages. In addition, 28 endochondral ossification related genes were found by searching for DEG libraries in B. gargarizans. Of the 28 genes, 10 genes were validated using RT-qPCR. CONCLUSIONS: The exquisite coordination of the 28 genes is essential for regulation of endochondral ossification during B. gargarizans metamorphosis. GENERAL SIGNIFICANCE: The present study will not only provide an invaluable genomic resource and background for further research of endochondral ossification in amphibians but will also aid in enhancing our understanding of the evolution of terrestrial vertebrates.

Artificial light at night decreases metamorphic duration and juvenile growth in a widespread amphibian.

Artificial light at night (ALAN) affects over 20% of the earth's surface and is estimated to increase 6% per year. Most studies of ALAN have focused on a single mechanism or life stage. We tested for indirect and direct ALAN effects that occurred by altering American toads' (Anaxyrus americanus) ecological interactions or by altering toad development and growth, respectively. We conducted an experiment over two life stages using outdoor mesocosms and indoor terraria. In the first phase, the presence of ALAN reduced metamorphic duration and periphyton biomass. The effects of ALAN appeared to be mediated through direct effects on toad development, and we found no evidence for indirect effects of ALAN acting through altered ecological interactions or colonization. In the second phase, post-metamorphic toad growth was reduced by 15% in the ALAN treatment. Juvenile-stage ALAN also affected toad activity: in natural light, toads retreated into leaf litter at night whereas ALAN toads did not change behaviour. Carry-over effects of ALAN were also present; juvenile toads that had been exposed to larval ALAN exhibited marginally increased activity. In this time frame and system, our experiments suggested ALAN's effects act primarily through direct effects, rather than indirect effects, and can persist across life stages.