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.

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.

Patterns of fluctuating asymmetry in the limbs of anurans.

It has been hypothesized that fluctuating asymmetry (FA) may provide an indication of the functional importance of structures within an organism, with structures that more strongly impact fitness being more symmetric. Based on this idea, we predicted that for tetrapods in which the forelimbs and hindlimbs play an unequal role in locomotion, the less functionally important limb set should display higher levels of FA. We conducted a multispecies test of this hypothesis in anurans (frogs and toads), whose saltatory locomotor mode is powered by the hindlimbs. We also tested whether FA in the forelimbs, which play a more important role during landing, differed between families that differ in the degree of forelimb use in locomotion (Bufonidae vs. Ranidae). We calculated FA from the lengths of humeri and femora measured from disarticulated skeletal specimens of four anuran taxa (Bufonidae: Anaxyrus americanus, Rhinella marina; Ranidae: Lithobates catesbeianus, Lithobates clamitans). Our findings were consistent with the hypothesis that natural selection for increased locomotor performance may influence patterns of FA seen in vertebrate limbs, with all species displaying lower mean FA in the hindlimbs. More subtle functional roles between the forelimbs of bufonids and ranids, however, did not elicit different levels of FA.

Ophthalmic findings in a captive population of Panamanian golden frogs: Atelopus zeteki.

PURPOSE: The aim of this study was to describe normal ocular morphology and establish parameters for select diagnostics in a population of Panamanian Golden Frogs (PGF) Atelopus zeteki maintained at the Detroit Zoo. METHOD: A total of 22 toads free of ocular disease were included in this study. Ophthalmic diagnostic tests included as follows: endodontic absorbent paper point tear test (EAPPTT), assessment of intraocular pressures (IOP), Rose Bengal stain, palpebral fissure width, blink rate and corneal cultures. Histology was performed on stored postmortem samples. RESULTS: Panamanian Golden Frogs have a distinct brow ridge and mobile third eyelid. Biomicroscopy revealed avascular, convex corneas with a shallow anterior chamber. Iris color ranged from yellow to green with dark pigmentation of the peripheral iris. Pupil shape was round. Posterior segment and fundus could not be visualized. Total number of blinks observed over a 10-minute period was one. Mean EAPPTT was 2 ± 1 mm and 3 ± 1 mm in males and females, respectively (P). Mean IOP using rebound tonometry in horizontal and vertical restraint positions were 21.6 ± 2.28 mm Hg and 17.45 ± 3.66 mm Hg, 21.85 ± 2.7 mm Hg and 17.5 ± 3.93 mm Hg in males and females, respectively. Mean palpebral fissure width was 5.02 ± 0.46 mm and 6.12 ± 0.39 mm in males and females, respectively (P). Enterococcus faecalis was isolated from one clinically normal toad. CONCLUSIONS: Tear production and blink rate were low and not significantly different between males and females. Intraocular pressure was significantly greater in male toads (P value <0.01). Females had a significantly larger palpebral fissure width when compared to males (P value <0.01) 5819. The ophthalmic parameters established in this study can aid in the evaluation and diagnosis of eye diseases in PGF.

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.

Chondroitin sulfate isolated from the secretion of the venom-producing parotoid gland of Brazilian bufonid.

The parotoid gland of bufonids is characterized as a specialized integument region, formed by different gland types. The secretion elaborated by the largest glandular alveoli has been related to animal chemical defense and is constituted by granular protein content, associated with a basophilic and alcianophilic material with features of glycoconjugates. This study aimed to identify and characterize the glycoconjugates in the secretion of the largest granular gland of the parotoid gland of Rinella icterica by histochemical and immunohistochemical techniques at light microscopy, biochemical methods, and nuclear magnetic resonance spectroscopy. Our results showed that the glycoconjugate content contains a mixture of chondroitin­6­sulfate (C6S) and chondroitin-non-sulfate (C0S). Thus, chondroitin sulfate probably plays an important role in gland physiology, probably protecting the protein content while inside the secretory portion.

Interplay among steroids, body condition and immunity in response to long-term captivity in toads.

Stressful experiences can promote harmful effects on physiology and fitness. However, stress-mediated hormonal and immune changes are complex and may be highly dependent on body condition. Here, we investigated captivity-associated stress effects, over 7, 30, 60, and 90 days on plasma corticosterone (CORT) and testosterone (T) levels, body index, and innate immunity (bacterial killing ability and phagocytosis of peritoneal cells) in toads (Rhinella icterica). Toads in captivity exhibited elevated CORT and decreased T and immunity, without changes in body index. The inter-relationships between these variables were additionally contrasted with those obtained previously for R. schneideri, a related species that exhibited extreme loss of body mass under the same captive conditions. While T and phagocytosis were positively associated in both species, the relationship between CORT and bacterial killing ability was dependent on body index alterations. While CORT and bacterial killing ability were positively associated for toads that maintained body index, CORT was negatively associated with body index in toads that lost body mass over time in captivity. In these same toads, body index was positively associated with bacterial killing ability. These results demonstrate that steroids-immunity inter-relationships arising from prolonged exposure to a stressor in toads are highly dependent on body condition.

Delayed effects and complex life cycles: How the larval aquatic environment influences terrestrial performance and survival.

Species with complex life cycles are susceptible to environmental stressors across life stages, but the carryover and latent effects between stages remain understudied. For species with biphasic life histories, such as pond-breeding amphibians, delayed effects of aquatic conditions can influence terrestrial juveniles and adults directly or indirectly, usually mediated through fitness correlates such as body size. We collected adult southern toads (Anaxyrus terrestris) from 2 source populations-a natural reference wetland and a metal-contaminated industrial wetland-and exposed their offspring to 2 aquatic stressors (a metal contaminant, copper [Cu], and a dragonfly predator cue) in outdoor mesocosms (n = 24). We then reared metamorphs in terraria for 5 mo to examine delayed effects of early life stage environmental conditions on juvenile performance, growth, and survival. Larval exposure to Cu, as well as having parents from a contaminated wetland, resulted in smaller size at metamorphosis-a response later negated by compensatory growth. Although Cu exposure and parental source did not affect larval survival, we observed latent effects of these stressors on juvenile survival, with elevated Cu conditions and metal-contaminated parents reducing postmetamorphic survival. Parental source and larval Cu exposure affected performance at metamorphosis through carryover effects on body size but, 1 mo later, latent effects of parental source and larval predator exposure directly (i.e., not via body size) influenced performance. The carryover and latent effects of parental source population and aquatic Cu level on postmetamorphic survival and juvenile performance highlight the importance of conducting studies across life stages and generations. Environ Toxicol Chem 2018;37:2660-2669. © 2018 SETAC.

Sensitive high-frequency hearing in earless and partially eared harlequin frogs (Atelopus).

Harlequin frogs, genus Atelopus, communicate at high frequencies despite most species lacking a complete tympanic middle ear that facilitates high-frequency hearing in most anurans and other tetrapods. Here, we tested whether Atelopus are better at sensing high-frequency acoustic sound compared with other eared and earless species in the Bufonidae family, determined whether middle ear variation within Atelopus affects hearing sensitivity and tested potential hearing mechanisms in Atelopus We determined that at high frequencies (2000-4000 Hz), Atelopus are 10-34 dB more sensitive than other earless bufonids but are relatively insensitive to mid-range frequencies (900-1500 Hz) compared with eared bufonids. Hearing among Atelopus species is fairly consistent, evidence that the partial middle ears present in a subset of Atelopus species do not convey a substantial hearing advantage. We further demonstrate that Atelopus hearing is probably not facilitated by vibration of the skin overlying the normal tympanic membrane region or the body lung wall, leaving the extratympanic hearing pathways in Atelopus enigmatic. Together, these results show Atelopus have sensitive high-frequency hearing without the aid of a tympanic middle ear and prompt further study of extratympanic hearing mechanisms in anurans.

Trait independence primes convergent trait loss.

The repeated, independent evolution of traits (convergent evolution) is often attributed to shared environmental selection pressures. However, developmental dependencies among traits can limit the phenotypic variation available to selection and bias evolutionary outcomes. Here, we determine how changes in developmentally correlated traits may impact convergent loss of the tympanic middle ear, a highly labile trait within toads that currently lack adaptive explanation. The middle ear's lability could reflect evolutionary trade-offs with other skull features under selection, or the middle ear may evolve independently of the rest of the skull, allowing it to be modified by active or passive processes without pleiotropic trade-offs with other skull features. We compare the skulls of 55 species (39 eared, 16 earless) within the family Bufonidae, spanning six hypothesized independent middle ear transitions. We test whether shared or lineage-specific changes in skull shape distinguish earless species from eared species and whether earless skulls lack other late-forming skull bones. We find no evidence for pleiotropic trade-offs between the middle ear and other skull structures. Instead, middle ear loss in anurans may provide a rare example of developmental independence contributing to evolutionary lability of a sensory system.

Characterization of the Adaptive Morphology of Japanese Stream Toad (Bufo torrenticola) Using Geometric Morphometrics.

The order Anura (frogs and toads) is a group of amphibians and contains over 6500 extant species living in a variety of environments. Each frog species evolved body form adaptive for living and breeding in their own habitats. In Japan, four taxa of Bufo are living: Western-Japanese common toad (Bufo japonicus japonicus), Eastern-Japanese common toad (B. japonicus formosus), Miyako toad (B. gargarizans miyakonis) and Japanese stream toad (B. torrenticola). The former three taxa breed in still water as many other species of Bufo do. In contrast, B. torrenticola breeds in running water such as streams in mountainous area. Corresponding to their breeding in a stream environment, both adult and larva of B. torrenticola acquired unique morphological characters. However, few study have explored differences in the body form between B. torrenticola and closely-related Bufo species quantitatively, remaining the details about the morphological adaptation to a stream environment in this toad species poorly understood. In this study, we quantitatively compared the shape of the foot and skull between the adult male of B. torrenticola and its close relative B. j. formosus using landmark-based geometric morphometrics. Our analyses revealed that B. torrenticola has relatively longer toe phalanges with relatively larger foot webs and relatively shorter metatarsals and a narrower and more streamlined skull, compared to closely-related B. j. formosus. These morphological characteristics are considered adaptive for their breeding in mountain torrents.

Two New Species of Ansonia from Thailand (Anura: Bufonidae).

The taxonomic status of two populations of stream toads of the genus Ansonia from Thailand, previously suspected to be specifically distinct on the basis of molecular data, are assessed using morphological and additional genetic data. In mtDNA phylogeny, each of the two Thai lineages, one from Pilok Subdistrict, Kanchanaburi, and another from Phuket Island, are separated from other congenerics from the Thai-Malay Peninsula by large genetic distances, comparable with those observed between other species in the genus. Although each of the two lineages is superficially similar to other species morphologically, they are distinguishable in several morphological traits and are considered to represent valid, independently evolving species. We therefore describe them as A. pilokensis sp. nov. and A. phuketensis sp. nov.

Effects of chronic cadmium exposure on metamorphosis, skeletal development, and thyroid endocrine disruption in Chinese toad Bufo gargarizans tadpoles.

The present study examined the effects of chronic cadmium (Cd) exposure on metamorphosis, body size, thyroid gland, and skeletal development of Chinese toad (Bufo gargarizans) tadpoles. Tadpoles were exposed to Cd concentrations at 0, 5, 10, 50, 100, and 500 µg/L from Gosner stages 26 to 46 of completion of metamorphosis. Our results showed that 100 and 500 µg/L of Cd concentrations increased mortality and decelerated metamorphosis rate. In addition, significant body size reduction at Gosner stage 42 was observed at 100 and 500 µg/L of Cd treatments (p < 0.01). Average body length and hind-limb length were significantly decreased in the 500 µg/L of Cd group (p < 0.05) but body mass was not significantly different at Gosner stage 46. Moreover, bone formation was delayed in high Cd concentration treatments (50, 100, and 500 µg/L) at both Gosner stage 42 and 46. Histopathological changes of the thyroid gland showed that follicular cell hyperplasia and malformation were induced by high Cd concentrations (50, 100, and 500 µg/L). Furthermore, real-time polymerase chain reaction analysis suggested that the larvae exposed to high-dose Cd exhibited a significant decrease in deiodinase (Dio2) and thyroid hormone receptor (TRß) mRNA levels at Gosner stage 42 and 46. Our investigation indicated that high-dose Cd caused metamorphic deceleration, body size reduction, and delayed skeletal development through disrupting the thyroid system in B. gargarizans larvae. Environ Toxicol Chem 2018;37:213-223. © 2017 SETAC.

Earless toads sense low frequencies but miss the high notes.

Sensory losses or reductions are frequently attributed to relaxed selection. However, anuran species have lost tympanic middle ears many times, despite anurans' use of acoustic communication and the benefit of middle ears for hearing airborne sound. Here we determine whether pre-existing alternative sensory pathways enable anurans lacking tympanic middle ears (termed earless anurans) to hear airborne sound as well as eared species or to better sense vibrations in the environment. We used auditory brainstem recordings to compare hearing and vibrational sensitivity among 10 species (six eared, four earless) within the Neotropical true toad family (Bufonidae). We found that species lacking middle ears are less sensitive to high-frequency sounds, however, low-frequency hearing and vibrational sensitivity are equivalent between eared and earless species. Furthermore, extratympanic hearing sensitivity varies among earless species, highlighting potential species differences in extratympanic hearing mechanisms. We argue that ancestral bufonids may have sufficient extratympanic hearing and vibrational sensitivity such that earless lineages tolerated the loss of high frequency hearing sensitivity by adopting species-specific behavioural strategies to detect conspecifics, predators and prey.

Structural cutaneous adaptations for defense in toad (Rhinella icterica) parotoid macroglands.

Toads have a pair of glandular accumulations on each side of the dorsal region of the head known as parotoid macroglands. These macroglands consist of secretory units (granular glands), each one capped with an epithelial plug. When threatened, toads point one of the parotoids toward the aggressor, and if the aggressor squeezes the parotoid with its teeth, jets of poison will come out of the secretory units and hit the predator's oral mucosa, thereby causing poisoning. Our study focused on the mechanism of parotoid function by comparing parotoids from toads naturally attacked by dogs with those manually compressed. We verified that the process of glandular emptying in response to dog bites is very similar to that following manual compression. We observed that the structure of the plug plays an essential role in the release of the poison jets. Our results suggest that the parotoids may act as "bulletproof vests," reducing the impact of the force exerted by predator attacks, and thus may function as a passive antipredator mechanism.

Histological changes, lipid metabolism and oxidative stress in the liver of Bufo gargarizans exposed to cadmium concentrations.

Chinese toad (Bufo gargarizans) were exposed to different concentrations of cadmium (5, 50, 100, 200 and 500 µg Cd L-1) from Gosner stage 3-42. Metamorphosis rate, body weight, total length and body length were measured. Histological alterations in thyroid gland and liver were examined. Changes in hepatocyte were also examined using Transmission electron microscopic. In addition, the mRNA expression of several genes involved in lipid metabolism, oxidative stress and thyroid hormones signaling pathways were also measured. Our results showed that 200 and 500 µg Cd L-1 decreased the metamorphosis rate and inhibited the body size of B. gargarizans larvae at G42. Moreover, histological examinations have clearly exhibited that cadmium caused liver damage. Ultrastructural examination revealed lipid accumulation and abnormal mitochondria. Exposure to 200 and 500 µg Cd L-1 significantly up-regulated mRNA expression of D2, SOD, GPx, ACC and FAE, but down-regulated mRNA expression of TRα, TRß, PPARα, ACOX, CPT and SCP. However, low Cd concentration (5, 50 and 100) exposure did not cause any effect in genes expression. Thus, we conclude that high Cd concentrations could affect the normal processes of lipid metabolism though increasing lipid synthesis and reducing the ability of fatty acid ß-oxidation, and disturb thyroid hormone pathways in liver, and induced oxidative stress. In addition, lipid metabolism might be regulated by THs. To our knowledge, the present study is the first to report the influence of cadmium on hepatic lipid metabolism in B. gargarizans and will greatly provide new insights into cadmium hepatotoxicity in amphibian.

Designation and description of a neotype of Sclerophrys maculata (Hallowell, 1854), and reinstatement of S. pusilla (Mertens, 1937) (Amphibia: Anura: Bufonidae).

Molecular analysis indicates that African material previously referred to Amietophrynus maculatus (Hallowell, 1854; now Sclerophrys maculata), is divisible into two distinct clades: a Western Clade from Cameroon westwards and an Eastern Clade from Central African Republic eastwards, and Uganda southwards to South Africa, extending to Angola-Namibia. Preliminary morphological and bioacoustic data support this division. The two clades are recognised here as two separate species. The Western species retains the name S. maculata, with Hallowell's designated type locality of Liberia. The Eastern Clade retains the name published by Mertens (1937), S. pusilla. It is noted that a type specimen of S. maculata cannot be traced and is presumed lost; the so-called syntypes in the Philadelphia Academy of Natural Sciences are not the material described by Hallowell. None of these have been designated as a neotype, consequently a specimen from Liberia in the collection of the Natural History Museum, London, is designated here as the neotype of S. maculata.

The phylogenetic relationships of a new Stream Toad of the genus Ansonia Stoliczka, 1870 (Anura: Bufonidae) from a montane region in Peninsular Malaysia.

Twelve species of Ansonia occur on the Thai-Malay peninsula, of which, five from Peninsular Malaysia, form a monophyletic group. One of these, A. jeetsukumarani, is endemic to the Titiwangsa Mountain Range, in which, we discovered a new population of Ansonia that is not A. jeetsukumarani or even its closest relative. Based on morphology, color pattern, and molecular phylogenetic analyses using the mitochondrial genes 12s and 16s rRNA, we have determined that this new species, A. smeagol sp. nov., forms the sister lineage to an upland, monophyletic group composed of A. jeetsukumarani, A. lumut, A. malayana, and A. penangensis. We have noted similar biogeographic patterns in other taxa from the Titiwangsa Mountain Range in a number of upland lineages in Peninsular Malaysia. We hypothesize that the phylogeographic structure of these upland populations is a result of stochastic processes stemming from interaction of climate-driven forest dynamics and life histories.

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 ß (ERß) 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 ERß 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. ERß 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.

Polyphyly of Asian Tree Toads, Genus Pedostibes Günther, 1876 (Anura: Bufonidae), and the Description of a New Genus from Southeast Asia.

The Asian Tree Toad genus Pedostibes, as currently understood, exhibits a conspicuously disjunct distribution, posing several immediate questions relating to the biogeography and taxonomy of this poorly known group. The type species, P. tuberculosus and P. kempi, are known only from India, whereas P. hosii, P. rugosus, and P. everetti are restricted to Southeast Asia. Several studies have shown that these allopatric groups are polyphyletic, with the Indian Pedostibes embedded within a primarily South Asian clade of toads, containing the genera Adenomus, Xanthophryne, and Duttaphrynus. Southeast Asian Pedostibes on the other hand, are nested within a Southeast Asian clade, which is the sister lineage to the Southeast Asian river toad genus Phrynoidis. We demonstrate that Indian and Southeast Asian Pedostibes are not only allopatric and polyphyletic, but also exhibit significant differences in morphology and reproductive mode, indicating that the Southeast Asian species' are not congeneric with the true Pedostibes of India. As a taxonomic solution, we describe a new genus, Rentapia gen. nov. to accommodate the Southeast Asian species.