Amphibians show a variety of reproductive modes and strategies. The cyclicity and continuity of reproduction can often be predicted from the annual gonadosomatic index trends in a species specific manner. This research aims to document the female and male gonadosomatic index profiles and to ascertain the reproductive cyclicity or continuity of Fejervarya limnocharis (Anura: Ranidae) caught in Mae Sot, Tak Province, Thailand. Frogs were collected monthly between November 2007 and October 2009 in rice fields and their surrounding areas in the study site. For each frog, total weight and ovarian/testicular weight were measured to obtain the female and male gonadosomatic index. The number of female individuals with mature eggs (postvitellogenic eggs) was also counted. The results showed that female frogs had two main surges of increased GSI in March and September of 2008. Alternatively, male frogs showed a more gradual increase and decrease in gonadosomatic index, thus the index remaining high throughout the year. This study concluded that while F. limnocharis in this area is essentially a continuous breeder, it is more optimized for a cyclic reproduction mode with two breeding cycles during the rainy season.
Glutathione-S-Transferase (GST) and metallothionein are important biomarker endpoints in studying the effect of Cd exposure. The purpose of this research was to study the correlation between hepatic GST and metallothionein with hepatic Cd in wild Fejervarya limnocharis exposed to environmental Cd. Results showed that frogs from contaminated sites had significantly higher hepatic metallothionein (3.58 mg/kg wet weight) and GST activity (0.259 μmol/min/mg total protein) than those from the reference site (2.36 mg/kg wet weight and 0.157 μmol/min/mg total protein respectively). There was a significantly positive correlation between hepatic Cd and GST activity (r = 0.802, p = 0.009) but not between hepatic Cd and metallothionein (r = 0.548, p = 0.139). The results concluded that while frogs from the contaminated site had higher GST and metallothionein, only GST showed significant positive correlation with hepatic Cd levels, indicating that hepatic GST activity may be used as a biomarker endpoint.
Background: The present study describes the morphology of sparganum (larva) of the Malaysian Spirometra spp. collected from naturally infected frogs (Rana cancrivora) from rice fields in Tanjung Karang, Malaysia.
Materials and Methods: Spargana of Spirometra spp. collected from naturally infected frogs (Rana cancrivora) were used for the morphological studies. Stretched on a metal ruler, measurements of the worm were recorded. Specimens were stained in Alum-carmine.
Results: The length of the body ranged from 11-50 mm and the width ranged from 0.5-1.5 mm. Specimens stained with Alum-carmine showed ridges (formation of segments) on the surface of the body, and no sexual organs in the body.
Conclusion: The Malaysian Spirometra spp. are similar in measurement and morphology to Spirometra erinacei but further studies are required for confirmation.
The tiger frog Hoplobatrachus rugulosus (Wiegmann 1834) is a large robust dicroglossid frog widely distributed in southern China, Malaysia, Myanmar, Vietnam and Thailand. The escaped bred tiger frog introduced from Thailand hybridized with Chinese native population may have affected the genetic diversity of local Chinese tiger frogs. However, previous microsatellite loci of this species do not offer enough information to construct the genetic map. Here, we reported 33 new microsatellite loci from transcriptome sequencing for H. rugulosus. Alleles ranged between 1 and 10 per locus and only one locus (HRT001) was monomorphic. The polymorphic information content, observed and expected heterozygosity were 0-0.794, 0-0.969 and 0-0.831, respectively. None of the loci was observed in linkage disequilibrium and two loci (HRT023 and HRT068) deviated from Hardy-Weinberg equilibrium after Bonferroni correction for multiple tests. These transcriptome-derived microsatellite markers will be usedto study the genetic divergence and construct the genetic map in H. rugulosus.
Mesocoelium malayanum sp. n. is described from the frog Rana macrodon, in Malaysia. Elongate body, broader anteriorly, measuring 1.900 (1.679-2.070) mm long by 0.404 (0.380-0.437) wide, tegument aspinose oral sucker 0.212 (0.200-0.228) by 0.202 (9.191-0.205), acetabulum 0.141 (0.132-0.150) by 0.139 (0.123-0.146), prepharynx present, oesophagus 0.115 (0.096-0.137), caeca reaching posterior 1/3 of body, anterior testis 0.097 (0.087-0.110) by 0.091 (0.087-0.100) dorsal to acetabulum, posterior testis 0.094 (0.087-0.101) by 0.092 (0.091-0.100), cirrus pouch 0.121 (0.111-0.130) by 0.047 (0.041-0.055), genital pore at left of midline of oesophagus just anterior to intestinal bifurcation, ovary 0.110 (0.091-0.127) by 0.089 (0.085-0.096) on left of body and posterior to acetabulum, vitelline glands with single follicles extending from intestinal bifurcation to ends of caeca, excretory vesicle I-shaped and eggs 0.040 (0.037-0.046) by 0.023 (0.022-0.024). Although morphologically related to M. maroccanum and M. meggitti, M. malayanum is considered to be a new species.
The present research is on a milky sap obtained from the Antiaris toxicaria tree (Moraceae) which is called Upas or Ipoh in Indonesia. The crude sap was administered to anesthetized rats, and changes in electrocardiogram (ECG) and systemic blood pressure was observed. Biologically and pharmacologically active components were extracted from the crude sap by means of water-acetone solution. Based on the strength of chemical qualitative detection tests of the sap extract (SE), cardiac glycosides are supposed to be the main components. The SE inhibited the Na+-, K+-ATPase (EC 3.6.1.3) which was partially purified from guinea pig heart muscle. When the SE and, concurrently, authentic ouabain were applied to isolated frog heart muscles, the fall of twitch tension was observed after the increased tension on mechanograms. These facts suggest that the main components of the milky sap are cardiac glycosides, and glycosides affect Na+, K+-ATPase activity of muscle membrane and heart muscle contraction.
Boesenbergia rotunda, traditionally used to relieve stomach, abdomen, joint, muscle, and rheumatic pain was also reported for its antinociceptive effect on a mouse model. However, the possible pain relief effect of Boesenbergia rotunda ethanolic extract (BREE) via the inhibition to the neural pain pathway remains to be elucidated. This study investigated the inhibitory effect of BREE on compound action potentials (CAPs) and the possible involvement of the opioid receptors. The changes in the CAPs amplitudes of the frog’s sciatic nerves were evaluated following the exposure to three different dosages of BREE (1, 3 and 10 mg/ml and morphine (3 mg/ml). In another set of experiment, the nerves were pretreated with a non-selective opioid receptor antagonist, naloxone (0.1 mg/ml), before exposing the nerve to BREE (1 mg/ml) to investigate the involvement of opioid receptors in the CAPs inhibitory mechanism. The outcome showed a reduction in the CAPs amplitudes when treated with BREE (1, 3 and 10 mg/ml) whereby the effect was reversible. The CAPs inhibition by BREE was absent when the opioid receptors were blocked. Taken together, these findings suggest that BREE-induced CAPs amplitude reduction involves the activation of opioid receptors.
Accurately delimiting species boundaries is a nontrivial undertaking that can have significant effects on downstream inferences. We compared the efficacy of commonly used species delimitation methods (SDMs) and a population genomics approach based on genomewide single-nucleotide polymorphisms (SNPs) to assess lineage separation in the Malaysian Torrent Frog Complex currently recognized as a single species (Amolops larutensis). First, we used morphological, mitochondrial DNA and genomewide SNPs to identify putative species boundaries by implementing noncoalescent and coalescent-based SDMs (mPTP, iBPP, BFD*). We then tested the validity of putative boundaries by estimating spatiotemporal gene flow (fastsimcoal2, ABBA-BABA) to assess the extent of genetic isolation among putative species. Our results show that the A. larutensis complex runs the gamut of the speciation continuum from highly divergent, genetically isolated lineages (mean Fst = 0.9) to differentiating populations involving recent gene flow (mean Fst = 0.05; Nm > 5). As expected, SDMs were effective at delimiting divergent lineages in the absence of gene flow but overestimated species in the presence of marked population structure and gene flow. However, using a population genomics approach and the concept of species as separately evolving metapopulation lineages as the only necessary property of a species, we were able to objectively elucidate cryptic species boundaries in the presence of past and present gene flow. This study does not discount the utility of SDMs but highlights the danger of violating model assumptions and the importance of carefully considering methods that appropriately fit the diversification history of a particular system.
The genus Rana, notably diversified in Oriental regions from China to Southeast Asia, includes a group of cascade frogs assigned to subgenera Odorrana and Eburana. Among them, R. ishikawae and the R. narina complex represent the northernmost members occurring from Taiwan to the Ryukyu Archipelago of Japan. Relationships of these frogs with the continental members, as well as the history of their invasions to islands, have been unclear. The taxonomic status of Odorrana and related genera varies among authors and no phylogenetic reassessment has been done. Using partial sequences of mitochondrial 12S and 16S rRNA genes, we estimated phylogenetic relationships among 17 species of the section Hylarana including Odorrana and Eburana, and related species from the Ryukyus, Taiwan, China, Thailand, Malaysia, and Indonesia. We estimate that (1) Odorrana is monophyletic and encompasses species of Eburana and R. hosii, which is now placed in Chalcorana, (2) the ancestor of R. ishikawae separated from other Rana in the middle to late Miocene prior to its entry to the Ryukyu Archipelago, (3) the ancestor of the R. narina complex later diversified in continental Asia, and invaded the Ryukyu Archipelago through Taiwan, (4) the R. narina complex attained its current distribution within the Ryukyus through niche segregations, and (5) vicariance of R. hosii between Malay Peninsula and Borneo occurred much later than the divergence events in the R. narina complex. Current subgeneric classification of Rana, at least of Southeast Asian members, requires full reassessment in the light of phylogenetic relationships.
A fanged frog Limnonectes kuhlii was once thought to be wide-ranging in Southeast Asia, but is now confined to its type locality Java through recent phylogenetic studies, which clarified heterospecific status of non-Javanese populations, and monophyly of Bornean populations. However, large genetic differences among Bornean populations suggest occurrence of cryptic species, which we test using dense geographic sampling. We estimated the phylogenetic relationships among samples of Bornean populations together with their putative relatives from the continental Southeast Asia, using 2517bp sequences of the 12S rRNA, tRNA(val), and 16S rRNA of mitochondrial DNA, and 2367bp sequences of the NCX1, POMC, and RAG1 of nuclear genes. In the mtDNA trees, Bornean L. kuhlii-like frogs formed a monophyletic group split into 18 species lineages including L. hikidai, with the deepest phylogenetic split separating L. cintalubang from the remaining species. Almost all of these lineages co-occur geographically, and two to three lineages were found syntopically in each locality. Co-occurrence of more than one lineage may be maintained by differential morphology and microhabitat selection. These syntopic lineages should be regarded as distinct species. Our results clearly indicate that taxonomic revision is urgent to clarify many evolutionary problems of Bornean L. kuhlii-like frogs.
he Chinese tiger frog Hoplobatrachus rugulosus is widely distributed in southern China, Malaysia, Myanmar, Thailand, and Vietnam. It is listed in Appendix II of CITES as the only Class II nationally-protected frog in China. The bred tiger frog known as the Thailand tiger frog, is also identified as H. rugulosus. Our analysis of the Cyt b gene showed high genetic divergence (13.8%) between wild and bred samples of tiger frog. Unexpected genetic divergence of the complete mt genome (14.0%) was also observed between wild and bred samples of tiger frog. Yet, the nuclear genes (NCX1, Rag1, Rhod, Tyr) showed little divergence between them. Despite this and their very similar morphology, the features of the mitochondrial genome including genetic divergence of other genes, different three-dimensional structures of ND5 proteins, and gene rearrangements indicate that H. rugulosus may be a cryptic species complex. Using Bayesian inference, maximum likelihood, and maximum parsimony analyses, Hoplobatrachus was resolved as a sister clade to Euphlyctis, and H. rugulosus (BT) as a sister clade to H. rugulosus (WT). We suggest that we should prevent Thailand tiger frogs (bred type) from escaping into wild environments lest they produce hybrids with Chinese tiger frogs (wild type).
Bukit Perangin Forest Reserve, Kedah Malaysia is a popular forest park well known for its clear rivers and streams, interesting sedimentary rock formations and a rich biodiversity in flora and fauna. However to date no report on the amphibians and reptiles of this forest has ever been published. Therefore, from January 2009 to May 2010, herpetofauna surveys were conducted at Bukit Wang Recreational Forest, which forms a part of the Bukit Perangin Forest Reserve. The results of the survey showed that a total of 15 species of amphibians belonging to 6 families and 15 species of reptiles
belonging to 7 families were found in the study area. These constitute the first records for the amphibians and reptiles in this forest. The herpetofauna checklist showed that the most abundant amphibians were from the family Ranidae while the most abundant reptiles were from the family Gekkonidae.
Studies have shown that some characters, such as morphological, biochemical and behavioural characteristics were shared among con-specifics of closely related frog species. It is hypothesized that closely related frogs would have similar vocal apparatus structure to produce similar call characteristics, in order to be recognized by con-specifics. The vocal apparatus structures of the frogs from Ranidae were, thus, investigated and calls were recorded from various locations in Sarawak, Borneo. The vocal apparatus was sliced and stained using haematoxylin and eosin staining method. The prepared slides were scanned using Dotslide System Olympus BX51. Captured images were further measured using Microscope Olympus BX51. The results showed that the vocal apparatus structure of ranids in Sarawak differed in length between pulvinar vocale and posterior margin (pvpm) or known as vocal cord stricter. This character was deemed to be the most significant character to discriminate the Sarawak ranids. It is suggested that histological analysis is also a good method for study of phenotypic variation of frog species, as shown by the vocal apparatus structure of the anurans. This study provides an alternative method to differentiate taxonomic identity among frog species.
Rakaman panggilan katak Borneo daripada genus Hylarana telah dijalankan di tujuh lokasi kajian di Sarawak, Malaysia. Sebanyak 12 individu daripada lima spesies katak Borneo telah berjaya dirakam dan dianalisis. Daripada 38 pembolehubah panggilan, hanya 27 daripadanya dapat menerangkan ciri-ciri panggilan daripada katak Hylarana Borneo. Saiz katak jantan mempengaruhi secara signifikan tenaga panggilan antara 90:50% amplitud puncak, tempoh nadi di antara 50%, nisbah tempoh panggilan untuk mencapai setengah modulasi frekuensi dan kadar ulangan not. Saiz katak Hylarana Borneo sedikit mempengaruhi frekuensi asas dan dominan panggilan. Di samping itu, taburan pembolehubah panggilan katak dan dendogram Euclid menyokong identiti taksonomi bagi setiap spesies katak Borneo Hylarana.
Recordings of frog calls from the genus Hylarana were conducted at seven study sites in Sarawak, Malaysia. The results showed that each species differs in terms of call characteristics, in which a high number of pulsed note belongs to H. baramica, while high repetition note belongs to the sibling species, H. glandulosa. Higher pitched and number of amplitude spectrum, however, belong to H. signata. Pulsed note and note repetition differed significantly among species, except for spectrum amplitude. Advertisement calls for each species were described in detail. This study provide a baseline data on advertisement call characteristics of frogs from the genus Hylarana for future studies of these frogs particularly and the Oriental frogs generally.
The sound produced by the anurans including toads can be a survival strategy for the selection of fittest males by their conspecific females and a form of effective prezygotic mechanism. Most of the published works on anuran sound production were on true frogs or ranids, yet little is known about sound characteristics of toads. Here we describe the properties of mating calls from six selected toad species from six study sites in Sarawak. Males' mating calls were recorded and analyzed using Sound Ruler Acoustic Analysis ver 0.9.6.0. Call characteristic were subsequently compared for toad species differentiation. The results showed that each species differ in call characteristics, with pulsating note, rate of note repetition and pitch being the most apparent characters. The highest number of pulse notes belongs to Duttaphrynus melanostictus while Ansonia spinulifer exhibited highest pitch and rate of note repetition. These species' differences in mating call characteristics provide an additional method for anuran species classification besides morphological and molecular DNA data. It is also a very useful guide during field survey as the sound is audible and identification can be made without the need of capturing the toads.
Sundapolystoma chalconotae. n. g., n. sp. (Polystomatidae, Polystomatinae) is proposed for a new polystomatid from the urinary bladder of Rana chalconota (Schlegel) in Peninsular Malaysia. This is the first species of polystomatid to be described from the amphibians of Peninsular Malaysia and the second for the Southeast Asian region. This new genus, as exemplified by S. chalconotae, differs from other polystomatids, and in particular Parapolystoma Ozaki, 1935 (P. bulliense (Johnston, 1912) Ozaki, 1935 and P. johnstoni Pichelin, 1995), in having a tubular uterus and a single diffuse testis. P. crooki Vande Vusse, 1976 is similar to S. chalconotae in having a similar type of uterus and testis, and is re-assigned as Sundapolystoma crooki (Vande Vusse, 1976) n. comb. S. chalconotae differs from S. crooki in having anchors with a longer outer root rather than a longer inner root and 7-8 genital spines compared to 9-13 in S. crooki.
Infection with Chloromyxum careni Mutschmann, 1999 was found in the Asian horned frog Megophrys nasuta from Malaysia and Indonesia. Kidney was the only organ infected. Coelozoic plasmodia up to 300 μm were localized in Bowman's space, embracing the glomerulus from all sides, or rarely in lumina of renal tubules. Plasmodia are polysporic, containing disporic pansporoblasts. Myxospores observed by light microscopy are colorless, variable in shape and size, measuring 6.0-8.5 × 5.0-6.5 μm, composed of two symmetrical valves joined by a meridian suture, containing four pyriform polar capsules 3.0-4.0 × 2.5-3.0 μm and a single sporoplasm. Each valve possesses 14-24 (median 21) fine longitudinal ridges clearly visible only in scanning electron microscopy. Rarely, atypical spores with a markedly pointed posterior pole and only 6-10 surface ridges are present in plasmodia together with typical spores. Both small subunit (SSU) and large subunit (LSU) rRNA gene sequences possess extremely long GU-rich inserts. In all SSU and LSU rDNA-based phylogenetic analyses, C. careni clustered as a distinct basal branch to the Myxobolus+Myxidium lieberkuehni clade, out of the marine Chloromyxum clade containing Chloromyxum leydigi, the type species of the genus. These morphological and phylogenetic data suggest erection of a new genus for the C. careni lineage, but we conservatively treat it as a Chloromyxum sensu lato until more information is available.
The synlophe of Batrachonema synaptospicula Yuen, 1965 collected from Rana limnocharis Boie, 1835 of peninsular Malaysia was found to be identical morphologically to that in the specimens from Rana narina Stejneger, 1901 of Okinawa, and R. limnocharis of Taiwan. In the midbody, 20-22 ridges are present, and the ridges increase gradually in size and are oriented from right to left in the dorsal and left ventral fields, whereas the right ventral ridges are small and almost perpendicular to the body wall. The orientation of ridges from right to left is considered to be a key characteristic of the genus Batrachonema. Because Amphibiophilus ranae Wang et al., 1978 and Amphibiophilus sp. from R. limnocharis of south China are regarded to be conspecific with B. synaptospicula, this nematode is surmised to be distributed widely in southeast and east Asia.
Frogs caught from two States (Selangor and Langkawi) in Malaysia were examined for spargana of Spirometra sp. Infected frogs usually show no marks of infection but some had swelling and bleeding at the infection site. The size and weight of the infected frogs did not correlate with the infection status. The infection status in relation to human health is discussed.