In vitro immunoreactivity and in vivo neutralization of Trimeresurus gracilis venom with antivenoms targeting four pit viper species.

Snakebite envenomation is a significant global health issue that requires specific antivenom treatments. In Taiwan, available antivenoms target a variety of snakes, but none specifically target Trimeresurus gracilis, an endemic and protected species found in the high mountain areas of Taiwan. This study evaluated the effectiveness of existing antivenoms against T. gracilis venom, focusing on a bivalent antivenom developed for Trimeresurus stejnegeri and Protobothrops mucrosquamatus (TsPmAV), as well as monovalent antivenoms for Deinagkistrodon acutus (DaAV) and Gloydius brevicaudus (GbAV). Our research involved in vivo toxicity testing in mice and in vitro immunobinding experiments using (chaotropic) enzyme-linked immunosorbent assays, comparing venoms from four pit viper species (T. gracilis, T. stejnegeri, P. mucrosquamatus, and D. acutus) with three types of antivenoms. These findings indicate that TsPmAV partially neutralized T. gracilis venom, marginally surpassing the efficacy of DaAV. In vitro tests revealed that GbAV displayed higher binding capacities toward T. gracilis venom than TsPmAV or DaAV. Comparisons of electrophoretic profiles also reveal that T. gracilis venom has fewer snake venom C-type lectin like proteins than D. acutus, and has more P-I snake venom metalloproteases or fewer phospholipase A2 than G. brevicaudus, T. stejnegeri, or P. mucrosquamatus. This study highlights the need for antivenoms that specifically target T. gracilis, as current treatments using TsPmAV show limited effectiveness in neutralizing local effects in patients. These findings provide crucial insights into clinical treatment protocols and contribute to the understanding of the evolutionary adaptation of snake venom, aiding in the development of more effective antivenoms for human health.

The Presence of Four Pathogenic Oral Bacterial Species in Six Wild Snake Species from Southern Taiwan: Associated Factors.

The oral cavity of snakes serves as a habitat for various microorganisms, some of which may include potential zoonotic pathogens posing risks to hosts and causing wound infections in snakebite victims. Clinical studies on snakebite cases in Taiwan have identified specific pathogens, such as Enterococcus faecalis (Gram-positive), Morganella morganii, Aeromonas hydrophila, and Pseudomonas aeruginosa (Gram-negative). However, the prevalence of these bacteria in the oral cavity of wild snakes remains largely unknown. This study investigated the occurrence of these bacteria in six wild snake species (Naja atra, Bungarus multicinctus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Boiga kraepelini, and Elaphe taeniura friesi) from southern Taiwan, along with factors influencing their presence. Oropharyngeal swab samples were collected from a substantial number of wild-caught snakes (n = 1104), followed by DNA extraction, polymerase chain reaction, and gel electrophoresis. The band positions of samples were compared with positive and negative controls to determine the presence of target bacteria in each sample. The overall occurrence rates were 67.4% for E. faecalis, 31.5% for M. morganii, 8.2% for A. hydrophila, and 7.7% for P. aeruginosa. Among snake species, B. kraepelini exhibited dominance in E. faecalis (93.4%), A. hydrophila (17.1%), and P. aeruginosa (14.5%), while male N. atra showed dominance in M. morganii (51.3%). The occurrence of E. faecalis was lowest in winter. The results of multiple logistic regression analyses suggest that factors such as species, sex, temperature, season, and coexisting pathogens may have a significant impact on the occurrence of target bacteria. These findings have implications for wildlife medicine and snakebite management.

Comparative analysis of Deinagkistrodon acutus venom from Taiwan and China utilizing chromatographic, electrophoretic, and bioinformatic approaches, along with ELISA employing a monospecific antivenom.

Deinagkistrodon acutus is a medically important pitviper inhabiting mainly South China and Taiwan. The hemorrhagic effects of its envenoming are compatible to its venom, which is abundant in metalloproteases (svMPs) and C-type lectin-like proteins. In this study, we investigated geographic variations in the venom of D. acutus collected from Taiwan and four Mainland Chinese provinces: Fujian, Jiangxi, Anhui, and Hunan. The variations were assessed through high-performance liquid chromatography, non-metric multidimensional scaling analysis, gel electrophoresis, and enzyme-linked immunosorbent assay (ELISA) with a monospecific antivenom (DaMAV) generated against the Taiwanese D. acutus venom, and discussed based on venom-protein sequences in databases and literature related to D. acutus venom. Additionally, the cross-reactivity of DaMAV against Crotalus horridus and Calloselasma rhodostoma venoms was investigated. We noted differential abundances of D. acutus venom metalloproteases, C-type lectin-like proteins, and phospholipase A2, along with point mutations and selective expression of serine protease isoforms. The ELISA results revealed that the venom from Taiwan was more reactive toward Taiwanese DaMAV than the four Mainland Chinese venoms, consistent with chromatographic profile differences, whereas C. horridus venom presented moderate cross-reactivity with DaMAV. The observed immunoreactivities of these venom with DaMAV can be attributed to the high prevalence of their PIII-svMPs, which are the dominant antigens, and the conservation of PIII-svMP epitopes.

Incidence and Risk Factors for Wound Infections after Trimeresurus stejnegeri Snakebites in Taiwan.

Snakebite envenomation is a neglected tropical disease. Taiwan, with its subtropical and Southeast Asian environment, provides suitable habitat for several venomous snake species. Trimeresurus stejnegeri, an arboreal pit viper, is the most common cause of venomous snakebite in Taiwan. Trimeresurus stejnegeri envenomation can cause local swelling, occasional ecchymosis, and wound infection. The primary treatment of T. stejnegeri envenomation is the binary antivenom, vacuum freeze-dried F(ab')2 fragments of equine antibodies, against T. stejnegeri and Protobothrops mucrosquamatus. This study aimed to analyze the incidence of post-envenomation wound infections caused by T. stejnegeri based on data collected over a decade from institutions affiliated with the Chang Gung Memorial Hospital in Taiwan. A total of 254 patients were enrolled in this study. Clinical and laboratory data, treatment information, and patient outcomes were extracted from electronic medical records. Wound infection was associated with delay in antivenom initiation (adjusted odds ratio: 3.987; 95% CI: 1.406-11.302). The infection rates were 20.5%, 12.5%, 31.3%, and 48.1% for antivenom administration within 2 hours, 2-4 hours, 4-6 hours, and > 6 hours, respectively. Therefore, early initiation of antivenom treatment (within 6 hours) is recommended. Morganella morganii was cultured from wounds of the patients, whereas Enterobacter cloacae and Enterococcus faecalis were cultured from both the oral cavity of snakes and the wounds of the patients. For post-envenomation patients who develop a local infection, empiric antibiotics such as third-generation cephalosporins, quinolones, and piperacillin/tazobactam are recommended because snakebite wound infections are often polymicrobial in nature.

Comparisons of the Oral Microbiota from Seven Species of Wild Venomous Snakes in Taiwan Using the High-Throughput Amplicon Sequencing of the Full-Length 16S rRNA Gene.

A venomous snake's oral cavity may harbor pathogenic microorganisms that cause secondary infection at the wound site after being bitten. We collected oral samples from 37 individuals belonging to seven species of wild venomous snakes in Taiwan, including Naja atra (Na), Bungarus multicinctus (Bm), Protobothrops mucrosquamatus (Pm), Trimeresurus stejnegeri (Ts), Daboia siamensis (Ds), Deinagkistrodon acutus (Da), and alpine Trimeresurus gracilis (Tg). Bacterial species were identified using full-length 16S rRNA amplicon sequencing analysis, and this is the first study using this technique to investigate the oral microbiota of multiple Taiwanese snake species. Up to 1064 bacterial species were identified from the snake's oral cavities, with 24 pathogenic and 24 non-pathogenic species among the most abundant ones. The most abundant oral bacterial species detected in our study were different from those found in previous studies, which varied by snake species, collection sites, sampling tissues, culture dependence, and analysis methods. Multivariate analysis revealed that the oral bacterial species compositions in Na, Bm, and Pm each were significantly different from the other species, whereas those among Ts, Ds, Da, and Tg showed fewer differences. Herein, we reveal the microbial diversity in multiple species of wild snakes and provide potential therapeutic implications regarding empiric antibiotic selection for wildlife medicine and snakebite management.

Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species.

Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A2, 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrhagic venom of T. gracilis appears to be especially rich in PI-class SVMPs and lacks basic phospholipase A2. We also cloned and sequenced the cDNAs encoding two CRISP and three VEGF variants from T. gracilis venom glands. Sequence alignments and comparison revealed that the PI-SVMP, kallikrein-like proteases, CRISPs, and VEGF-F of T. gracilis and Ovophis okinavensis are structurally most similar, consistent with their close phylogenetic relationship. However, the expression levels of some of their toxins were rather different, possibly due to their distinct ecological and prey conditions.

Full sequencing and comparison of five venom metalloproteases of Trimeresurus gracilis: The PI-enzyme is most similar to okinalysin but the PIII-enzyme is most similar to Crotalus venom enzymes.

The cDNAs encoding the Zn+2-metalloproteases (SVMPs) of Trimeresurus gracilis (abbreviated as Tgc), a pitviper endemic to Taiwan, were cloned from venom glands and sequenced. The amino-acid sequences of five novel SVMPs, including one P-III, three P-II and one P-I class enzymes, were thus deduced and subjected to BLAST-analyses. The P-III enzyme (designated as Tgc-PIII) is structurally most similar to the PIII-SVMPs of New World pitvipers but not similar to the PIII-SVMP of Ovophis okinavensis. Sequence-similarity analysis of 22 homologous PIII-SVMPs reveal three major structural subtypes of the pitviper PIII-SVMPs, which possibly have different substrate specificities. In addition, Tgc-PIII and the PI-class SVMP (named Tgc-MP) were isolated from the venom and verified by mass spectrometry. All the three deduced sequences of PII-SVMPs (Tgc-PIIs) contain an abnormal Zn+2-binding-site in their catalytic-domain, and an identical "long-disintegrin" domain. The predicted 85-residues disintegrin, gracilisin, bears high similarities to some long-disintegrins of the New-World pitvipers and salmosin3. By BLAST search and comparison, Tgc-MP is 96% similar to okinalysin, the hemorrhagic PI-SVMP of O. okinavensis, rather than any other PI-SVMPs in the databanks. Our results confirm the fast evolution of Tgc-SVMPs as well as their structural similarities to different SVMP-classes of the New-World pitvipers and of O. okinavensis, respectively. The implications of our findings are discussed along with our previous sequence comparisons of venom phospholipases A2 and ten venom serine proteases of Tgc.

Oral Bacteria and Their Antibiotic Susceptibilities in Taiwanese Venomous Snakes.

Wound infections after venomous snakebites are clinically important. Information regarding the nature and antibiotic susceptibilities of snake oral bacterial flora could support empiric antibiotic therapy. Wild venomous snakes were collected from southern Taiwan: a total of 30 each of Bungarus multicinctus, Naja atra, Protobothrops mucrosquamatus, and Trimeresurus stejnegeri; 3 Deinagkistrodon acutus; and 4 Daboia siamensis. The species and antibiotic susceptibilities of their oral bacteria were determined. Aerobic gram-negative bacteria, especially Pseudomonas aeruginosa and Proteus vulgaris, were the most abundant. Proteus vulgaris were more abundant in B. multicinctus, N. atra, and P. mucrosquamatus than in T. stejnegeri (40%, 43.3%, and 40% vs. 13.3%, respectively). The gram-negative species were less susceptible to first- and second-generation cephalosporins and ampicillin-sulbactam than to third-generation cephalosporins, fluoroquinolones, carbapenems, or piperacillin-tazobactam. The most abundant aerobic gram-positive species cultured was Enterococcus faecalis, which was more abundant in N. atra than in other snakes (p < 0.001) and was highly susceptible to ampicillin, high-level gentamicin, penicillin, teicoplanin, and vancomycin. Bacteroides fragilis and Clostridium species were the most common anaerobic bacteria. The anaerobic organisms were highly susceptible to metronidazole and piperacillin. As a reference for empiric antimicrobial therapy, third-generation cephalosporins, fluoroquinolones, carbapenems, or piperacillin-tazobactam can be initiated in venomous snakebites wound infections.

Case Report: Symptoms and Prognosis of Trimeresurus gracilis Envenomation.

Trimeresurus gracilis is a pit viper inhabiting the high mountains of Taiwan. No specific antivenom against T. gracilis is available, and we did not find any prior published case reports on its clinical envenomation. We present two patients with T. gracilis envenomation who were both bitten on the index finger and were administered bivalent hemotoxic antivenom against Trimeresurus stejnegeri and Protobothrops mucrosquamatus. In case 1, the patient was administered seven vials of antivenom within 2 days of envenomation. She received surgical intervention on day 26 and recovered from the wound 3 months after envenomation, but her left index finger was slightly and irreversibly bent. In case 2, the patient was administered 12 vials of antivenom within 10 hours after envenomation, received surgical intervention on day 1, and underwent debridement and reconstruction surgery on day 7 after envenomation. The wounds healed 1.5 months after envenomation, and no deformity occurred. Laboratory data showed elevated D-dimer levels and prothrombin times. The cross-neutralizing ability of bivalent hemotoxic antivenom against T. stejnegeri and P. mucrosquamatus appeared insufficient to treat the local effects of T. gracilis envenomation. The deformity of the finger of the patient in case 1 might have been caused by the lower dose, later administration of antivenins, and a delay in the necessary surgery.

Sequence determination and bioinformatic comparison of ten venom serine proteases of Trimeresurus gracilis, a Taiwanese endemic pitviper with controversial taxonomy.

Trimeresurus gracilis (Tgc) is endemic to Taiwan and shown to be closely related with Ovophis okinavensis by previous phylogenetic analyses, but their taxonomic status remain controversial. Here, we cloned and sequenced ten of its venom serine-proteases (designated as Tgc-vSPs). All the Tgc-vSPs conserve the catalytic triads, six appear to be kallikrein-like (KNs) and four are plasminogen-activator homologs (PAHs and PAs). They are studied under four structural categories: (1) highly similar Tgc-KN1, Tgc-KN2 and Tgc-KN3, with four predicted N-glycosylation sites; (2) Tgc-KN4, with a single N -glycosylation site; (3) Tgc-KN5 and Tgc-KN6, with two distinct N-glycosylation sites; (4) Tgc-PAH1/PAH2, TgcPA3, and Tgc-PA4, with two conserved N-glycosylation sites. Additionally, Tgc-KN1, Tgc-KN4 and Tgc-PAH1 were purified by reversed-phase HPLC and identified by peptide-mass-fingerprinting. Results of BLAST and sequence alignments reveal that Tgc-KN1∼3 and Tgc-KN6 are most like the vSPs of rattlesnakes, while the sequences of Tgc-KN4, KN5 and Tgc-PAH1/PAH2 match closely to the partial sequences of three O. okinavensis vSPs. Thus, our results reveal non-overlapping similarities of Tgc-vSPs to the O. okinavensis vSPs and vSPs of the New World pitvipers. In addition, molecular phylogenetic analyses of the plasminogen-activator like vSPs reveal separate evolution of two clusters of the enzymes with distinct functions.

Personal Experience of Daboia siamensis Envenomation.

Reports of envenomation induced by Daboia siamensis, a medically important venomous snake in Taiwan, are rare, and species identification might not be definitive. This article reports the complete course of a definite D. siamensis bite. The patient in this report was one of the authors who was bitten on the right palm near the base of the index finger by D. siamensis. The patient experienced local effects, neurological manifestations, and acute kidney injury. The laboratory analysis revealed elevated D-dimer and coagulopathy. The patient was administered 8 vials of antivenom and did not undergo surgical intervention or endotracheal tube intubation, but serum sickness occurred 8 days after antivenom administration. The horse immunoglobulin produced by the Centers for Disease Control, R. O. C. (Taiwan), against D. siamensis was effective and safe in the treatment of the patient. However, the best antivenom administration strategy remains unclear and requires further study.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Evolution of nuchal glands, unusual defensive organs of Asian natricine snakes (Serpentes: Colubridae), inferred from a molecular phylogeny.

A large body of evidence indicates that evolutionary innovations of novel organs have facilitated the subsequent diversification of species. Investigation of the evolutionary history of such organs should provide important clues for understanding the basis for species diversification. An Asian natricine snake, Rhabdophis tigrinus, possesses a series of unusual organs, called nuchal glands, which contain cardiotonic steroid toxins known as bufadienolides. Rhabdophis tigrinus sequesters bufadienolides from its toad prey and stores them in the nuchal glands as a defensive mechanism. Among more than 3,500 species of snakes, only 17 Asian natricine species are known to possess nuchal glands or their homologues. These 17 species belong to three nominal genera, Balanophis, Macropisthodon, and Rhabdophis. In Macropisthodon and Rhabdophis, however, species without nuchal glands also exist. To infer the evolutionary history of the nuchal glands, we investigated the molecular phylogenetic relationships among Asian natricine species with and without nuchal glands, based on variations in partial sequences of Mt-CYB, Cmos, and RAG1 (total 2,767 bp). Results show that all species with nuchal glands belong to a single clade (NGC). Therefore, we infer that the common ancestor of this clade possessed nuchal glands with no independent origins of the glands within the members. Our results also imply that some species have secondarily lost the glands. Given the estimated divergence time of related species, the ancestor of the nuchal gland clade emerged 19.18 mya. Our study shows that nuchal glands are fruitful subjects for exploring the evolution of novel organs. In addition, our analysis indicates that reevaluation of the taxonomic status of the genera Balanophis and Macropisthodon is required. We propose to assign all species belonging to the NGC to the genus Rhabdophis, pending further study.

Species Identification of Fragmented or Faded Shed Snake Skins by Light Microscopy.

Few convenient/expeditious methods for identifying the species of shed snake skins in specific areas have been developed. The scales on shed snake skins are permeable to light and can be examined by light microscopy (LM), which is of higher availability-especially for wild animal researchers and citizen scientists-than conventional approach which examines the scale microstructures by scanning electron microscopy. We collected and examined a total of 801 shed samples or scale specimens from 53 snake species in Taiwan and adjacent islands, and developed the first guide to identify the fragmented or faded shed skins of most snake species by LM. Morphological characters of scales can be examined by LM include the apical notch, apical pits, apical lobes, keels, scale symmetry, unpigmented spots (mechanoreceptor-like organs), interscale follicles, cross/longitudinal micro-ridge, oberhautchen cells, rows of spines, light/tiny dots, and other microstructures. The microstructures on the scale specimens prepared by the stripped method and the impression method were similar to those on shed skins when examined by LM. We investigated the variations of scale morphology associated with ontogeny, body region, and position on scales, discussed the character evolution of snake scale morphology, and certified that the interscale follicles and the unpigmented spots could also be useful characters for shed skin identification. The methods and results of this study could be applied to identify squamate skins/sloughs and even fecal remnants.

An Improved Technique for Obtaining Accurate and Precise Morphometric Data on Snakes.

Morphometric measurements are basic but essential data in morphological and ecological research. It is thus beneficial to develop a safe, convenient, multipurpose device/technique to determine body length and physical characteristics of living snakes with high accuracy/precision and low stress, and to enable the probing of snakes and collection of tissue samples. To meet these requirements, we developed an improved technique, which we named the Confining-Box Method (CBM). On measuring the body lengths of a total of 72 live snakes, we found significant differences in the accuracy and precision of measurements among the squeeze-box method, the anesthesia method, and the CBM, as well as between vertebral line and the mid-ventral line measurements. Body lengths, as measured along the vertebral line and corrected for perspective errors, using the CBM, did not differ significantly from the standard lengths measured by hand using anesthesia. The squeeze-box method produced substantial negative bias and/or less precision in ventral measurements, especially for large snakes (> 1.2 m in snout-vent length). The differences between measurers were much smaller than among the different techniques. Compared to the anesthesia technique, CBM is a fast and simple method that is also safer for snakes. Unlike the traditional squeeze-box technique, CBM can record both dorsal and ventral body images of snakes simultaneously and enable researchers to measure accurate and/or precise total length (from dorsal images), tail length (from ventral images), and snout-vent length using images from a single photography session. CBM could therefore become a standard to improve measurement consistency, allowing improved data comparison in future studies.

Correction to: Development of 21 polymorphic microsatellite markers for the black-banded sea krait, Laticauda semifasciata (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species.

Unfortunately, one of the co-author's family name has been incorrectly published in the original online publication. The correct family name should be Tsai.

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, Laticauda semifasciata (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species.

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The cross-species amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.

Phylogenetic relationships of three representative sea krait species (genus Laticauda; elapidae; serpentes) based on 13 mitochondrial genes.

To investigate the phylogenetic relationships of the genus Laticauda to related higher taxa, we compared the sequences of four mitochondrial genes (12S rRNA, 16S rRNA, ND4, Cytb) from three Laticauda species (L. colubrina, L. laticaudata, and L. semifasciata) with those of 55 Asian and Australo-Melanesian elapid species. We also characterized the complete mitogenomes of the three Laticauda species and compared the sequences of 13 mitochondrial genes from Laticauda species with five terrestrial elapid and one viperid species to estimate phylogenetic relationships and divergence times. Our results showed that the genus Laticauda is paraphyletic to terrestrial elapids and diverged from the Asian elapids approximately 16.23 Mya. The mitogenomes of the three Laticauda species commonly encoded 13 proteins, 22 tRNAs, 12S and 16S rRNAs and two control regions and ranged from 17,170 and 17,450 bp in size. The L. colubrina mitogenome was more similar to that of L. laticaudata than that of L. semifasciata. The divergence time among the three Laticauda clades was estimated at 8-10 Mya, and a close phylogenetic relationship between L. colubrina and L. laticaudata was found. Our results contribute to our understanding of the evolutionary history of sea kraits.

Northward dispersal of sea kraits (Laticauda semifasciata) beyond their typical range.

Marine reptiles are declining globally, and recent climate change may be a contributing factor. The study of sea snakes collected beyond their typical distribution range provides valuable insight on how climate change affects marine reptile populations. Recently, we collected 12 Laticauda semifasciata (11 females, 1 male) from the waters around southern South Korea-an area located outside its typical distribution range (Japan, China including Taiwan, Philippines and Indonesia). We investigated the genetic origin of Korean specimens by analyzing mitochondrial cytochrome b gene (Cytb) sequences. Six individuals shared haplotypes with a group found in Taiwan-southern Ryukyu Islands, while the remaining six individuals shared haplotypes with a group encompassing the entire Ryukyu Archipelago. These results suggest L. semifasciata moved into Korean waters from the Taiwan-Ryukyu region via the Taiwan Warm Current and/or the Kuroshio Current, with extended survival facilitated by ocean warming. We highlight several contributing factors that increase the chances that L. semifasciata establishes new northern populations beyond the original distribution range.

Species Identification of Shed Snake Skins in Taiwan and Adjacent Islands.

Tein-Shun Tsai and Jean-Jay Mao (2017) Shed snake skins have many applications for humans and other animals, and can provide much useful information to a field survey. When properly prepared and identified, a shed snake skin can be used as an important voucher; the morphological descriptions of the shed skins may be critical for taxonomic research, as well as studies of snake ecology and conservation. However, few convenient/ expeditious methods or techniques to identify shed snake skins in specific areas have been developed. In this study, we collected and examined a total of 1,260 shed skin samples - including 322 samples from neonates/ juveniles and 938 from subadults/adults - from 53 snake species in Taiwan and adjacent islands, and developed the first guide to identify them. To the naked eye or from scanned images, the sheds of almost all species could be identified if most of the shed was collected. The key features that aided in identification included the patterns on the sheds and scale morphology. Ontogenetic differences and intraspecific variation in the patterns of sheds were evident in some snake species, and the proportion of young snakes with patterned shed skins was larger than that of adults. The retention of markings on the ventral side of the body (especially the ventral head) during sloughing was much lower than that on the dorsal side. We hope that this pioneering work will not only encourage other researchers to develop similar keys for their country, but also promote local schools, organizations, and citizen scientists to conduct snake inventories.