Zinc-dependent prothrombin activator from Rhabdophis tigrinus venom with extended substrate specificity.

Polymerase chain reaction (PCR)-based species identification methods could enhance snakebite surveillance, especially in regions where multiple venomous snake species with similar clinical presentations coexist. We developed species-specific PCR primers targeting mitochondrial cytochrome b gene sequences for five snake species commonly found in Japan: Elaphe climacophora, Elaphe quadrivirgata, Gloydius blomhoffii, Rhabdophis tigrinus, and Protobothrops flavoviridis. The PCR method successfully detected snake DNA from tissue samples with high specificity, showing minimal crossreactivity between species. Only P. flavoviridis primers showed crossreaction with E. climacophora DNA at the target size, whereas G. blomhoffii primers produced weak amplification of E. climacophora DNA at a different size than expected. Some nonspecific bands were detected with human DNA, but these were substantially weaker and different in size compared with specific amplification products. The method also successfully detected snake DNA from noninvasive samples, including oral swabs, samples from venom collection containers (where snakes had deposited saliva during venom extraction procedures), and bite sites on dead mice. The minimum detectable DNA concentrations ranged from 0.02 to 4.5 pg/µL across different species. Although our current method requires separate PCR reactions for each species and needs further validation with clinical specimens, it provides a foundation for molecular-based snake species identification. This approach could particularly benefit regions in which accurate species identification is crucial for appropriate antivenom selection, effective clinical management, and well-informed interventional research. Future studies should focus on validating this method with clinical specimens and developing multiplex PCR systems for more practical surveillance applications.

Yamakagashi (Rhabdophis tigrinus) is a widely distributed snake species in Japan. Yamakagashi causes venom-induced consumption coagulopathy (VICC) when the amount of infused venom is high, and bites can be fatal if antivenom treatment is delayed. However, yamakagashi antivenom is an unapproved treatment, and its storage capacity is limited, preventing its prompt administration. Therefore, we investigated the application of commercially available drugs, namely tranexamic acid and antithrombin III, in the treatment of VICC caused by yamakagashi venom in a rat model. Furthermore, we investigated the combination of each drug with recombinant thrombomodulin α. Administration of tranexamic acid or antithrombin III alone failed to extend rat survival or correct changes in blood coagulation markers, such as prothrombin time, fibrinogen concentrations, and D-dimer levels, in yamakagashi venom-treated rats. However, combined administration of recombinant thrombomodulin α and tranexamic acid extended rat survival and partially restored blood coagulation markers. Therefore, the combination of recombinant thrombomodulin α and tranexamic acid might represent a useful therapeutic regimen for yamakagashi venom exposure.

Introduction Among medically important non-front-fanged colubroid snakes in Asia, the bites from Rhabdophis subminiatus (red-necked keelback) and Rhabdophis tigrinus (the yamakagashi) can cause severe systemic hematotoxic effects. Clinical data from these envenomations are limited. Rhabdophis siamensis (Siamese red-necked keelback), which was previously classified as a subspecies of Rhabdophis subministus, inhabits Thailand. This study aimed to describe the clinical effects of Rhadophis species envenomation in Thailand, including data on treatments and outcomes. Methods This 15-year retrospective study examined data from the Ramathibodi Poison Center from 2008 to 2022. The objectives were to summarize the clinical characteristics, treatments, and outcomes following Rhabdophis siamensis bites. Results Fifty-three patients (50.9% males, median age 37 years, range 1–79 years) were included. Fang marks were observed in 84.9%. Thirteen patients experienced local effects, including wound swelling (eight patients) and bleeding at the bite site (eight patients). Coagulopathy, thrombocytopenia, and systemic bleeding occurred in nine, five, and three patients, respectively. All patients with thrombocytopenia exhibited coagulopathy. Wound swelling and bleeding at bite sites were present in patients with systemic effects (coagulopathy), more frequently than in those who did not have them. Non-specific hematotoxic antivenom was administered to nine patients, and three patients received blood components. The median length of hospital stay was 1 day (range 1–14). No deaths were reported. Discussion Local and systemic effects following Rhabdophis siamensis bites were not frequently observed. Systemic bleeding rarely occurred. The antivenoms available in Thailand are not produced to neutralize the venom Rhadophis species snakes. We believe that they are unlikely to be effective and should not be used for this type of snakebite. Conclusions Local and systemic hematotoxic effects were observed in some patients. Systemic bleeding occurred in a few patients. No fatalities were observed.

Snake envenomation, a neglected tropical disease, presents complex and species-dependent systemic pathophysiology. Here, we conduct an integrated proteomic and in vivo toxicological assessment of venoms from ten medically significant Chinese snakes. Our proteomic analysis delineated the venom composition of the ten snake species. Viperidae venoms were rich in snake venom metalloproteinases and phospholipase A2, consistent with their hemorrhagic and myotoxic effects, while elapidae venoms were dominated by three-finger toxin and phospholipase A2. Furthermore, we identified that the colubrid Rhabdophis tigrinus possesses a unique venom arsenal primarily composed of three-finger toxin (33.1%) and phospholipase A2 (30.8%). In vivo toxicity assessment in mice via four administration routes (subcutaneous, intramuscular, intraperitoneal, intravenous) demonstrated that venom lethality was highly route-dependent, with elapid venoms (particularly Bungarus multicinctus, intravenous median lethal dose = 0.16mg/kg) being an order of magnitude more potent than viperid venoms. Notably, the injection route critically modulated the toxicological manifestations; for instance, intramuscular injection accentuated local tissue damage, while intravenous injection exacerbated coagulopathy. This multi-omics study systematically links the specific toxin arsenals of these ten venoms to distinct pathological phenotypes observed via different clinically relevant routes, providing a valuable resource for understanding venom toxicity mechanisms and guiding clinical management of snakebite.

IntroductionThe gut microbiota is an emerging frontier in animal research, and researchers are increasingly transparent about its importance to animal health. Reptiles, particularly snakes, have not received the same attention given to other vertebrates, and the composition of their wild gut microbiome remains understudied.MethodsIn this study, the HiSeq high-throughput sequencing platform was used to sequence and analyze the 16S rRNA V4 region of the gut microbiota of three species (Gonyosoma coeruleum, Rhabdophis pentasupralabralis, Rhabdophis tigrinus).ResultsThis study investigated alpha diversity analysis and showed that the gut microbiota richness of RP was significantly higher than that of the other two snakes. The dominant genus of Gonyosoma coeruleum (GC) and Rhabdophis tigrinus (RT) is Cetobacterium, while Enterobacteriaceae; g_uncultured is the dominant genus of Rhabdophis pentasupralabralis (RP). Tree clustering based on Bray-Curtis distances and Jaccard similarity coefficients indicated that the gut microbiota composition of RP and RT was more similar. The unique diet of RP promotes a diverse, competitive gut microbiota, while GC and RT displayed more stable networks linked to shared dietary habits. The functional heat map showed that the predicted functions of the gut microbes of the three snake species were different. These findings suggest that dietary preferences exert a stronger influence on gut microbial composition and function than host genetic background, and distantly related species with similar diets exhibit convergent gut microbiota characteristics.

Abstract Colour pattern variations in animals range from a small number of discrete morphs to continuous variations. However, the possibility that the degree itself is shaped by bias produced by limited observations cannot be excluded. Through a community science approach, also known as citizen science, we refined the definitions of the colour pattern morphs of the Japanese natricine snake, Rhabdophis tigrinus. Despite the limited number of geographical observations, R. tigrinus has been classified into six distinct colour pattern morphs over the past decades: three region-specific morphs, two hypopigmented morphs, and one hyperpigmented morph. However, our newly collected community science images of R. tigrinus revealed that 123 colour pattern combinations of R. tigrinus have emerged across the Japanese Archipelago. The community science images with detailed geographical information also provided insights into the anti-predatory functions of R. tigrinus colour patterns through an association between blotch size and habitat temperature via the flicker-fusion effect. Our results highlight the advantages of using community science data in overcoming observational biases and investigating intraspecific colour pattern variations across a wide geographical range.

Studies examining the oxygen dependency of embryonic survival, growth, and differentiation have been conducted for decades in a diverse array of animal taxa but including only one oviparous snake, the viperine water snake Natrix maura. Here, we describe a study incubating eggs of the tiger keelback Rhabdophis tigrinus lateralis (Colubridae) under four oxygen conditions, hypoxia (7% and 11% O2), normoxia (21% O2), and hyperoxia (31% O2), for different lengths or at different stages of incubation. The length of hypoxic exposure is important in affecting embryonic development in R. t. lateralis, with prolonged hypoxic exposure retarding embryonic growth and differentiation, increasing embryonic mortality and deformity, reducing hatchling size and mass, and altering hatchling body shape relative to normoxic controls. Embryonic tiger keelbacks are most susceptible to hypoxia late in development, as revealed by the fact that a 5-day exposure of eggs to hypoxia of 7% O2 reduced embryo mass and hatchling mass if it occurred at late stages of incubation. Hyperoxia of 31% O2 did not enhance development of R. t. lateralis embryos, only affecting hatchling head width, which slightly differed between hyperoxic hatchlings and their normoxic siblings. This study demonstrates the importance of avoiding hypoxic exposure at late stages of embryonic development in snakes.

Introduction of a snake trematode of the genus Ochetosoma in eastern Japan

Leucism, in which pigmentation is lost over part or the entire body of an animal, has a range of possible genetic causes. Here, we report leucism in an individual tiger keelback (Rhabdophis tigrinus) found on Jeung Island, Shinan-gun, Jeollanam-do, South Korea, during a survey of the distribution of reptiles in the area. The individual was observed sunbathing in the bushes next to a pond. This individual exhibited ecdysis, thus it considered that have normal feeding activity. Our report represents the first observation of leucism in R. tigrinus, and thus, further analysis is needed of this phenotype to more clearly understand its impact on the species and its natural history.

症例は16歳，男性．山でヘビを捕まえた際に右手背に咬傷を負った．創部の腫脹は増悪し，止血が得られないため受傷2時間後に当院を受診した．来院時は2箇所の牙痕から出血を認め，圧迫を行っても止血は困難であった．血液検査で著明な線溶系の亢進を認めたが，血小板数の低下は認めなかった．播種性血管内凝固症候群（DIC）をきたしていたため，入院加療の方針とした．トロンボモジュリン製剤を投与したが，受傷翌日の血液検査でDICは増悪した．著明な線溶亢進を伴うDICであることから，ヤマカガシ咬傷と診断した．対症療法では不充分であると考え，臨床研究に参加し抗毒素血清を使用した．抗毒素血清投与後直ちに，創部の止血を得た．抗毒素血清投与翌日には凝固系も改善傾向を認め，投与後3日でDICを離脱，投与後5日で退院となった．急速に進行する重症DICをきたしたヤマカガシ咬傷は稀であり，若干の文献的考察を加えて報告する．

Exotic (non-native) snakebite envenomation in Japan: A review of the literature between 2000 and 2022

The snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads and stores them in the nuchal glands as defensive toxins. It has previously been shown that there are individual differences in the total quantity of BDs stored in the nuchal glands of adult R. tigrinus and that BD quantities and profiles of R. tigrinus exhibit geographic variation. However, no previous study has examined the total quantity of BDs as a percentage of body mass (relative BD quantity) and the concentration of BDs in the nuchal gland fluid (BD gland concentration). In addition, intrinsic factors that are associated with relative BD quantity and BD concentration have not been examined within a single population. We collected 158 adult snakes from an area of central Japan from May to October and analyzed their BD quantities by UV analysis. We assessed individual differences in BD quantity, relative BD quantity and BD gland concentration. We found that 1) in approximately 60% of the 158 individuals, the BD gland concentration was greater than 50%; 2) body length and body condition are positively correlated with relative BD quantity and BD gland concentration; 3) even in a single population, individual differences of BD quantity are large, and are greater in females than in males; and 4) relative BD quantity and BD gland concentration of females during the gestation season are lower than those during the non-gestation season.

Long-term field studies have provided evidence of worldwide declines in vertebrate populations, including amphibians and reptiles. However, studies investigating the decline in snake populations are scarce. As obligate predators, snakes play important roles in local ecosystems and are in turn affected by their food resources. In the present study we investigated the seasonal abundance of snakes and their food habits in a rice paddy field and compared the results of the present study with those of a previous study that was conducted at the same site approximately 30 years ago. Our results showed that Elaphe quadrivirgata, a dietary generalist snake that had been one of the dominant species at the site, has drastically declined and that Rhabdophis tigrinus and Gloydius blomhoffii have remained as dominant species. Furthermore, in the present study, most of the stomach contents of R. tigrinus consisted of the Japanese tree frog, Dryophytes japonicus, whereas three frog species, D. japonicus, Rana japonica, and Pelophylax porosus porosus, had been the main prey of R. tigrinus 30 years ago. In addition, fewer individuals of R. tigrinus and G. blomhoffii contained food in their stomachs than 30 years ago. Our findings imply that a change in prey availability may have affected the guild structure of snakes at this study site, although further investigation is necessary to verify the reasons for the decline of the dietary generalist, E. quadrivirgata.

The paper presents the case of discovery of 60 eggs of the tiger keelback (Rhabdophis tigrinus) in the vicinity of the Dunay settlement (Primorsky Krai, Russian Federation) under a wooden railway sleeper on July 27, 2022. The authors suggest that it was a collective clutch of eggs from three females. Incubation in artificial conditions, from the discovery of this collective clutch to the release of juveniles from the eggs, lasted 17–20 days. Given the synchronicity of the hatching of young snakes, it is assumed that the eggs were laid by different females almost simultaneously. The youth emerging from the eggs had a mass of 1.60–4.78 g, a body length of 152.4–211.3 mm and a tail of 28.4–48.1 mm.

Rhabdophis tigrinus (R. tigrinus) is a common colubrid snake that possesses a series of paired sac-like nuchal glands behind the head. When pressure is applied to the nuchal area, the thin skin over the nuchal glands can rupture and release secretions. In Japan, 19 cases of ophthalmia caused by the nuchal gland secretion of R. tigrinus have been reported. However, only one case has been documented in an English report. A 72-year old woman was sprayed by the nuchal gland fluid of R. tigrinus in her right eye. She presented with symptoms of eye pain and blurred vision. A slit-lamp examination revealed diffuse superficial keratitis, corneal stromal edema with Descemet membrane folds, and conjunctival injection. The best-corrected visual acuity (BCVA) of her right eye was 0.6. She was prescribed 0.5% moxifloxacin and 0.1% fluorometholone eye drops four times a day, and the symptoms resolved without sequelae within 5 days. The BCVA in the right eye improved to 1.0. In previous reports, ophthalmic examinations revealed conjunctivitis, keratitis, and corneal edema with Descemet membrane folds. Topical antibiotics and corticosteroid were prescribed in most cases, and eyes healed within 5-7 days without any sequelae. While corneal edema may resolve spontaneously in a few days when inhibition of the toxin has ceased, the use of topical steroids is recommended, as it can increase the activity of Na/K pumps that remained functional, thereby accelerating recovery. In fact, our patient used a topical steroid and recovered without sequelae.

Yamakagashi (Rhabdophis tigrinus) inhabits Japan widely, and incidents involving its bites occur every year. Its bite causes disseminated intravascular coagulation when the amount of infused venom is high, and it can be fatal if treatment with Yamakagashi antivenom is delayed. Although Yamakagashi antivenom is used for treating Yamakagashi bites, it is an unapproved drug and its capacity for storage is limited. Hence, it is difficult to administer to patients promptly. As a therapeutic agent for this bite, we investigated the application of recombinant thrombomodulin alpha, a commercially available disseminated intravascular coagulation therapeutic agent. Its therapeutic effect on Yamakagashi venom was confirmed in a coagulation system of human plasma using in vitro Yamakagashi venom as well as a rat experimental model of disseminated intravascular coagulation using in vivo Yamakagashi venom. The administration of recombinant thrombomodulin alpha induced an effect that prolonged the blood coagulation time of Yamakagashi venom in vitro, and the drug was administered in vivo within 0.5 h after the administration of Yamakagashi venom to save rats. Blood coagulation markers such as platelet count, prothrombin time, fibrinogen concentration, and D-dimer levels recovered to normal values in rats. Therefore, recombinant thrombomodulin alpha may be used as a therapeutic agent for Yamakagashi bites.

BackgroundYamakagashi venom is a prothrombin activator, leading to disseminated intravascular coagulation. We report a fatal case of severe coagulopathy from head trauma assumed to be caused by a yamakagashi bite.Case presentationAn 80-year-old man fell and developed systemic tonic–clonic convulsions. Head computed tomography revealed brain contusion and acute subdural hematoma. Physical examination revealed two bite marks with persistent bleeding on the right lower leg. The patient stated that he had been bitten by some creature 3 days prior, but the bite was left untreated. Laboratory tests showed fibrinogen levels below the detection limit. Although eighteen units of fresh frozen plasma were administered for coagulopathy, fibrinogen levels did not improve. He died about 18 h after a head injury.ConclusionIn this case of a yamakagashi bite with active bleeding due to trauma, early administration of yamakagashi antivenom should be considered to control coagulopathy.

The Red Keelback (Pseudagkistrodon rudis Boulenger, 1906) is widely distributed in the southern of China. The complete mitochondrial genome (mitogenome) of P. rudis was determined for the first time by using next-generation sequencing. The size of assembled mitogenome for P. rudis was 19,150 bp, which included 13 protein coding genes (PCGs), 22 tRNAs, two rRNAs and two control regions (d -loop1 and d -loop2). The Bayesian tree showed that P. rudis and Rhabdophis tigrinus have a closed relationship. These results can provide data for phylogeny and molecular classification of the genus.

The Japanese natricine snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads in the nuchal glands as defensive toxins. A previous study showed that R. tigrinus in captivity converts dietary BDs when it sequesters them. However, it is unknown whether the dietary BDs are actually converted and the modified products accumulated under natural conditions. It is also unknown to what extent the BD profile of ingested toads is reflected in that of the snake. We collected 123 snakes from throughout Japan, analyzed their BD profiles by liquid chromatography/mass spectrometry, and identified 15 BDs from R. tigrinus by nuclear magnetic resonance analyses. We also compared their BD profiles using hierarchical cluster analysis (HCA). HCA exhibited two main clusters associated with their collection locations: eastern and western regions of the Japanese main islands. These results, coupled with previous findings on the BDs of Japanese toads, suggest that 1) R. tigrinus converts toad-derived BDs into other compounds under natural conditions; 2) there are both universal and regionally-specific conversions of dietary BDs by R. tigrinus; and 3) geographic variation in toad BD profiles is partially reflected in the variation of snake BD profiles.