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

[[alternative]]Feeding and thermal selection of Chinese green tree viper, Trimeresurus s. stejnegeri

[[abstract]]The Chinese green tree vipers (Trimeresurus s. stejnegeri) are nocturnal and ambush prey in arboreal habitats. They feed mainly on amphibians in field, but rodents, shrews, lizards, geckos, and birds were also consumed. Several topics about this species have been studied, but no studies have focused on the behavior and physiology of feeding. In this dissertation, all subjects were associated with the feeding of this snake, and the relations of these subjects to arboreality, nocturnality, and ambush-feeding habitat were concerned. In Chapter 1, I found Trimeresurus s. stejnegeri held on the prey after capturing it, which should be adapted to the less aggressive prey (i.e., frog), lower prey size, and avoidance the inconvenience for tracing the released prey from a twig. I fed the snake with frog (Rana limnocharis) and mouse (Mus musculus) in the laboratory (22 ± 1 oC), under three sensory deprivation conditions (intact cues, visual cue blocked, visual and infrared thermal cues blocked). The feeding behavior was recorded by videotape-recording method or naked eyes. The snake withdrawal and pulled up the prey from the ground following catching it. The prey was hanged and the body tilted, like a lever with the snake-biting site as the fulcrum. Mostly, the snake gradually moved its jaw to the upper end of the prey body and ingested it. That is, the capturing site, which should be decided before attacking the prey, determined the ingestion direction The snake ingested mouse mainly from head, as in other snakes; but the pattern was not dominant in feeding frog. More proportion of frogs was stroked on the posterior end than that of mice. The ratio of prey ingested from anterior to posterior side was 55 : 19 and 29 : 22 for mouse and frog, respectively, under intact sensory condition. When the visual and infrared cues were blocked, the ingested direction ratio, 14 : 4 and 14 : 9 for mouse and frog respectively, did not shift significantly from above. The snakes spent more time feeding on frogs than on mice. On feeding mice, the spending time to launch successful strikes (T1) was lowest at intact cues groups and significantly increased when the visual and/or thermal cues were blocked. On feeding frogs, however, T1 did not differ significantly among the sensory deprivation conditions. Visual and thermosensory cues seem to be less important on feeding frogs (but not on feeding mice) for T. s. stejnegeri. Besides, on feeding frogs but not mice, the time from capturing prey to start moving the jaws (T2) was significantly longer when ingesting prey from the anterior end. On feeding mice but not frogs, the time from moving the jaws to start flicking the tongue following the ingestion (T3) was longer when feeding prey from the posterior end. The feeding time (T2, T3) decreased when the sensory cues were blocked. In Chapter 2 and 3, I measured the temperature selection of adult males and females in a linear thigmothermal gradient and checked the degree of instrumental interferences. I conducted three experiments to study the possible effect of thermocouples, the influence of seclusion, and the presence of water on the temperature-selecting behavior of the snake. Thermocouples might change a snake’s preferred temperature (Tp) by causing it to lift its prehensile tail from the gradient floor or affecting its movement. With the videotape-recording method, the snake presented postprandial thermopily only when seclusion sites and water were provided in the gradient. In the absence of seclusion sites and water, the fasting and postprandial body temperature (Tb) of males was 23.0 ± 1.2 oC and 24.7 ± 1.2 oC, respectively. With seclusion sites and water, the fasting and postprandial Tb of males was 22.5 ± 1.0 oC (the set point Tset = 20.3 ~ 24.3 oC) and 27.8 ± 0.6 oC (Tset = 26.5 ~ 28.8 oC), respectively. The fasting and postprandial Tb of non-reproductive females (N = 16) was 21.2 ± 1.4 oC (Tset = 20.6 ~ 23.8 oC) and 24.8 ± 1.5 oC (Tset = 25.0 ~ 26.3 oC), respectively. Preferred temperature of females was higher after feeding or during pregnancy. Preferred temperature of pregnant snakes (N = 5) was 27.4 ± 2.0 oC. In Chapter 4, I investigated the combined effect of meal size (below 30%) and temperature (15~35 oC) on the aerobic metabolism, digestive efficiency, and digestive rate in this study. As other sit-and-wait foraging species, the snake had lower resting metabolic rate (0.033 ± 0.002 ml O2/g/hr at 25 oC). But it showed less difference at 20 and 25 oC (Q10 = 1.58). Respiratory quotient significantly increased at the anterior part of digestion period except at 15 oC. Specific dynamic action (SDA), peak VO2 and scope of peak VO2 increased with meal size, while temperature had little effect on SDA and SDA coefficient. Similar with other crotalids, the SDA coefficient was lower (15.8 ± 0.6%) in this study. With regression analysis, I found SDA in T. s. stejnegeri responded latterly and less sharply than other sit-and-wait feeding snakes. The metabolizable energy coefficient (= 0.66~0.89) was lowest at 15 oC and tended to peak at the postprandial Tset of the snake. In addition, I investigated the digestive rate from three aspects, included gut passage time (gut movement), gastric digesting time (Tbone; chemical digestion), and the timing of SDA (digestive metabolism). The final defecation time (PTe) and Tbone, but not the first defecation time, increased with food ration. PTe was less than two weeks at above 25 oC, but was larger than one month at 15 oC. The time to peak VO2, Tbone, and duration of SDA represented about 20%, 50%, and 80% of total digestion process (PTe), respectively. In Chapter 5, I tested whether temperature selection makes maximal net energy gain in Chinese green tree viper. I used multiple linear regressions, which expressing the effects of snake mass, mouse mass as well as temperature on digestion-associated variables, to simulate the monthly maximal net energy gain (Enet) under certain feeding frequencies and activity levels. With the energy budget model, I found some dominant trends. Enet peaked at lower temperature when snakes had less food ration. At high feeding frequency, selecting high body temperature, which closes to the postprandial Tp of the snake, could get higher Enet. When feeding frequencies lower down, Enet could be negative at 10% meal size, and the superiority or necessity to select high postprandial temperature disappear because of broad range of B80 at 20 ~ 30% meal size. Owing to the potential low feeding frequency in wild, T. s. stejnegeri may not get relative maximal Enet by postprandial behaviors. I have suggested potential reasons about why the snake selects high postprandial temperatures still under less energetic benefits. In conclusion, many innovated ideas and investigations have been mentioned in this dissertation. Several characters (nocturnality, arboreal habitats and ambush-feeding behavior) of Chinese green tree vipers may have important influences on the results. Whether these attributes occurred in wild as well as in other arboreal and/or ambush-feeding snakes is worth to study in future researches.

[[alternative]]A study on the reproductive cycle of Chinese green tree viper, Trimeresurus s. stejnegeri, in northern Taiwan

[[abstract]]I studied the reproductive cycle of Chinese green tree vipers, Trimeresurus s. stejnegeri in northern Taiwan by examining seasonal changes in the morphology and histology of their reproductive organ and the hormonal activity. From March 1996 to August 1997, I collected a total of 288 snakes with an average of 7 female and 9 male snakes per month. Based on the size of follicles and the presence of corpora lutea, I classified mature female snakes into 7 stages. The snakes in the first stage had only primary follicles of which the maximal length is less than 5 mm. The snakes that belonged to stage 2 to stage 6 had secondary follicles with the length ranged between 5- 10 mm, 10- 15 mm, 15- 20 mm, 20- 25 mm and above 25 mm respectively. The last stage snakes had both primary follicles and corpora lutea. I found estrogen level was highest in the snakes of stage 5 while that in stage 6 had the peak concentration of progesterone in serum. The numbers of reproductive females which belong to stage 2 to 7 versus that of nonreproductive females, classified in first stage, was close to unity. This suggested that female T. s. stejnegeri reproduced biennially rather than annually. The mass of fat body correlated negatively with the process from first stage to last stage. Fat reserves may become the limiting factor of reproductive frequency. Female Chinese green tree vipers had seasonal cyclic changes in morphology and histology of reproducitve organs as well as hormonal activities. Their timing of vitellogenesis belongs to postnuptial type (type II) vitellogenesis, which is common in the viperids.According to the morphological difference of seminiferous epithelium, I classified male Chinese green tree vipers into five stages, which were early recrudescence, late recrudescence, spermiogenesis, early regression and late regression. Early recrudenscence stage started from March and could last till May. Spermiogenesis stage occurred mainly in summer and autum. While in winter, I found most male snakes belonged to regression stages. Other parameters, such as diameter of seminiferous tubule, renal sexual segment and fat body mass also revealed seasonal cylcic changes. However the peak of each cycling may not be the same. For example, the peak of renal sexual segment diameter, which correlate well with copulation was later than that of seminiferous tubule diameter. The same as many other viperids, male Chinese green tree vipers had postnuptial spermaotgenesis cycle.

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

Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries.

Phospholipases A2 (PLA2s) were purified from the Trimeresurus stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition to the six previously documented PLA2 isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular masses and partial sequences of most of the purified PLA2s matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2 with Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius besides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species

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