Ming Chung Tu

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.

Sea Snakes (Laticauda spp.) Require Fresh Drinking Water: Implication for the Distribution and Persistence of Populations

Dehydration and procurement of water are key problems for vertebrates that have secondarily invaded marine environments. Sea snakes and other marine reptiles are thought to remain in water balance without consuming freshwater, owing to the ability of extrarenal salt glands to excrete excess salts obtained either from prey or from drinking seawater directly. Contrary to this long‐standing dogma, we report that three species of sea snake actually dehydrate in marine environments. We investigated dehydration and drinking behaviors in three species of amphibious sea kraits (Laticauda spp.) representing a range of habits from semiterrestrial to very highly marine. Snakes that we dehydrated either in air or in seawater refused to drink seawater but drank freshwater or very dilute brackish water (10%–30% seawater) to remain in water balance. We further show that Laticauda spp. can dehydrate severely in the wild and are far more abundant at sites where there are sources of freshwater. A more global examination of all sea snakes demonstrates that species richness correlates positively with mean annual precipitation within the Indo–West Pacific tropical region. The dependence of Laticauda spp. on freshwater might explain the characteristically patchy distributions of these reptiles and is relevant to understanding patterns of extinctions and possible future responses to changes in precipitation related to global warming. In particular, metapopulation dynamics of the Laticauda group of sea snakes are expected to change in relation to projected reductions of tropical dry‐season precipitation.

Water exchange and permeability properties of the skin in three species of amphibious sea snakes (Laticauda spp.).

SUMMARY Evolutionary transitions between different environmental media such as air and water pose special problems with respect to skin permeability because of the dramatic changes in the driving gradients and nature of water exchange processes. Also, during the transitional periods prior to complete adaptation to a new medium, the skin is exposed to two very different sets of environmental conditions. Here, we report new data for transepidermal evaporative water loss (TEWL) and cutaneous resistance to evaporative water loss (Rs) of sea snakes that are transitional in the sense of being amphibious and semi-terrestrial. We investigated three species of sea kraits (Elapidae: Laticaudinae) that are common to Orchid Island (Lanyu), Taiwan. Generally, Rs of all three species is lower than that characteristic of terrestrial/xeric species of snakes measured in other taxa. Within Laticauda, Rs is significantly greater (TEWL lower) in the more terrestrial species and lowest (TEWL highest) in the more aquatic species. Previously reported losses of water from snakes kept in seawater exhibit a reversed trend, with lower rates of loss in the more aquatic species. These data suggest selection for adaptive traits with respect to increasing exposure to the marine environment. Thus, a countergradient of traits is reflected in decreased TEWL in aerial environments and decreased net water efflux in marine environments, acting simultaneously in the three species. The pattern for TEWL correlates with ultrastructural evidence for increased lipogenesis in the stratum corneum of the more terrestrial species. The skin surfaces of all three species are hydrophobic. Species differences in this property possibly explain the pattern for water efflux when these snakes are in seawater, which remains to be investigated.

Prey envenomation does not improve digestive performance in Taiwanese pit vipers (Trimeresurus gracilis and T. stejnegeri stejnegeri)

It has been a common belief that snake venom may help in the digestion of its prey, although direct examples and supporting evidence have not been sufficient. To address this, the present study examined whether preinjecting natural amounts of pit viper venom into experimental mice may accelerate their digestion by the snakes or gain energy benefit as compared to the control without the envenomation. Live adults of two Asian pit viper species Trimeresurus gracilis and T. stejnegeri stejnegeri, which inhabit the cold and warm environment respectively, were the subjects studied herein. A natural dose of 1.2 mg of each of the pit viper venom in phosphate-buffered saline (PBS) was injected into the mouse (about 10% of the snake mass) before it was being fed to the same species of vipers, while the pit vipers in control group were given mouse injected with sterile PBS. The snakes were kept at 14 degrees C or 24 degrees C, and parameters of gut passage time, costs of digestion, and/or digestive efficiency were measured. The results did not support the hypotheses that envenomation facilitates prey digestion. The venom in fact caused longer first defecation time and lower assimilation energy at 14 degrees C. Besides, the time to reach the oxygen consumption peak, and the first defecation time of T. s. stejnegeri were longer than that of T. gracilis.

Abundance of sea kraits correlates with precipitation

Recent studies have shown that sea kraits (Laticauda spp.) – amphibious sea snakes – dehydrate without a source of fresh water, drink only fresh water or very dilute brackish water, and have a spatial distribution of abundance that correlates with freshwater sites in Taiwan. The spatial distribution correlates with sites where there is a source of fresh water in addition to local precipitation. Here we report six years of longitudinal data on the abundance of sea kraits related to precipitation at sites where these snakes are normally abundant in the coastal waters of Lanyu (Orchid Island), Taiwan. The number of observed sea kraits varies from year-to-year and correlates positively with previous 6-mo cumulative rainfall, which serves as an inverse index of drought. Grouped data for snake counts indicate that mean abundance in wet years is nearly 3-fold greater than in dry years, and this difference is significant. These data corroborate previous findings and suggest that freshwater dependence influences the abundance or activity of sea kraits on both spatial and temporal scales. The increasing evidence for freshwater dependence in these and other marine species have important implications for the possible impact of climate change on sea snake distributions.

Reproductive Biology of the Sea Snake, Laticauda semifasciata, in Taiwan

Four species of sea snakes, Laticauda semifasciata, L. laticaudata, L. colubrina and Emydo- cephalus ijimae occur near Orchid Island on the southeastern coast of Taiwan. We describe here aspects of the reproductive biology of the most abundant species, L. semifasciata, based on a sample of 243 snakes collected between April 1986 and March 1987. Eight meristic and morphometric characters were examined for sexual dimorphism. The two best indicators were caudal width and the number of subcaudal scales. Minimum snout-vent lengths at sexual maturity for male and female snakes were 70 and 80 cm, respectively. The breeding season for female snakes was prolonged and peaked between August and November. There was no seasonal fluctuation in the weight or dimensions of the testes; sperm were found within the vas deferens throughout the year. Mating took place sometime before ovulation. Three to seven eggs (mean, 4) comprise the clutch. There are indications that some females may breed more than once per year. Eggs were laid in muddy water within tidal caves where they would not be inundated by sea water; moisture levels during incubation were maintained by fresh water dripping from condensation on the cave roof. The incubation period was 4 to 5 months.

Habitat Selection by Sea Kraits (Laticauda spp.) at Coastal Sites of Orchid Island, Taiwan

Three species of amphibious sea kraits (Laticauda spp.) spend variable time at sea and require fresh water for water balance. Both the rate of cutaneous evaporative water loss and the extent of terrestriality are known to differ among them. Laticauda semifasciata has the greatest rate of water loss and the least extent of terrestriality, whereas L. colubrina exhibits the reverse and L. laticaudata is intermediate. These sea kraits tend to be more abundant at places where there are sources of fresh water, but other factors also influence their distribution. To further clarify the habitat requirements, we investigated the abundance of each species of sea krait at six different habitats and the availability of each type of habitat on Orchid Island, Taiwan. The six habitats were high coral reef without fresh water (HR) and with fresh water (HRF); low coral reef without fresh water (LR) and with fresh water (LRF); sand or gravel coast, which has no coral reef, without fresh water (NR) and with fresh water (NRF). The extent of safety judged from the relative availability of retreat sites, from high to low, was HR, LR, and NR among these habitats. More than 75% of individuals counted for each species were found in HRF. We found no sea kraits in NRF and NR. The most available habitat was LR, but no L. laticaudata or L. semifasciata were found in this habitat. We found 3.3% and 16.7% of L. colubrina in LR and HR, respectively. For L. colubrina, the second abundant habitat was HR, whereas for L. laticaudata and L. semifasciata, the second abundant habitat was LRF. We conclude that both safety (availability of retreat sites) and fresh water are important to the habitat selection of sea kraits. Compared with other species, L. colubrina is characterized by a greater extent of terrestrial habit and possibly greater variety of access to sources of fresh water.

Bioenergetic modeling reveals that Chinese green tree vipers select postprandial temperatures in laboratory thermal gradients that maximize net energy intake

With bioenergetic modeling, we tested the hypothesis that reptiles maximize net energy gain by postprandial thermal selection. Previous studies have shown that Chinese green tree vipers (Trimeresurus s. stejnegeri) have postprandial thermophily (mean preferred temperature T(p) for males =27.8 degrees C) in a linear thigmothermal gradient when seclusion sites and water existed. With some published empirical models of digestion associated factors for this snake, we calculated the average rate (E(net)) and efficiency (K(net)) of net energy gain from possible combinations of meal size, activity level, and feeding frequency at each temperature. The simulations consistently revealed that E(net) maximizes at the T(p) of these snakes. Although the K(net) peaks at a lower temperature than E(net), the value of K(net) remains high (>=0.85 in ratio to maximum) at the peak temperature of E(net). This suggested that the demands of both E(net) and K(net) can be attained by postprandial thermal selection in this snake. In conclusion, the data support our prediction that postprandial thermal selection may maximize net energy gain.

Comparison of the osmoregulatory capabilities among three amphibious sea snakes (Laticauda spp.) in Taiwan

BackgroundThe three species of amphibious sea snakes (Laticauda semifasciata, L. laticaudata, and L. colubrina) in Taiwan were described as having different habitat affinities from terrestrial to marine. In this study, the osmoregulatory capabilities of three species were compared to test if their capabilities were associated with different habitat affinities.ResultsThe sea snakes were transferred from a terrestrial environment to freshwater (FW) or seawater (SW) for 1 week, and then, Na+/K+-ATPase (NKA) activities of the salt gland (sublingual glands) and kidneys, the water content of the muscles, the body fluid osmolality, and Na+, Cl−, and K+ concentrations were measured. Results showed that the body fluid osmolality, Na+ and Cl− levels, and muscle water content of most marine species, L. semifasciata, remained relatively constant, and the NKA activity of its salt gland was approximately threefold higher than those of L. laticaudata and L. colubrina. In both L. semifasciata and L. laticaudata, NKA activities of the salt glands were higher in SW than in FW; however, no significant change was found in L. colubrina (the most terrestrial species).ConclusionsThis study suggests that the NKA activity of the sublingual gland is associated with salt excretion, and the three species possess different osmoregulatory strategies which are associated with their habitat affinities.

Locomotor performance of three sympatric species of sea kraits (Laticauda spp.) from Orchid Island, Taiwan

BackgroundAssuming that locomotion has a strong influence on animals’ fitness, we hypothesized that better locomotor performance would be associated with the most frequently utilized habitat. Laticauda colubrina, Laticauda laticaudata, and Laticauda semifasciata have different amphibious habits and microhabitat preferences at Orchid Island, Taiwan. We investigated the morphology and locomotor performance of the three sympatric species of sea krait. The measurements of body size, tail area, and body shape were compared in our study. Data on crawling and swimming speeds were gathered to investigate locomotor performance in terrestrial and aquatic environments.ResultsWe found significant differences in the locomotor performances among the three species. L. colubrina was the most terrestrial species in habits and sprinted significantly faster than the others during terrestrial locomotion. On the other hand, L. semifasciata was the most aquatic species, and it swam significantly faster than the other two species. These results are consistent with our hypothesis that sea kraits move well in their respective primary environments. With respect to the highly aquatic L. semifasciata, its laterally compressed body form, large body size, and large area of compressed tail are considered to be beneficial to swimming in an aquatic environment.ConclusionsMore data are required to understand the superior terrestrial locomotion of L. colubrina, but this species may benefit from its more-cylindrical body form compared to L. semifasciata and from its greater muscle mass compared to L. laticaudata. L. laticaudata was intermediate in habits but exhibited the poorest performance in both swimming and terrestrial locomotion. The reasons for this remain unclear.