Microbial communities are thermally more sensitive in warm-climate lizards compared with their cold-climate counterparts.

Environmental temperature affects the composition, structure, and function of the gut microbial communities in host animals. To elucidate the role of gut microbiota in thermal adaptation, we designed a 2 species × 3 temperatures experiment, whereby we acclimated adult males of two agamid lizard species (warm-climate Leiolepis reevesii and cold-climate Phrynocephalus przewalskii) to 20, 28, and 36°C for 2 weeks and then collected their fecal and small-intestinal samples to analyze and compare the microbiota using 16S rRNA gene amplicon sequencing technology. The fecal microbiota displayed more pronounced interspecific differences in microbial community than the small-intestinal microbiota in the two species occurring in thermally different regions. The response of fecal and small-intestinal microbiota to temperature increase or decrease differed between the two species, with more bacterial taxa affected by acclimation temperature in L. reevesii than in P. przewalskii. Both species, the warm-climate species in particular, could cope with temperature change by adjusting the relative abundance of functional categories associated with metabolism and environmental information processing. Functional genes associated with carbohydrate metabolism were enhanced in P. przewalskii, suggesting the contribution of the fecal microbiota to cold-climate adaptation in P. przewalskii. Taken together, our results validate the two hypotheses tested, of which one suggests that the gut microbiota should help lizards adapt to thermal environments in which they live, and the other suggests that microbial communities should be thermally more sensitive in warm-climate lizards than in cold-climate lizards.

Lessons from the diet: Captivity and sex shape the gut microbiota in an oviparous lizard (Calotes versicolor).

Studies have indicated that the abundance and community structure of gut microbiota are altered by diet. In this study, next-generation sequencing of the 16S rRNA gene amplicon was performed to evaluate variations in the gut microbiota of wild and captive individuals of both sexes of Calotes versicolor. The results showed that there was a significant sex difference in microbial community structure for wild C. versicolor, Bacteroide was the dominant genus in wild females (WF), whereas Ochrobactrum was the dominant genus in wild males (WM). Acinetobacter and Hymenobacter were the dominant genera in WF, while Clostridium was the dominant genus in captive females (CF). The results indicated that differences in diet between wild and captive C. versicolor also resulted in variations in gut microbiota. Thus, it was not surprising that captivity and sex shape the gut microbiota in C. versicolor. In summary, the fundamental information presented about the gut microbiota of both sexes of wild (and captive females) C. versicolor, indicates that the artificial environments are not suitable for the wild C. versicolor.

Incubating green turtle (Chelonia mydas) eggs at constant temperatures: Hatching success, hatchling morphology and post-hatch growth.

Past studies applying constant-temperature incubation of eggs have involved all species of sea turtles, but rarely can we find a single one incubating eggs at three or more temperatures. Here, we incubated green turtle (Chelonia mydas) eggs from Ganquan Island, South China Sea, at five constant temperatures (26, 28, 30, 32 and 34 °C) to determine hatching success, incubation length and hatchling phenotype at each test temperature and temperatures optimal for embryonic development. Temperature affected hatching success, incubation length and all seven examined hatchlings traits, and clutch origin affected three (head length, fore-flipper length and hind-flipper length) of the seven. Hatching success was lowest at 34 °C and none of hatchlings hatched at this temperature was normal and survived over one week. The rate of embryonic development and the rate of post-hatch growth both were lowest at 26 °C. Given that low survival and growth rates can translate into reduced individual fitness, we conclude that both 26 °C and 34 °C are unsuitable for incubation of C. mydas eggs. Post-hatch growth was fastest in hatchlings incubated at 30 °C, and eggs of C. mydas incubated at temperatures around 30 °C are more likely to produce mixed sexes. Accordingly, we conclude that temperatures within the range from 28 °C to 32 °C are generally optimal for embryonic development of C. mydas.

Venom of the Annulated Sea Snake Hydrophis cyanocinctus: A Biochemically Simple but Genetically Complex Weapon.

Given that the venom system in sea snakes has a role in enhancing their secondary adaption to the marine environment, it follows that elucidating the diversity and function of venom toxins will help to understand the adaptive radiation of sea snakes. We performed proteomic and de novo NGS analyses to explore the diversity of venom toxins in the annulated sea snake (Hydrophis cyanocinctus) and estimated the adaptive molecular evolution of the toxin-coding unigenes and the toxicity of the major components. We found three-finger toxins (3-FTxs), phospholipase A2 (PLA2) and cysteine-rich secretory protein (CRISP) in the venom proteome and 59 toxin-coding unigenes belonging to 24 protein families in the venom-gland transcriptome; 3-FTx and PLA2 were the most abundant families. Nearly half of the toxin-coding unigenes had undergone positive selection. The short- (i.p. 0.09 µg/g) and long-chain neurotoxin (i.p. 0.14 µg/g) presented fairly high toxicity, whereas both basic and acidic PLA2s expressed low toxicity. The toxicity of H. cyanocinctus venom was largely determined by the 3-FTxs. Our data show the venom is used by H. cyanocinctus as a biochemically simple but genetically complex weapon and venom evolution in H. cyanocinctus is presumably driven by natural selection to deal with fast-moving prey and enemies in the marine environment.

Venom-gland transcriptomic, venomic, and antivenomic profiles of the spine-bellied sea snake (Hydrophis curtus) from the South China Sea.

BACKGROUND: A comprehensive evaluation of the -omic profiles of venom is important for understanding the potential function and evolution of snake venom. Here, we conducted an integrated multi-omics-analysis to unveil the venom-transcriptomic and venomic profiles in a same group of spine-bellied sea snakes (Hydrophis curtus) from the South China Sea, where the snake is a widespread species and might generate regionally-specific venom potentially harmful to human activities. The capacity of two heterologous antivenoms to immunocapture the H. curtus venom was determined for an in-depth evaluation of their rationality in treatment of H. curtus envenomation. In addition, a phylogenetic analysis by maximum likelihood was used to detect the adaptive molecular evolution of full-length toxin-coding unigenes. RESULTS: A total of 90,909,384 pairs of clean reads were generated via Illumina sequencing from a pooled cDNA library of six specimens, and yielding 148,121 unigenes through de novo assembly. Sequence similarity searching harvested 63,845 valid annotations, including 63,789 non-toxin-coding and 56 toxin-coding unigenes belonging to 22 protein families. Three protein families, three-finger toxins (3-FTx), phospholipase A2 (PLA2), and cysteine-rich secretory protein, were detected in the venom proteome. 3-FTx (27.15% in the transcriptome/41.94% in the proteome) and PLA2 (59.71%/49.36%) were identified as the most abundant families in the venom-gland transcriptome and venom proteome. In addition, 24 unigenes from 11 protein families were shown to have experienced positive selection in their evolutionary history, whereas four were relatively conserved throughout evolution. Commercial Naja atra antivenom exhibited a stronger capacity than Bungarus multicinctus antivenom to immunocapture H. curtus venom components, especially short neurotoxins, with the capacity of both antivenoms to immunocapture short neurotoxins being weaker than that for PLA2s. CONCLUSIONS: Our study clarified the venom-gland transcriptomic and venomic profiles along with the within-group divergence of a H. curtus population from the South China Sea. Adaptive evolution of most venom components driven by natural selection appeared to occur rapidly during evolutionary history. Notably, the utility of commercial N. atra and B. multicinctus antivenoms against H. curtus toxins was not comprehensive; thus, the development of species-specific antivenom is urgently needed.

Characterization of the complete mitochondrial genome of the many-lined sun skink (Eutropis multifasciata) and comparison with other Scincomorpha species.

Characterizating the complete mitochondrial genome (mitogenome) of an organism allows detailed genomic studies in systematics and evolution. The present study decodes the mitogenome (17,062 bp) of the many-lined sun skink, Eutropis multifasciata, using next-generation sequencing. To compare the diversity of mitogenomic structure and investigate intraspecific evolutionary relationships among the Asian Scincomorpha, the mitogenomes of 46 other species were examined concurrently. Within the group, the size of mitogenomes varied predominantly in the length at their control regions. The Ka/Ks ratios of 12 protein codon genes (PCGs) were lower than 1.00, demonstrating that they were under relaxed or moderate purifying selection. However, the ND5 had a Ka/Ks ratio >1, and was considered to be under positive selection. Currently there are two superfamilies in Scincomorpha (i.e. Scincoidea and Lacertoidea), but phylogenetic analysis using Bayesian Inference and Maximum-Likelihood Estimations produced phylogenetic trees with three clades in Scincomorpha ((Scincoidea + Lacertoidea (part)) + Gymnophthalmidae)).

Dietary Correlates of Oral and Gut Microbiota in the Water Monitor Lizard, Varanus salvator (Laurenti, 1768).

Numerous studies have demonstrated that food shapes the structure and composition of the host's oral and gut microbiota. The disorder of oral and gut microbiota may trigger various host diseases. Here, we collected oral and gut samples from wild water monitor lizards (Varanus salvator) and their captive conspecifics fed with bullfrogs, eggs, and depilated chicken, aiming to examine dietary correlates of oral and gut microbiota. We used the 16S rRNA gene sequencing technology to analyze the composition of the microbiota. Proteobacteria and Bacteroidota were the dominant phyla in the oral microbiota, and so were in the gut microbiota. The alpha diversity of microbiota was significantly higher in the gut than in the oral cavity, and the alpha diversity of oral microbiota was higher in captive lizards than in wild conspecifics. Comparing the relative abundance of oral and gut bacteria and their gene functions, differences among different animal groups presumably resulted from human contact in artificial breeding environments and complex food processing. Differences in gene function might be related to the absolute number and/or the taxonomic abundance of oral and gut microorganisms in the wild and the water environment. This study provides not only basic information about the oral and gut microbiota of captive and wild water monitor lizards, but also an inference that feeding on frogs and aquatic products and reducing human exposure help water monitor lizards maintain a microbiota similar to that in the wild environment.

Lineage diversification and niche evolution in the Reeves' Butterfly Lizard Leiolepis reevesii (Agamidae).

We used mitochondrial cytochrome b and ND4 genes and 9 microsatellite loci to determine genetic diversity, population structure, evolutionary history, and migration patterns within the Reeves' butterfly lizard Leiolepis reevesii (Agamidae). Considering molecular-based phylogeographical lineages, we then performed niche equivalency and similarity tests between divergent lineages. Phylogenetic analyses based on mitochondrial DNA (mtDNA) data revealed 2 lineages (A and B) diverging ≈0.84 million years ago and, respectively, restricted to the northern and southern portions of the Wuzhishan and Yinggeling mountain ranges. Lineage B contains individuals from southern Hainan; Lineage A includes individuals from all other localities and can be further divided into 3 clusters according to microsatellite data. The null hypothesis that the 2 lineages shared identical niches was rejected in all niche equivalency tests, indicating niche shifts during genetic divergence. Similarity tests provided evidence of niche conservatism, suggesting that the 2 lineages share more characteristics of their niche spaces than randomly expected. The niche similarity and equivalency tests indicated a complex niche pattern in which both lineages share a main portion of their ecological spaces. The climatic niche of Lineage B represented a marginal and specialized fraction of the entire ecological space of the climatic niche of Lineage A, with warmer conditions. Isolation caused by orogenesis and subsequent niche divergence, together with local adaptation, may have led to genetic differentiation and further lineage sorting in L. reevesii.

Preparation and detection of sea snake antisera raised in rabbits.

Antivenoms are currently the most effective medication used in the treatment of snakebites. However, there were relatively few studies on preparation of antivenoms targeting sea snakes, especially common sea snakes in China. In this study, we sought to prepare and detect mono- and bispecific antisera raised in rabbits against venoms of two sea snakes, Hydrophis cyanocinctus and H. curtus. The results of enzyme-linked immunosorbent assay showed that the rabbit antisera generally showed clearly detectable immunological cross-reactions after the third immunization and indicated that the strength of cross-reactions increased with an increase in the immunizing dose. Proteins within the H. cyanocinctus and H. curtus venoms showed similar profiles and were mainly concentrated in the low-molecular-weight region (8-25 kDa). Western blotting results revealed that the bands of these low-molecular weight proteins were dense and showed strong immunogenicity. Although we detected comparatively few bands of the high-molecular-weight proteins, these also showed strong immunogenicity. Our results indicate that both mono- and bispecific antisera both can neutralize H. cyanocinctus and H. curtus venoms, and in this regard, the monospecific H. curtus and bispecific antiserum were found to be superior to the H. cyanocinctus antiserum. Given the increasing frequency of snakebites worldwide, we believe that the findings of this study will have high practical applicability.

The geographical diversification in varanid lizards: the role of mainland versus island in driving species evolution.

Monitor lizards (Varanidae) inhabit both the mainland and islands of all geological types and have diversified into an exceptionally wide range of body sizes, thus providing an ideal model for examining the role of mainland versus island in driving species evolution. Here we use phylogenetic comparative methods to examine whether a link exists between body size-driven diversification and body size-frequency distributions in varanid lizards and to test the hypothesis that island lizards differ from mainland species in evolutionary processes, body size, and life-history traits (offspring number and size). We predict that: 1) since body size drives rapid diversification in groups, a link exists between body size-driven diversification and body size-frequency distributions; 2) because of various environments on island, island species will have higher speciation, extinction, and dispersal rates, compared with mainland species; 3) as a response to stronger intraspecific competition, island species will maximize individual ability associated with body size to outcompete closely-related species, and island species will produce smaller clutches of larger eggs to increase offspring quality. Our results confirm that the joint effect of differential macroevolutionary rates shapes the species richness pattern of varanid lizards. There is a link between body size-driven diversification and body size-frequency distributions, and the speciation rate is maximized at medium body sizes. Island species will have higher speciation, equal extinction, and higher dispersal rates compared with mainland species. Smaller clutch size and larger hatchling in the island than in mainland species indicate that offspring quality is more valuable than offspring quantity for island varanids.

Next-generation sequencing yields the complete mitogenome of the blue-banded sea-snake (Squamata: Elapidae).

We report the complete mitogenome of Hydrophis cyanocinctus, which is 17,750 bp in size and includes 13 protein-coding (PCGs), 2 rRNA genes, 22 tRNA genes, and 2 control regions. PCGs, with 13 genes, is 11,427 bp in length. All PCGs use ATN as the typical start codon except COX1 with GTG; the TAG was found as the stop codon in ND1 and ND2, the AGA was found as the stop codon in COI and ND6, the TAA was found as the stop codon in ATP8, ATP6, ND4L, ND5 and Cytb; while other 3 PCGs stop with a single T. Phylogeny reconstructed using the Bayesian inference (BI) method with 13 PCGs indicates the presence of H. cyanocinctus at the root of Laticaudinae.

Next-generation sequencing yields the complete mitogenome of the Shaw's sea-snake (Squamata: Elapidae).

We report the complete mitogenome of Hydrophis curtus, which is 17,702 bp in size and includes 13 protein-coding (PCGs), two rRNA genes, 22 tRNA genes, and two control regions. PCGs, with 13 genes, are 11,261 bp in length. All PCGs use as the start codon ATN except ND1 (CTA) and COX1 (GTG); ATP8, ATP6, ND4L, ND5, and Cytb use the typical stop codon TAA; but COX2 and ND4 with a single T-. Phylogeny recon-structed using the Bayesian inference (BI) method with 13 PCGs indicates that H. curtus at the root of Laticaudinae.

The complete mitochondrial genome of Acanthosaura lepidogaster (Squamata: Agamidae).

In this paper, we report the complete mitochondrial genome of Acanthosaura lepidogaster (Squamata, Agamidae), which is a circular molecule of 16 899 bp in size and consists of 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a control region. The overall base composition is as follows: T (22.8%), C (30.5%), A (32.3%), and G (14.4%). We constructed a phylogeny that included for 10 species of Leiolepidinae lizards and one outgroup Leiocephalus personatus constructed in BEAST, based on 15 mitochondrial genes (12S, 16S, ND1, ND2, COI, COII, ATP8, ATP6, COIII, ND3, ND4L, ND4, ND5, ND6, and cytochrome b). The topology of the phylogenetic tree is broadly similar to that mentioned by Pyron et al.

Climatic correlates of female and male reproductive cycles and plasma steroid hormone levels in the many-lined sun skink Eutropis multifasciata.

Gonadal activity and plasma steroid hormone (testosterone and 17ß-estradiol) levels in males and females of a viviparous skink (Eutropis multifasciata) were investigated. Changes in the hormone profiles were then correlated to changes in environmental factors such as temperature and rainfall and were found to vary seasonally in both sexes. Gonadal activity, calculated using testicular mass and volume, seminiferous tubule diameter and epithelial height in males and, in females, ovary mass and largest follicular volume also varied seasonally. Peak spermiogenesis was in synchrony with maximal vitellogenic activity, but reproductive synchronicity among females was low. Ovary mass and largest follicular volumes were negatively related to air temperature and rainfall. Testicular mass and volume were not related to air temperature, but both were negatively related to rainfall. Rainfall explained a greater proportion of variation in vitellogenic activity than temperature. As for the climatic correlates of seasonal variation in plasma steroid hormones, we found only in males that the plasma level of testosterone was negatively related to rainfall. Taken together, our data show that male and female reproductive activities are more tightly correlated with rainfall than temperature in E. multifasciata.

Phylogeography and population structure of the Reevese's Butterfly Lizard (Leiolepis reevesii) inferred from mitochondrial DNA sequences.

Butterfly lizards of the genus Leiolepis (Agamidae) are widely distributed in coastal regions of Southeast Asia and South China, with the Reevese's Butterfly Lizard Leiolepis reevesii having a most northerly distribution that ranges from Vietnam to South China. To assess the genetic diversity within L. reevesii, and its population structure and evolutionary history, we sequenced 1004 bp of cytochrome b for 448 individuals collected from 28 localities covering almost the whole range of the lizard. One hundred and forty variable sites were observed, and 93 haplotypes were defined. We identified three genetically distinct clades, of which Clade A includes haplotypes mainly from southeastern Hainan, Clade B from Guangdong and northern Hainan, and Clade C from Vietnam and the other localities of China. Clade A was well distinguished and divergent from the other two. The Wuzhishan and Yinggeling mountain ranges were important barriers limiting gene exchange between populations on the both sides of the mountain series, whereas the Gulf of Tonkin and the Qiongzhou Strait were not. One plausible scenario to explain our genetic data is a historical dispersion of L. reevesii as proceeding from Vietnam to Hainan, followed by a second wave of dispersal from Hainan to Guangdong and Guangxi. Another equally plausible scenario is a historically widespread population that has been structured by vicariant factors such as the mountains in Hainan and sea level fluctuations.

Influence of pregnancy on locomotor and feeding performances of the skink, Mabuya multifasciata: why do females shift thermal preferences when pregnant?

It has been documented in the many-lined sun skink (Mabuya multifasciata) that pregnant females select lower body temperatures (27.6-30.8 degrees C) than do nonpregnant females and adult males (29.7-35.7 degrees C). We therefore used the skink to test the hypothesis that the maximization of reproductive benefits should be achieved in pregnant females by shifting thermal preferences towards the levels optimal for embryonic development but entailing relatively small costs of reproduction. Data on adult males showed that temperatures maximizing swimming stamina (indicative of locomotor endurance) fell within the range of body temperatures selected by nonpregnant females and adult males. Data on swimming stamina and feeding performance of pregnant females, nonpregnant females and adult males measured at 26 and 30 degrees C showed that: (1) pregnancy impaired locomotor and feeding performances, but such impairments did not persist after parturition; (2) the degree of locomotor impairment during pregnancy was greater at 26 degrees C than at 30 degrees C, but the degree of feeding impairment during pregnancy was greater at 30 degrees C than at 26 degrees C. Pregnant females of M. multifasciata selecting body temperatures at about 29 degrees C could not only produce good-quality offspring in a relatively short gestation length but also reduce reproductive costs associated with locomotor and feeding impairments to some extent. Thus, data from M. multifasciata validate the above hypothesis.