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Taiwania cryptomerioides Hayata is an endangered relict plant belonging to Taxodiaceae, and it is also an endemic plant to China. The decay-resistant of Taiwania timber can provide highly quality wood for building and furniture. Plenty of regenerative of leaves of T. cryptomerioides also has been used as a resource for the discovery of new dimeric diterpenoids. In a search for structurally diverse dimeric diterpenoids and potent bioactive isolates, ten new heterodimeric diterpenoids, taiwaniadducts K-T (1-4, 6, 8-11, 14), along with five known ones (5, 7, 12, 13, 15), were isolated from the leaves of T. cryptomerioides. These new compounds were defined by comprehensive spectroscopic analyses, putative biosynthetic pathways, and the value of optical. Biologically, anti-multidrug resistance (MDR) activities of compounds were evaluated. Compounds 4 and 10 exerted a 9.18-fold potentiation effect on bortezmib (BTZ) susceptibility at a tested concentration (20 μM) better than the positive control verapamil. The research of the leaves of T. cryptomerioides not only added the new data to the structural diversity and activities of dimeric diterpenoids but also could provide support for the medical and industrial application of the leaves of this endangered relict plant.

Brevinins exhibit a wide range of structural features and strong biological activities. Brevinin-2, derived from several amphibians, has shown antimicrobial activities. However, little is known about the wound-healing activity of brevinin-2. In this study, brevinin-2 cDNA was identified from the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus) and it comprises a signal peptide, a propeptide, and a mature peptide. Sequence alignment with brevinin-2 derived from other amphibians showed variability of the mature peptide, and the presence of a C-terminal cyclic heptapeptide domain (Cys-Lys-Xaa4-Cys) in the mature peptide. Dark-spotted frog brevinin-2 belonged to the brevinin-2 cluster and was closely related to brevinin-2HB1 from Pelophylax hubeiensis. Synthetic dark-spotted frog brevinin-2 mature peptide (brevinin-2PN) exhibited antibacterial activity against several pathogens by destroying cell membrane integrity and hydrolysis of genomic DNA. Brevinin-2PN exhibited significant wound-healing activity by accelerating the healing of human skin fibroblast cell scratches, influencing cell migration, and stimulating gene expression of growth factors.

Vocalization plays an important role in the communication of anurans. In this study, the advertisement calls of male Fejervarya multistriata obtained in Lishui, Zhejiang, China during the breeding season were recorded. Their note number (NN), note duration (ND), note interval (NI), call duration (CD), pitch (PIT), call intensity (CIT) and dominant frequency (DF) were analyzed. The calls of F. multistriata are composed of one to 38 notes, and calls composed of fewer than ten notes have the highest frequency. Male frogs produced calls ranging from 1201 Hz to 3357 Hz with two DFs (1412.49 Hz and 2953.89 Hz). By comparing the differences among individual calls, it was found that the within-individual coefficients of variation (CVW) and among-individual coefficients of variation (CVA) for NN, NI, CD, PIT and DF were more than 10%, whereas that of CIT was less than 5%. The CVA/CVW ratios indicate that ND is important for sexual selection, whereas NN, NI CD, PIT, CIT, and DF are important for individual recognition. Phylogenetic generalized least squares analysis showed that phylogenetic signals affect DF vs. snout-vent length (SVL) and CD of anurans in China, and accounting for phylogenetic signals, DF was negatively correlated with SVL. DF was found to be higher in anurans that breed in flowing water than in those that breed in stagnant water, after eliminating the effects of phylogeny and SVL. Therefore, we conclude that phylogenetic effects, SVL, and the water type of breeding habitats have a combined impact on the advertisement calls in anurans.

The β-defensins are important components of the vertebrate innate immune system. While mammalian β-defensins have wide-ranging antibacterial and immunomodulatory activities, those of amphibians remain largely uncharacterised. In this study, β-defensin cDNA was identified from the skin transcriptome of the Chinese spiny frog Quasipaa spinosa. This β-defensin (QS-BD) consists of a signal and a mature peptide. Sequence alignments with other amphibian β-defensins showed conservation of the functional mature peptide and that its closest relative is β-defensin from Zhangixalus puerensis. Synthetic QS-BD showed antibacterial activity against Vibrio vulnificus, Vibrio harveyi, Streptococcus iniae, and Aeromonas hydrophila. QS-BD showed bactericidal activity by destroying the cell membrane integrity, but did not hydrolyse genomic DNA. QS-BD treatment promoted respiratory bursts and upregulated the expression of interleukin-1β and tumour necrosis factor-α in the murine leukemic monocyte/macrophage cell line RAW264.7. This is the first demonstration of immunomodulatory activity by an amphibian β-defensin.

The difference in body size and/or shape between males and females, called sexual size dimorphism, is widely accepted as the evolutionary consequence of the difference between reproductive roles. To study the mating pattern, female reproduction and sexual size dimorphism in a population of Microhyla fissipes, amplexus pairs were collected, and the snout-vent length of males and females, female reproductive traits and fertilization rate were measured. If the body size of amplexed females is larger than that of amplectant males, this is referred to as a female-larger pair, a phenomenon that was often observed for M. fissipes in this study. Interestingly, snout-vent length of males in male-larger pairs was greater than that in female-larger pairs, however the post-spawning body mass, clutch size, egg dry mass and clutch dry mass did not differ between both types of pairs. Snout-vent length of males was positively related to that of females in each amplexus pair. After accounting for the snout-vent lengths of females, we showed that snout-vent lengths of males in male-larger pairs were greater than those of females in female-larger pairs. The snout-vent length ratio of males and females was not related to fertilization rate in each amplexus pair. The mean fertilization rate was not different between both amplexus pairs. These results suggest that (1) M. fissipes displays female-biased sexual size dimorphism and has two amplexus types with size-assortative mating; (2) the snout-vent length ratio of males and females in each amplexus type was consistent with the inverse of Rensch’s rule, and was driven by the combined effect of sexual selection and fecundity selection; (3) females with a larger body size were preferred by males due to their higher fecundity, while the body size of males was not important for fertilization success.

NK-lysins (NKLs) are a family of multifunctional antimicrobial peptides that have activity against various microorganisms. However, the immunomodulatory activity of NKL in fish remains unclear. In this study, the cDNA sequence of barbel steed (Hemibarbus labeo) NKL gene was cloned. Barbel steed NKL amino acid sequence comprised a signal peptide and a mature peptide. The saposin B domain in the mature peptide has six conserved cysteines that form three disulfide bonds. Phylogenetic analysis showed that the barbel steed NKL was most closely related to that of the common carp (Cyprinus carpio) NKL. Differential expression analysis showed that the barbel steed NKL gene was expressed in all tested tissues, with the highest expression in the spleen. In response to Aeromonas hydrophila infection, NKL was significantly upregulated in the liver, spleen, head kidney, and gill. The barbel steed NKL showed strong antibacterial activity against Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, and Listeria monocytogenes. However, NKL had no antibacterial activity against the pathogenic bacteria A. hydrophila. Lactate dehydrogenase release assays showed that NKL damaged the V. parahaemolyticus cell membrane. NKL significantly increased barbel steed survival rate after A. hydrophila infection and upregulated IL-1β and TNF-α expression in the spleen and head kidney. NKL induced monocyte/macrophage chemotaxis and enhanced the respiratory burst and proinflammatory cytokine expression. Our study shows that fish NKL exhibits immunomodulatory effects and protects the host from pathogenic infections independent of direct bacterial clearance.

Cathelicidins are an important antimicrobial peptide family and are expressed in many different vertebrates. They play an important role in the innate immune system of the host. However, amphibian cathelicidins are poorly understood. In this study, the cDNA of the cathelicidin gene was obtained from the skin transcriptome of tiger frog (Hoplobatrachus rugulosus). The predicted amino acid sequence of tiger frog cathelicidin (HR-CATH) comprises a signal peptide, a cathelin domain, and a mature peptide. The HR-CATH amino acid sequence alignment with other frog cathelicidins showed that the functional mature peptide is highly variable in amphibians, whereas the cathelin domain is conserved. A phylogenetic tree analysis showed that HR-CATH is most closely related to cathelicidin-NV from Nanorana ventripunctata. HR-CATH was chemically synthesized and its in vitro activity was determined. It had high antibacterial activity against Vibrio parahaemolyticus, Staphylococcus aureus, and the pathogenic bacterium Aeromonas hydrophila. HR-CATH damaged the cell membrane integrity of A. hydrophila according to a lactate dehydrogenase release assay and was able to hydrolyze the genomic DNA from A. hydrophila in a dose-dependent manner. Furthermore, in RAW264.7 cells (mouse leukemic monocyte/macrophage cell line), HR-CATH induced chemotaxis and enhanced respiratory burst. Our study shows that amphibian cathelicidin has antimicrobial activity and an immunomodulatory effect on immune cells.

The complete mitochondrial DNA (mtDNA) for Pareas stanleyi was determined in this study. The length of complete mtDNA was 18,932 bp, including 13 protein-coding genes (PCGs) (COI-III, ND1-6, ND4L, ...

We report the complete mitochondrial genome (mtDNA) of Pareas formosensis (Squamata: Colubridae). This circular mtDNA is 17,703 bp in size and consists of 13 protein-coding genes, 22 transfer RNAs,...

We reported the partial mitochondrial genome (mitogenome) for Hyla sanchiangensis (Anura: Hylidae), a arboreal frog and endemic in China. The length of partial mitogenome of H. sanchiangensis was 15,664 bp, and contained PCGs (COX1-3, ND1-6, ND4L, ATP6, ATP8 and CYTB), 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding regions of a L-strand replication origin and a partial loop region. The overall base composition of the sequence is 29.91% A, 29.86% T, 14.58% G, and 25.65% C, with a total A + T content of 59.77%. The result of phylogenetic analysis showed that H. sanchiangensis formed a clade with other species belonging to the genus of Hyla. This mitogenome data could help in evolutionary biology and population genetics of the Hylid species.

We reported the complete mitochondrial genome (mitogenome) of broad-folded frog (Hylarana latouchii). This mitogenome is 17,007 bp in size and consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one non-coding sequence (D-loop). The total composition was 58.54% A + T and 41.46% G + C (T: 29.31%, C: 27.33%, A: 29.23%, and G: 14.13%). The phylogenetic analysis revealed that H. latouchii formed a clade with other two species of genus Hylarana. This mitogenomic sequence of H. latouchii provides useful data to study its population genetics and phylogeography.

Eukaryotic and prokaryotic cell genomes exhibit multiple microsatellites. In this study, we characterized microsatellites in genomes and genes of Nanorana parkeri and Xenopus laevis. This characterization was used for gene ontology (GO) analysis of coding sequences (CDS). Compared to the genome of N. parkeri, the genome of X. laevis is larger and contains more number of microsatellites, but the diversity of both species are similar. Trinucleotide repeats in the genome of N. parkeri and dinucleotide and tetranucleotide repeats inthe genome of X. laevis were the most diverse. In both the species, diversity of microsatellites was highest in intergenic regions, followed by intron and exon regions, and lowest in coding regions. Microsatellites in CDS are thus subject to higher selective pressure. Many microsatellites are concentrated upstream and downstream of genes in both species, suggesting suppression of repeats in the middle of protein-CDS. Repeats are enriched in regions near gene termini purely due to the biophysical constraints of protein structure. In GO analysis, two and five unique GO terms, only found in N. parkeri and X. laevis, respectively, indicate advantageous mutations during species evolution. Biological process, cellular component and molecular function ontology reflected in the GO analysis predicted that the microsatellites located in CDS can alter protein function and may provide a molecular basis for species adaptation to new and changing environments.

Background Species-specific advertisement calls are the main mechanism of transmitting information between individuals in anuran amphibians and are therefore indispensable for anuran survival and reproduction. Survey methods that monitor these calls can be used for rapid species recognition, behavioral experiments, and conservation monitoring. In this study, we described in detail 10 call parameters from three sympatric species in the genus Microhyla and analyzed the differences in call parameter among these species to provide a basis for systematic monitoring, acoustic analysis and taxonomic study of this genus. Methods The quantitative analyses of temporal and spectral call parameters were used in our study for the advertisement calls of three sympatric Microhyla species (M. beilunensis, M. fissipes and M. heymonsi) in Zhejiang Province, East China. Results Our results showed the following: (1) Significant differences existed among the three sympatric Microhyla species in call duration (CD), call interval (CI), number of pulses (NP), pulse rate, call intensity (CIT), dominant frequency (DF) and frequency of the first to fourth formants (F1, F2, F3 and F4). (2) Some spectral parameters (DF, F1 and F3) were negatively correlated with the body size of the vocalizing individuals in each species. (3) The coefficients of variation within individuals (CVw) for CIT, DF and F1–F4 were smaller than 5%, whereas the CVW for CI was larger than 10% in each species. (4) The principal component analysis and discriminant function analysis showed that call parameters could distinguish the three Microhyla species. (5) The phylogenetic generalized least squares analysis showed that phylogenetic relationships affected CD and NP against snout-vent length (SVL), DF and NP against CD, and NP against DF, but not of DF against SVL; based on the phylogenetic analysis, CD and NP were not related to SVL, but DF was negatively related to SVL.

Acoustic signalling is the most important form of communication in anuran amphibians. Here we recorded and analysed the calls of 18 male Guenther’s frogs (Hylarana guentheri) from the wild during t...

Paramesotriton aurantius (Caudata: Salamandridae) is a new species that found in southeastern China. Its complete mitochondrial genome (mitogenome) sequence was 16,313 bp in length with with A + T contents of 60.9%, and contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, one control region (D-loop), and one non-coding region. Our molecular tree showed that P. aurantius was positioned near P. hongkongensis, and formed a clade with other Paramesotriton species. The first complete mitogenome sequence of P. aurantius could provided fundamental data for resolving phylogenetic and genetic problems related to genus Paramesotriton.

The black-spotted stout newt (Pachytriton brevipes) is widely distributed in the mountains of southeastern China. In this study, we sequenced and determined the complete mitochondrial genome (mitog...

We reported the partial mitochondrial genome (mitogenome) for Hyla sanchiangensis (Anura: Hylidae), a arboreal frog and endemic in China. The length of partial mitogenome of H. sanchiangensis was 1...

In this paper, a planar nitrogen-based atmospheric-pressure dielectric barrier discharge (DBD) jet with enhanced UV emission and radical generation, and much lower power consumption and gas temperature using shorter electrodes as compared with a previously developed DBD jet is presented. The highest UV intensity (five times larger) and radical generation (two times larger) in the DBD jet region are found to occur when the electrode length is 1 cm. Corresponding jet gas temperature and power consumption are 31 °C and 39 W, respectively. The 1-cm DBD jet was used to treat polylactide film for enhancing its hydrophilic properties. The results show that a contact angle of 28.4° could be obtained with a residence time of 4 s. The resulting 1-cm DBD jet may provide an efficient surface treatment tool for delicate industrial and biomedical devices/materials.

In this paper, we compare the helium-based atmospheric pressure plasma jets (APPJs) with power-ground (PG) and power-floating (PF) configurations by using optical analysis. We find that the PG jet traps plasma between two electrodes (discharge region); thus, the hydroxyl radical intensity is significantly strong in discharge region; however, the plasma intensity in downstream region is relatively weak as compared with that of PF configuration. In contrast, using the APPJ with PF configuration, the hydroxyl radical and nitric oxide are approximately two and three times stronger, respectively, in the downstream region, even though the gas temperature and power consumption are lower. The PF jet successfully eradicates S. mutans and decreases water contact angle by ~40 within 15-s treatment, which demonstrates higher efficient than that of PG jet, and shows highly potential for dental filling demand.

In this paper, we want to develop and characterize a round argon-based nonthermal atmospheric-pressure plasma jet (APPJ) with an application of sterilization. We measured the basic properties of APPJ by an alcohol thermometer, an optical emission spectrometer (OES), a high-voltage probe and a Rogowski coil. The plasma jet temperature at the tube exit is lower than 37 °C, which will not induce damage to living tissue. The plasma absorbed power is about 2.7 W. The OES data indicate that this APPJ has a relatively high intensity of hydroxyl radicals (OH) in postdischarge region. Furthermore, we applied this device to inactivate the bacteria ( E. coli and B. subtilis ) and endospore (B. subtilis endospore). In addition, the trace amount of oxygen addition (0.04%) into working gas can not only enhance the OH radicals but also speed up the killing of bacteria and endospore. The results of D value and scanning electron microscope images show its great potential for the use of general sterilization in the future.

Background Species-specific advertisement calls are the main mechanism of transmitting information between individuals in anuran amphibians and are therefore indispensable for anuran survival and reproduction. Survey methods that monitor these calls can be used for rapid species recognition, behavioral experiments, and conservation monitoring. In this study, we described in detail 10 call parameters from three sympatric species in the genus Microhyla and analyzed the differences in call parameter among these species to provide a basis for systematic monitoring, acoustic analysis and taxonomic study of this genus. Methods The quantitative analyses of temporal and spectral call parameters were used in our study for the advertisement calls of three sympatric Microhyla species (M. beilunensis, M. fissipes and M. heymonsi) in Zhejiang Province, East China. Results Our results showed the following: (1) Significant differences existed among the three sympatric Microhyla species in call duration (CD), call interval (CI), number of pulses (NP), pulse rate, call intensity (CIT), dominant frequency (DF) and frequency of the first to fourth formants (F1, F2, F3 and F4). (2) Some spectral parameters (DF, F1 and F3) were negatively correlated with the body size of the vocalizing individuals in each species. (3) The coefficients of variation within individuals (CVw) for CIT, DF and F1–F4 were smaller than 5%, whereas the CVW for CI was larger than 10% in each species. (4) The principal component analysis and discriminant function analysis showed that call parameters could distinguish the three Microhyla species. (5) The phylogenetic generalized least squares analysis showed that phylogenetic relationships affected CD and NP against snout-vent length (SVL), DF and NP against CD, and NP against DF, but not of DF against SVL; based on the phylogenetic analysis, CD and NP were not related to SVL, but DF was negatively related to SVL.

Leptobrachella (Anura: Megophryidae) is currently composed of seventy-one species distributed in China and Southeast Asia (Frost 2018). Fourteen species of Leptobrachella are currently known from southern China: L. alpina (Fei, Ye & Li, 1990), L. laui (Sung, Yang & Wang, 2014), L. liui (Fei & Ye, 1990), L. mangshanensis (Hou, Zhang, Hu, Li, Shi, Chen, Mo & Wang, 2018), L. maoershanensis (Yuan, Sun, Chen, Rowley & Che, 2017), L. oshanensis (Liu, 1950), L. purpura (Yang, Zeng & Wang, 2018), L. tengchongensis (Yang, Wang, Chen & Rao, 2016), L. wuhuangmontis (Wang, Yang & Wang, 2018), L. yingjiangensis (Yang, Zeng & Wang, 2018), L. yunkaiensis (Wang, Li, Lyu & Wang, 2018), L. ventripunctata (Fei, Ye & Li, 1990), L. pelodytoides (Boulenger, 1893) and L. sungi (Lathrop, Murphy, Orlov & Ho, 1998) and the first eleven species are endemic of this region (AmphibiaChina 2018). Many new species in this genus were discovered based on molecular, morphometric and bioacoustic data in recent years (Frost 2018). In comparison to molecular and morphological data, bioacoustics is especially useful in species identification of Leptobrachella in field surveys (Rowley et al. 2016). Although the advertisement calls of many Leptobrachella species were described along with the species descriptions (e.g., Rowley et al. 2016), the advertisement calls of only three Chinese endemic species (L. alpinus; L. laui and L. oshanensis) are known (Jiang et al. 2002; Matsui 2006; Xu et al. 2005). Here, we describe the advertisement calls of L. liui, which inhabits rocky streams in southeastern China (Fujian, Jiangxi and Zhejinag) and is morphologically diagnosed by the combination of a set of external color traits (Fig. 1).

This study investigated the dehumidification effect of silica gel and sodium polyacrylate desiccants coated on fin and tube heat exchangers under various humidity and temperature conditions. In the system, the two desiccant-coated heat exchangers are periodically operated in mass equilibrium states between dehumidification and regeneration processes. The corresponding cyclic switching times between the two processes are investigated to determine suitable switching times that will further improve the operational efficiency of the system. The experimental results show that while water at a temperature of 50°C was supplied during the regeneration process, the sorption ability was greater and the vapor was discharged in a shorter period of the regeneration process for the sodium polyacrylate desiccant than for silica gel. Thus, the moisture sorption ability of sodium polyacrylate in the system is superior to that of silica gel under the same operational conditions. Suitable cyclic switching times for both des...

Hylarana guentheri is a lowland aquatic species and widely distributed throughout southern China and central Vietnam. In this study, the 25 novel polymorphic microsatellite loci were isolated from partial genome using the Hiseq 2000 platform. One population from Wukeng Village, Zhejiang Province, China were genotyped (n = 30). The results showed that the mean number of observed alleles per locus was 13 (with a range from 6 to 25), observed and expected heterozygosity values ranged from 0.067 to 0.967 and from 0.274 to 0.921 respectively. One locus (HG22) showed significant departure from Hardy–Weinberg equilibrium and no linkage disequilibrium was found among any pairwise loci. These highly informative microsatellite markers should be useful for population genetic analyses for H. guentheri and other species in genus of Hylarana.

In this study, we used an argon-based round atmospheric-pressure plasma jet (APPJ) for enhancing wound healing in streptozotocin (STZ) induced diabetic rats. The APPJ was characterized by optical emission spectroscopy. We induced Type 1 and Type 2 diabetes in rats with different amounts of STZ combined with normal and high-fat diets, respectively. The wound area ratio of all the plasma-treated normal and diabetic groups was greatly reduced (up to 30%) compared with that of the untreated groups during healing. Histological analysis revealed faster re-epithelialization, collagen deposition, less inflammation, and a complete skin structure in the plasma-treated groups was found as compared with the untreated control groups. In addition, the new blood vessels of plasma-treated tissues decreased more than untreated tissues in the middle (Day 14) and late (Day 21) stages of wound healing. The plasma-treated wounds demonstrated more transforming growth factor beta (TGF-β) expression in the early stage (Day 7), whereas they decreased in the middle and late stages of wound healing. The levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) increased after plasma treatment. In addition, plasma-treated water had a higher concentration of hydrogen peroxide, nitrite and nitrate when the plasma treatment time was longer. In summary, the proposed argon APPJ based on the current study could be a potential tool for treating diabetic wounds.

We compared the morphology of the erythrocytes of five anurans, two toad species - Bufo gargarizans (Cantor, 1842) and Duttaphrynus melanostictus (Schneider, 1799) and three frog species - Fejervarya limnocharis (Gravenhorst, 1829), Microhyla ornata (Duméril & Bibron, 1841), and Rana zhenhaiensis (Ye, Fei & Matsui, 1995). We then reconstructed the ancestral state of erythrocyte size (ES) and nuclear size (NS) in amphibians based on a molecular tree. Nine morphological traits of erythrocytes were all significantly different among the five species. The results of principal component analysis showed that the first component (49.1% of variance explained) had a high positive loading for erythrocyte length, nuclear length, NS and ratio of erythrocyte length/erythrocyte width; the second axis (28.5% of variance explained) mainly represented erythrocyte width and ES. Phylogenetic generalized least squares analysis showed that the relationship between NS and ES was not affected by phylogenetic relationships although there was a significant linear relationship between these two variables. These results suggested that (1) the nine morphological traits of erythrocytes in the five anuran species were species-specific; (2) in amphibians, larger erythrocytes generally had larger nuclei.

In reptiles, habitat selection is the process whereby suitable habitat is selected that optimizes physiological functions and behavioral performance. Here, we used the brown forest skink (Sphenomorphus indicus) as a model animal and examined whether the frequency of active individuals, environmental temperature, illumination of activity area, and habitat type vary with different age classes. We surveyed the number of active individuals and measured environmental variables at Baiyunshan Mountain in Lishui, Zhejiang, China. We found no difference in the activity frequency of adult and juvenile S. indicus; the activity pattern of active individuals was bimodal. The mean environmental temperature selected by adults was higher than that selected by juveniles. The environmental temperature of active areas measured at 0900-1000 h and 1100-1200h was higher than at 1400-1500h; illumination of the active area at 1000-1200h was also higher than at 1400h-1600 h. The number of active individuals, the environmental temperature and illumination of activity areas showed pairwise positive correlation. There was a difference in habitat type between juveniles and adults whereby juveniles prefer rock habitats. We predict that active S. indicus select optimal habitats with different environmental temperatures and types to reach the physiological needs particular to their age classes.

The Chinese tiger frog Hoplobatrachus rugulosa is one of the most common frog species sold in China as food. To examine the combined effects of food supply and larval density on survival, growth and metamorphosis of H. rugulosa tadpoles, we conducted a two (food supplies) × three (larval densities) factorial experiment. Tadpoles in one feeding treatment always had free access to food, and tadpoles in the other feeding treatment had free access to food for 24 h every 4 days. Larval densities were set at 6, 12 and 18 tadpoles per liter water, respectively. We found that: (1) metamorphosis rate decreased as larval density increased, but it did not differ between the two feeding treatments; (2) metamorphosis time and its variance increased as density increased, and insufficient food supply resulted in longer and more variable larval periods; (3) high larval density led to the production of smaller froglets, so did insufficient food supply; and (4) larval density has a more pronounced role in affecting metamorphosis rate, and food supply has a more pronounced role in affecting metamorphosis time. Based on our results, the most important thing that should be done by farmers is to avoid the rearing of H. rugulosa tadpoles under food × density conditions greatly reducing larval survival, growth and developmental synchrony rather than to rear them under conditions substantially increasing the costs due to the increased payments for food and space.