Toxicity comparison and risk assessment of two chlorinated organophosphate flame retardants (TCEP and TCPP) on Polypedates megacephalus tadpoles.

Tris(2-chloroethyl) phosphate (TCEP) and tris(1­chloro-2-propyl) phosphate (TCPP) are widely used as chlorinated organophosphate flame retardants (OPFRs) due to their fire-resistance capabilities. However, their extensive use has led to their permeation and pollution in aquatic environments. Using amphibians, which are non-model organisms, to test the toxic effects of OPFRs is relatively uncommon. This study examined the acute and chronic toxicity differences between TCEP and TCPP on Polypedates megacephalus tadpoles and evaluated the potential ecological risks to tadpoles in different aquatic environments using the risk quotient (RQ). In acute toxicity assay, the tadpole survival rates decreased with increased exposure time and concentrations, with TCEP exhibiting higher LC50 values than TCPP, at 305.5 mg/L and 70 mg/L, respectively. In the chronic assay, prolonged exposure to 300 µg/L of both substances resulted in similar adverse effects on tadpole growth, metamorphosis, and hepatic antioxidant function. Based on RQ values, most aquatic environments did not pose an ecological risk to tadpoles. However, the analysis showed that wastewater presented higher risks than rivers and drinking water, and TCPP posed a higher potential risk than TCEP in all examined aquatic environments. These findings provide empirical evidence to comprehend the toxicological effects of OPFRs on aquatic organisms and to assess the safety of aquatic environments.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties.

BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.

The complete mitochondrial genome of the Baishanzu horned toad Boulenophrys baishanzuensis (Anura: Megophryidae).

The mitochondrial genome (mitogenome) of Boulenophrys baishanzuensis (Anura: Megophryidae) was sequenced by the Illumina platform. The assembled circular mitogenome of B. baishanzuensis had a total length of 17,040 bp, with a GC content of 41.25%. It consisted of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a D-loop region. The majority of the PCGs were encoded by the H-strand, while one PCG (nad6) and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser2, tRNA-Glu, and tRNA-Pro) were encoded in the L-strand. Phylogenetic analysis revealed that the newly sequenced species formed a clade with other Boulenophrys species, while the genus Boulenophrys itself formed a sister group with the genus Atympanophrys.

Overexpression of cucumber CYP82D47 enhances resistance to powdery mildew and Fusarium oxysporum f. sp. cucumerinum.

Cytochrome P450s are a large family of protein-encoding genes in plant genomes, many of which have not yet been comprehensively characterized. Here, a novel P450 gene, CYP82D47, was isolated and functionally characterized from cucumber (Cucumis sativus L.). Quantitative real-time reverse-transcription polymerase chain reaction analysis revealed that CYP82D47 expression was triggered by salicylic acid (SA) and ethephon (ETH). Expression analysis revealed a correlation between CYP82D47 transcript levels and plant defense responses against powdery mildew (PM) and Fusarium oxysporum f. sp. cucumerinum (Foc). Although no significant differences were observed in disease resistance between CYP82D47-RNAi and wild-type cucumber, overexpression (OE) of CYP82D47 enhanced PM and Foc resistance in cucumber. Furthermore, the expression levels of SA-related genes (PR1, PR2, PR4, and PR5) increased in CYP82D47-overexpressing plants 7 days post fungal inoculation. The levels of ETH-related genes (EIN3 and EBF2) were similarly upregulated. The observed enhanced resistance was associated with the upregulation of SA/ETH-signaling-dependent defense genes. These findings indicate the crucial role of CYP82D47 in pathogen defense in cucumber. CYP82D47-overexpressing cucumber plants exhibited heightened susceptibility to both diseases. The study results offer important insights that could aid in the development of disease-resistant cucumber cultivars and elucidate the molecular mechanisms associated with the functions of CYP82D47.

Characterization and Comparison of the Two Mitochondrial Genomes in the Genus Rana.

The mitochondrial genome (mitogenome) possesses several invaluable attributes, including limited recombination, maternal inheritance, a fast evolutionary rate, compact size, and relatively conserved gene arrangement, all of which make it particularly useful for applications in phylogenetic reconstruction, population genetics, and evolutionary research. In this study, we aimed to determine the complete mitogenomes of two morphologically similar Rana species (Rana hanluica and Rana longicrus) using next-generation sequencing. The entire circular mitogenome was successfully identified, with a length of 19,395 bp for R. hanluica and 17,833 bp for R. longicrus. The mitogenomes of both species contained 37 genes, including 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and one control region; mitogenome size varied predominantly with the length of the control region. The two synonymous codon usages in 13 PCGs showed that T and A were used more frequently than G and C. The ratios of non-synonymous to synonymous substitutions of all 13 PCGs were <1 in the Rana species, indicating that the PCGs were under purifying selection. Finally, phylogenetic relationship analyses suggested that R. hanluica and R. longicrus were classified in the R. japonica group. Our study provides valuable reference material for the taxonomy of the genus Rana.

Host defence peptide LEAP2 contributes to antimicrobial activity in a mustache toad (Leptobrachium liui).

BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) is essential in host immunity against harmful pathogens and is only known to act as an extracellular modulator to regulate embryonic development in amphibians. However, there is a dearth of information on the antimicrobial function of amphibian LEAP2. Hence, a LEAP2 homologue from Leptobrachium liui was identified, characterized, and chemically synthesized, and its antibacterial activities and mechanisms were determined. RESULTS: In this study, LEAP2 gene (Ll-LEAP2) cDNA was cloned and sequenced from the Chong'an Moustache Toad (Leptobrachium liui). The predicted amino acid sequence of Ll-LEAP2 comprises a signal peptide, a mature peptide, and a prodomain. From sequence analysis, it was revealed that Ll-LEAP2 belongs to the cluster of amphibian LEAP2 and displays high similarity to the Tropical Clawed Frog (Xenopus tropicalis)'s LEAP2. Our study revealed that LEAP2 protein was found in different tissues, with the highest concentration in the kidney and liver of L. liui; and Ll-LEAP2 mRNA transcripts were expressed in various tissues with the kidney having the highest mRNA expression level. As a result of Aeromonas hydrophila infection, Ll-LEAP2 underwent a noticeable up-regulation in the skin while it was down-regulated in the intestines. The chemically synthesized Ll-LEAP2 mature peptide was selective in its antimicrobial activity against several in vitro bacteria including both gram-positive and negative bacteria. Additionally, Ll-LEAP2 can kill specific bacteria by disrupting bacterial membrane and hydrolyzing bacterial gDNA. CONCLUSIONS: This study is the first report on the antibacterial activity and mechanism of amphibian LEAP2. With more to uncover, the immunomodulatory functions and wound-healing activities of Ll-LEAP2 holds great potential for future research.

Antimicrobial peptide hepcidin contributes to restoration of the intestinal flora after Aeromonas hydrophila infection in Acrossocheilus fasciatus.

Hepcidin is a cysteine-rich antimicrobial peptide that serves an important role in the immunity system of fishes. It exhibits antibacterial, antifungal, antiviral, and antitumor activities. However, the exact role of fish hepcidin in the regulation of the intestinal flora still remains a mystery. In our study, we sequenced and characterized hepcidin from the liver of Acrossocheilus fasciatus. Phylogenetic tree analysis showed that A. fasciatus hepcidin and Gobiocypris rarus hepcidin were the most closely related, and both belonged to the fish HAMP1 cluster. Studies conducted on in vivo tissue distribution showed that the expression of hepcidin was highest in healthy A. fasciatus liver. Aeromonas hydrophila infection was confirmed by the increased expression of pro-inflammatory cytokine genes and bacterial loads in A. fasciatus tissues. After A. hydrophila infection, hepcidin expression significantly increased in the liver, spleen, and head kidney. In vitro antibacterial assays showed that hepcidin exhibits strong broad spectrum antibacterial activity. Furthermore, we examined the regulatory effect of hepcidin on the intestinal flora and found that A. fasciatus hepcidin restored the reduced diversity and compositional changes in intestinal flora caused by A. hydrophila infection. Our results suggest that hepcidin could regulate the intestinal flora in fishes; however, the underlying mechanisms need to be explored in greater detail.

Transcriptomic analysis of intermuscular bone development in barbel steed (Hemibarbus labeo).

Intermuscular bones (IBs), which are little, bony spicules in muscle, are embedded in lower teleosts' myosepta. Despite the importance of studying IB development in freshwater aquaculture species, the genes associated with IB development need to be further explored. In the present study, we identified four stages of IB development in barbel steed (Hemibarbus labeo), namely stage 1: IBs have not emerged, stage 2: a few small IBs have emerged in the tail, stage 3: longer IBs gradually emerged in the tail and stage 4: all of the IBs in the tail are mature and long, via Alizarin red staining. Subsequently, we used the HiseqXTen platform to sequence and de novo assemble the transcriptome of epaxial muscle (between 35th and 40th myomere) of barbel steed at 29 days (stage 1) and 42 days (stage 3) after hatching. A total of 190,814 unigenes were obtained with an average length and N50 of 648 bp and 1027 bp, respectively. We found 2174 differentially expressed genes (DEGs) between stages 1 and 3, of which 378 and 1796 were up- and down-regulated, respectively. Functional enrichment analysis showed that several DEGs functioned in ossification, positive regulation of osteoblast differentiation, osteoblast differentiation, and BMP signaling pathway, and were further enriched in signal pathway, including osteoclast differentiation, TGF-ß signaling pathway, cytokine-cytokine receptor interaction, Jak-STAT signaling pathway, and other KEEG pathways. In conclusion, we identified genes that may be related to IB development, such as kazal type serine peptidase inhibitor domain 1 (KAZALD1), extracellular matrix protein 1 (ECM1), tetranectin, bone morphogenetic protein 1 (bmp1), acid phosphatase 5 (ACP5), collagen type XI alpha 1 chain (COL11A1), matrix metallopeptidase 9 (MMP9), pannexin-3 (PANX3), sp7 transcription factor (Sp7), and c-x-c motif chemokine ligand 8 (CXCL8), by comparing the transcriptomes of epaxial muscle before and after IB ossification. This study provided a theoretical basis for identifying the molecular mechanisms underlying IB development in fish.

Comprehensive assessment of the ecological risk of exposure to triphenyl phosphate in a bioindicator tadpole.

The toxicity of triphenyl phosphate (TPhP) to aquatic organisms in surface waters has been demonstrated; However, an understanding of toxicity profiles of TPhP in amphibians is limited. Therefore, the adverse effects and threshold concentrations of TPhP on metamorphosis, growth, locomotion, and hepatic antioxidants of Gosner stage 25 Polypedates megacephalus tadpoles under long-term (35 d) exposure to six TPhP concentrations until complete metamorphosis were assessed. Additionally, the overall effect of using integrated multiple biomarkers were determined to demonstrate the potential ecological risks of waterborne TPhP at environmentally relevant concentrations in amphibian tadpoles. With increasing TPhP concentrations, physical parameters (snout-vent length, body mass, condition factor, and hepatic somatic index), jumping distance, hepatic catalase, and superoxide dismutase activities decreased, whereas metamorphosis time and malondialdehyde content increased. The threshold concentration of TPhP that affected the tadpole biomarker, except for metamorphosis rate and jumping distance, was 50-400 µg/L. Furthermore, the standardized scores of the examined integrated biomarkers in the six TPhP concentrations were visualized using radar plots and calculated as the integrated biomarker responses (IBRs). The varying TPhP concentrations had different scores in the radar plots, and the threshold for affecting the IBR value was 10 µg/L, which was close to the TPhP concentration in surface waters. Additionally, IBR values were strongly positively correlated with the TPhP concentrations. These findings indicate that environmentally relevant exposure to waterborne TPhP can pose an ecological risk to amphibian tadpoles. This study can serve as a reference and assist in the formulation of relevant policies and strategies to control TPhP pollution in water bodies.

Microbial Diversity of the Chinese Tiger Frog (Hoplobatrachus rugulosus) on Healthy versus Ulcerated Skin.

The Chinese tiger frog (Hoplobatrachus rugulosus) is extensively farmed in southern China. Due to cramped living conditions, skin diseases are prevalent among unhealthy tiger frogs which thereby affects their welfare. In this study, the differences in microbiota present on healthy versus ulcerated H. rugulosus skin were examined using 16S rRNA sequences. Proteobacteria were the dominant phylum on H. rugulosus skin, but their abundance was greater on the healthy skin than on the ulcerated skin. Rhodocyclaceae and Comamonadaceae were the most dominant families on the healthy skin, whereas Moraxellaceae was the most dominant family on the ulcerated skin. The abundance of these three families was different between the groups. Acidovorax was the most dominant genus on the healthy skin, whereas Acinetobacter was the most dominant genus on the ulcerated skin, and its abundance was greater on the ulcerated skin than on the healthy skin. Moreover, the genes related to the Kyoto Encyclopedia of Genes and Genomes pathways of levels 2-3, especially those genes that are involved in cell motility, flagellar assembly, and bacterial chemotaxis in the skin microbiota, were found to be greater on the healthy skin than on the ulcerated skin, indicating that the function of skin microbiota was affected by ulceration. Overall, the composition, abundance, and function of skin microbial communities differed between the healthy and ulcerated H. rugulosus skin. Our results may assist in developing measures to combat diseases in H. rugulosus.

Complete mitochondrial genome of Takydromus kuehnei (Squamata: Takydromus) and its phylogenetic analysis.

We sequenced and annotated the complete mitochondrial genome (mitogenome) of Takydromus kuehnei Van Denburgh, 1909 (Squamata: Takydromus). This mitogenome was 17,224 bp long and encoded 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, one non-coding regions of an L-strand replication origin and a displacement loop region. The overall nucleotide composition was 32.8% of A, 13.8% of G, 24.8% of T, and 30.5% of C. Phylogenetic analysis using maximum likelihood method validated the taxonomic status of T. kuehnei, exhibiting the close relationship with the species from the genus Takydromus.

The complete mitochondrial genome of Rana wuyiensis (Anura, Ranidae) and its phylogenetic analysis.

The complete mitochondrial genome (mitogenome) of Rana wuyiensis (Anura, Ranidae) was sequenced and annotated. The circular complete mitogenome was 17,779 bp in length (OL467321) and constituted of 37 genes including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs), and a D-loop region. The result of Bayesian phylogenetic analysis indicated that R. wuyiensis exhibits a close relationship with R. johnsi. R. wuyiensis was recently discovered and therefore holds a lot of potential for future research studies. Our study added a new mitochondrial data and provided valuable reference material to the taxonomy of Rana.

Ecotoxicity assessment of triphenyl phosphate (TPhP) exposure in Hoplobatrachus rugulosus tadpoles.

Triphenyl phosphate (TPhP), a widely used aromatic organophosphate flame retardant, is known to accumulate in organisms through water, air, and soil, consequently, causing toxicity. This study is the first to evaluate the acute and sub-chronic toxicities of TPhP to amphibians. In the acute toxicity analysis, the 96-h median lethal concentration (LC50) for GS35 Hoplobatrachus rugulosus tadpoles was 2.893 mg/L, and the 10% effect concentration (EC10) was 289 µg/L. After two weeks of exposure to low TPhP concentrations, the survival and metamorphosis rates of H. rugulosus tadpoles decreased, and the metamorphosis time was prolonged as the TPhP concentration increased. The threshold concentration that affected tadpole survival and metamorphosis time was 50 µg/L and 100 µg/L, respectively. No significant differences were observed in the condition factor and hepatic somatic index of the tadpole after metamorphosis; however, tadpole body mass and TPhP concentration were negatively correlated. Further, TPhP inhibited the expressions of Cu-Zn sod and cat, thereby reducing the activities of superoxide dismutase and catalase in the tadpole liver. The threshold for affecting gene expression and enzymatic activity was 100 µg/L. These findings provide significant insights on the stress ecology of aquatic organisms.

Complete mitochondrial genome of the Red Keelback (Pseudagkistrodon rudis Boulenger, 1906).

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.

Analyzing the gonadal transcriptome of the frog Hoplobatrachus rugulosus to identify genes involved in sex development.

BACKGROUND: The tiger frog (Hoplobatrachus rugulosus) is listed as a national Class II protected species in China. In the context of global warming, the sex ratio of amphibians will be affected, and the development of the population will be limited. Therefore, considering the potential for a decrease in the number of amphibians, studying sex evolution and molecular regulation of gonadal development in H. rugulosus, phenomenon that are currently unclear, is of great significance. RESULTS: Here, H. rugulosus was used to explore the mechanisms regulating gonadal development in amphibians. Illumina HiSeq 3000 was used to sequence the gonadal transcriptome of male and female H. rugulosus at two growth stages to identify genes related to gonadal development and analyze expression differences in the gonads. This analysis indicated that cyp17α, hsd3ß, hsd11ß1, cyp19α, and hsd17ß12 perform vital functions in sex development in amphibians. Specifically, the expression of cyp3α, cyp17α, hsd3ß, hsd11ß1, sox2, sox9, sox30, soat, cyp19α, hsd17ß12, and hspα1s was correlated with gonadal development and differentiation in H. rugulosus, as determined using the quantitative reverse transcriptase-polymerase chain reaction. CONCLUSION: Significant differences were found in the gonadal gene expression levels in H. rugulosus of both sexes, and we identified a steroid hormone synthesis pathway in this species and analyzed related gene expression, but the changes during sex differentiation were still unclear. To our knowledge, this report presents the first analysis of the H. rugulosus gonadal transcriptome and lays the foundation for future research.

The first complete mitochondrial DNA of the Chinese short-limbed skink (Ateuchosaurus chinensis Gray, 1845) determined by next-generation sequencing.

The complete mitochondrial DNA (mtDNA) for the Chinese short-limbed skink (Ateuchosaurus chinensis Gray, 1845) was described by using next-generation sequencing. The total length of mtDNA was 16,840 bp, which contained 13 PCGs (COI-III, ND1-6, ND4L, ATP6, ATP8, and CYTB), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region (D-loop). The Bayesian inference tree showed that A. chinensis was a sister taxon to other scincid lizards in genera of Scincella, Isopachys, Sphenomorphus and Tropidophorus. The complete mtDNA of A. chinensis will be an important genetic resource to the studies of conservation and restoration of A. chinensis.

Characterization of the complete mitochondrial genome of the Chong'an Moustache Toad, Leptobrachium liui (Pope, 1947) with a phylogenetic analysis of Megophryidae.

The Chong'an Mustache Toad, Leptobrachium liui (Pope, 1947) is a Chinese endemic species, inhabiting the mountain streams with rich vegetation in southeastern China. The first complete mitochondrial genome (mitogenome) of L. liui was assembled using the data of whole-genome sequencing. The size of the complete mitogenome for L. liui was 17,190 bp, which included 13 PCGs, 23 tRNAs with two concatenated tRNAMet genes, 2 rRNAs, a non-coding region, and a D-loop. The Bayesian tree shows that L. liui was positioned near L. leishanense within the genus Leptobrachium.

Two complete mitochondrial genomes of the black-spotted stout newt (Pachytriton brevipes) and their relative phylogenetics relationship with other Salamandridae.

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 (mitogenomes) of two P. brevipes samples collected in Mount Wuyi. The assembled mitogenomes were 16,298 bp and 16,301 bp in length, and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, one non-coding region, and one control region. The phylogenetic analysis indicated the two samples of black-spotted stout newt grouped together and are the sister group of P. feii.

The complete mitochondrial genome of an Asian warty newt, Paramesotriton aurantius (Caudata: Salamandridae).

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.

Complete mitochondrial genome of the Bingzhi's stout newt (Pachytriton granulosus Chang, 1933) and its phylogenetic placement.

The Bingzhi's stout newt (Pachytriton granulosus Chang, 1933) is distributed in mountainous areas of Zhejiang, China. The first complete mitochondrial genome (mitogenome) of P. granulosus was determined by next-generation sequencing. The size of the assembled mitogenome for P. granulosus was 16,293 bp, which included 13 protein coding genes, 22 tRNAs, 2 rRNAs, a non-coding region, and a control region (D-loop). The phylogenetic analysis using Bayesian Inference validated the taxonomic status of P. granulosus, showing the close relationship with the other two species from the genus Pachytriton.