Potential antagonistic relationship of fgf9 and rspo1 genes in WNT4 pathway to regulate the sex differentiation in Chinese giant salamander (Andrias davidianus).

Farmed chinese giant salamander (Andrias davidianus) was an important distinctive economically amphibian that exhibited male-biased sexual size dimorphism. Fgf9 and rspo1 genes antagonize each other in Wnt4 signal pathway to regulate mammalian gonadal differentiation has been demonstrated. However, their expression profile and function in A. davidianus are unclear. In this study, we firstly characterized fgf9 and rspo1 genes expression in developing gonad. Results showed that fgf9 expression level was higher in testes than in ovaries and increased from 1 to 6 years while rspo1 expression was higher in ovaries than in testes. In situ hybridization assay showed that both fgf9 and rspo1 genes expressed at 62 dpf in undifferentiated gonad, and fgf9 gene was mainly expressed in spermatogonia and sertoli cells in testis while strong positive signal of rspo1 was detected in granular cell in ovary. During sex-reversal, fgf9 expression was significantly higher in reversed testes and normal testes than in ovaries, and opposite expression pattern was detected for rspo1. When FH535 was used to inhibit Wnt/ß-catenin pathway, expression of rspo1, wnt4 and ß-catenin was down-regulated. Conversely, expression of fgf9, dmrt1, ftz-f1 and cyp17 were up-regulated. Furthermore, when rspo1 and fgf9 were knocked down using RNAi technology, respectively. We observed that female biased genes were down regulated in ovary primordial cells after rspo1 was knocked down, while the opposite expression profile was observed in testis primordial cells after fgf9 was knocked down. These results suggested that fgf9 and rspo1 played an antagonistic role to regulate sex differentiation in the process of the gonadal development and provided a foundation for further functional characterizations. The data also provided basic information for genome editing breeding to improve the Chinese giant salamander farming industry.

A high-density genetic map construction and sex-related loci identification in Chinese Giant salamander.

BACKGROUND: The Chinese giant salamander Andrias davidianus is an important amphibian species in China because of its increasing economic value, protection status and special evolutionary position from aquatic to terrestrial animal. Its large genome presents challenges to genetic research. Genetic linkage mapping is an important tool for genome assembly and determination of phenotype-related loci. RESULTS: In this study, we constructed a high-density genetic linkage map using ddRAD sequencing technology to obtain SNP genotyping data of members from an full-sib family which sex had been determined. A total of 10,896 markers were grouped and oriented into 30 linkage groups, representing 30 chromosomes of A. davidianus. The genetic length of LGs ranged from 17.61 cM (LG30) to 280.81 cM (LG1), with a mean inter-locus distance ranging from 0.11(LG3) to 0.48 cM (LG26). The total genetic map length was 2643.10 cM with an average inter-locus distance of 0.24 cM. Three sex-related loci and four sex-related markers were found on LG6 and LG23, respectively. CONCLUSION: We constructed the first High-density genetic linkage map and identified three sex-related loci in the Chinese giant salamander. Current results are expected to be a useful tool for future genomic studies aiming at the marker-assisted breeding of the species.

Effect of Different Positions During Surgical Preparation With Combined Spinal-Epidural Anesthesia for Elective Cesarean Delivery: A Randomized Controlled Trial.

BACKGROUND: The intraoperative 15° left-tilt position during cesarean delivery has more recently been questioned regarding its effect on fetal acid-base balance and is a frequent source of complaints by surgeons. We hypothesized that a 30° left-tilt position during surgical preparation could improve the acid-base balance of the fetus compared with the 15° left-tilt or supine position during surgical preparation. METHODS: Women undergoing elective cesarean delivery under combined spinal epidural anesthesia were randomized to a supine position, 15° left-lateral tilt position or 30° left-lateral tilt position; the position was changed to supine before the incision. Anesthetic management was standardized and included fluid loading with 10 mL/kg of normal saline followed by colloid loading. Hypotension (systolic blood pressure [SBP] reduction >20% baseline value or SBP <90 mm Hg) was treated with boluses of phenylephrine or ephedrine according to maternal heart rate. The primary outcome was umbilical arterial blood pH and the secondary outcomes included maternal SBP within 15 minutes after induction of anesthesia, the amount of vasoactive drug administered before end of the surgery, and the incidence of hypotension during cesarean delivery. RESULTS: Seventy-five patients were included. After testing by analysis of variance, there was no significant difference in the umbilical arterial pH among the 3 groups (supine group: 7.31 ± 0.03 vs 15° group: 7.30 ± 0.04 vs 30° group: 7.31 ± 0.02, P = .28). The 30° group required significantly less phenylephrine (P = .007) and ephedrine (P = .005) before the end of surgery than the supine group; however, the only benefit observed in the 15° group was that the mean SBP at 3 minutes after spinal injection was significantly improved compared with the supine group. CONCLUSIONS: Compared with the supine position, the 30° left-tilt position during surgical preparation did not significantly improve the fetal acid-base status, but it significantly reduced the use of phenylephrine and ephedrine and reduced the incidence of hypotension; however, these benefits were not observed in the 15° left-tilt group.

Genetic and codon usage bias analyses of major capsid protein gene in Ranavirus.

The Ranavirus (one genus of Iridovidae family) is an emerging pathogen that infects fish, amphibian, and reptiles, and causes great economical loss and ecological threat to farmed and wild animals globally. The major capsid protein (MCP) has been used as genetic typing marker and as target to design vaccines. Herein, the codon usage pattern of 73 MCP genes of Ranavirus and Lymphocystivirus are studied by calculating effective number of codons (ENC), relative synonymous codon usage (RSCU), codon adaptation index (CAI), and relative codon deoptimization index (RCDI), and similarity index (SiD). The Ranavirus are confirmed to be classified into five groups by using phylogenetic analysis, and varied nucleotide compositions and hierarchical cluster analysis based on RSCU. The results revealed different codon usage patterns among Lymphocystivirus and five groups of Ranavirus. Ranavirus had six over-represented codons ended with G/C nucleotide, while Lymphocystivirus had six over-represented codons ended with A/T nucleotide. A comparative analysis of parameters that define virus and host relatedness in terms of codon usage were analyzed indicated that Amphibian-like ranaviruses (ALRVs) seem to possess lower ENC values and higher CAIs in contrast to other ranaviruses isolated from fishes, and two groups (FV3-like and CMTV-like group) of them had received higher selection pressure from their hosts as having higher relative codon deoptimization index (RCDI) and similarity index (SiD). The correspondence analysis (COA) and Spearman's rank correlation analyses revealed that nucleotide compositions, relative dinucleotide frequency, mutation pressure, and natural translational selection shape the codon usage pattern in MCP genes and the ENC-GC3S and neutrality plots indicated that the natural selection is the predominant factor. These results contribute to understanding the evolution of Ranavirus and their adaptions to their hosts.

Effect of continuous intraoperative infusion of methoxamine on renal function in elderly patients undergoing gastrointestinal tumor surgery: a randomized controlled trial.

BACKGROUND: Acute renal injury (AKI) caused by hypotension often occurs in elderly patients after gastrointestinal tumor surgery. Although vasoactive drugs can increase effective filtration pressure, they may increase renal vascular resistance and reduce renal blood flow. The effect of methoxamine on renal function is not clear. METHODS: After obtaining written informed consent, 180 elderly patients undergoing elective gastrointestinal tumor surgery were randomly allocated into two groups: M group (continuous infusion of methoxamine at 2 µg/kg/min) and N group (continuous infusion of normal saline). The patients' mean arterial pressure was maintained within 20% of baseline by a continuous infusion of methoxamine or normal saline. Maintenance fluid was kept at 5 mL/kg/h. According to Kidney disease improve global outcome (KDIGO) guidelines, creatinine was measured at 1, 2 and 7 days after operation, and urine volume at 6, 12 and 24 h after operation was measured to evaluate the occurrence of AKI. 162 patients were included in the final data analysis. RESULTS: Significant differences in the incidence of postoperative Acute kidney injury (M group: 7.5%; N group: 18.3%; P < 0.05), the frequency of hypotension (M group: 1 [1-3]; N group: 3 [1-5]; P < 0.05), and the duration of intraoperative hypotension (M group: 2[0-10]; N group: 10 [5-16]; P < 0.05) were identified between the groups. Multivariate logistic regression analyses demonstrated that preoperative creatinine and the frequency of intraoperative hypotension were the common factors leading to the occurrence of postoperative AKI. The results of Cox multivariate analysis showed that age and AKI were independent risk factors for 30-day death. CONCLUSION: Compared with the intraoperative continuous infusion of placebo and methoxamine, continuous infusion of 2 µg/kg/min methoxamine reduced the incidence of postoperative AKI and other clinical complications in elderly patients undergoing gastrointestinal surgery by raising blood pressure and improved the prognosis of patients. TRIAL REGISTRATION: Trial registration: Chinese Clinical Trial Registry, ChiCTR1900020536, registered 7 January, 2019.

Genome-wide RAD sequencing to identify a sex-specific marker in Chinese giant salamander Andrias davidianus.

BACKGROUND: Chinese giant salamander Andrias davidianus is an endangered species. The success of artificial breeding provides a useful way to protect this species. However, the method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism. RESULTS: We used restriction-site-associated DNA (RAD) sequencing to isolate a sex-specific genetic marker in A. davidianus to expand knowledge of the sex determination mechanism. Four male and four female specimens were subjected to RAD sequencing, which generated 934,072,989 reads containing approximately 134.4 Gb of sequences. The first round of comparison of the assembled sequence against the opposite sex raw reads revealed 19,097 female and 17,994 male unmatched sequences. Subsequently, 19,097 female sequences were subjected to a BLAST search against male genomic data, which revealed 308 sequences unmapped to the male genome. One hundred of these were randomly selected and validated by PCR in five male and five female specimens, and four putative sex-specific sequences were produced. Further validation was performed by PCR in another 24 females and 24 males, and all female individuals exhibited the expected specific bands, while the males did not. To apply the sex-specific marker, three specimens reversed from genetic female to physiological male were found in a group exposed to elevated temperature, and 13 individuals reversed from genetic male to physiological female were obtained in a 17ß-estradiol exposed group. CONCLUSION: This is the first report of a sex-specific marker in A. davidianus and may have potential for elucidation of its sex determination mechanism and, hence, its conservation.

Characterization and function of the T-box 1 gene in Chinese giant salamander Andrias davidianus.

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx1 was knocked down by tbx1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh, cyp26a, dmrt1, and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.

Identification of critical sex-biased genes in Andrias davidianus by de novo transcriptome.

The Chinese giant salamander Andrias davidianus is a protected amphibian with high nutritional and economic value. Understanding its sex determination mechanism is important for improving culture techniques and sex control in breeding. However, little information on the characterization of critical genes involved in sex is available. Herein, sequencing of ovary and test produced 40,783,222 and 46,128,902 raw reads, respectively, which were jointly assembled into 80,497 unigenes. Of these, 36,609 unigenes were annotated, of which 8907 were female-biased and 10,385 were male-biased. Several sex-related pathways were observed, including the Wnt signaling pathway. After elevated temperature and estrogen exposure, neomale and neofemale specimens were identified by a female-specific marker for the first time. RT-qPCR analysis showed the expression profile of ten selected sex-biased genes to be exhibited consistently in male and neomale and in female and neofemale, with the exception of the Amh and TfIIIa genes. Results suggested that these genes may play important roles in A. davidianus sex determination and gonad development. This provides a basis for further investigation of the molecular mechanisms of sex determination in amphibians.

Evolution and multiple origins of zona pellucida genes in vertebrates.

Animal egg coats are composed of different glycoproteins collectively named zona pellucida (ZP) proteins. The characterized vertebrate genes encoding ZP proteins have been classified into six subfamilies, and exhibit low similarity to the ZP genes characterized in certain invertebrates. The origin and evolution of the vertebrate ZP genes remain obscure. A search against 97 representative metazoan species revealed various numbers (ranging from three to 33) of different putative egg-coat ZP genes in all 47 vertebrates and several ZP genes in five invertebrate species, but no putative ZP gene was found in the other 45 species. Based on phylogenetic and synteny analyses, all vertebrate egg-coat ZP genes were classified into eight ZP gene subfamilies. Lineage- and species-specific gene duplications and gene losses occurred frequently and represented the main causes of the patchy distribution of the eight ZP gene subfamilies in vertebrates. Thorough phylogenetic analyses revealed that the vertebrate ZP genes could be traced to three independent origins but were not orthologues of the characterized invertebrate ZP genes. Our results suggested that vertebrate egg-coat ZP genes should be classified into eight subfamilies, and a putative evolutionary map is proposed. These findings would aid the functional and evolutionary analyses of these reproductive genes in vertebrates.

Phenylephrine vs ephedrine in cesarean delivery under spinal anesthesia: A systematic literature review and meta-analysis.

BACKGROUND: In the past 20 years, many studies compared phenylephrine with ephedrine to prevent or treat hypotension in elective or emergency cesarean delivery and parturients with pre-eclampsia. A meta-analysis of the abovementioned trials is needed. METHODS: Several databases (PubMed, Embase, Web of Science and Cochrane Library) were searched from inception to April 2018 for trials comparing phenylephrine with ephedrine in cesarean delivery. The primary outcome is the incidence of maternal hypotension. RESULTS: Thirty-six trials (2439 patients) with elective cesarean delivery, three trials (400 patients) with emergency cesarean delivery and three trials (192 patients) with parturients with pre-eclampsia were included and analyzed. The incidence of hypotension did not differ in the elective surgery group (relative risk 0.83, 95% CI 0.66 to 1.05), emergency surgery group (relative risk 1.02, 95% CI 0.87 to 1.19) and pre-eclamptic parturients group (relative risk 0.93, 95% CI 0.63 to 1.37). The phenylephrine group had a higher incidence of bradycardia and lower incidences of tachycardia and nausea or vomiting in all three patient groups. The phenylephrine group also had lower fetal acidosis rate, higher umbilical artery and vein pH values and less base excess in the elective surgery. The abovementioned outcomes were similar in the emergency surgery group and the pre-eclampsia group. Publication bias for hypotension was detected. However, the trim and fill method demonstrated that the publication bias had little impact on hypotension. Trial sequential analysis of hypotension in elective surgery showed that this meta-analysis lacked a sufficient cumulative sample size and that further studies should be included. CONCLUSION: Phenylephrine and ephedrine were both effective in maintaining hemodynamic balance. Newborns benefited more from phenylephrine in elective cesarean delivery, but not in emergency cesarean delivery or in parturients with pre-eclampsia. More trials should be included to achieve more conclusive results.

Goal-directed fluid therapy versus conventional fluid therapy in colorectal surgery: A meta analysis of randomized controlled trials.

OBJECTIVES: This meta-analysis was conducted to compare the effects of goal-directed fluid therapy (GDFT) versus conventional fluid therapy (CFT) in colorectal surgery on patients' postoperative outcome and to detect whether the results differ between studies with the Enhanced Recovery After Surgery (ERAS) protocol and those without, between studies using different devices for GDFT, or between different surgical approaches (laparoscopy or laparotomy). METHODS: The Cochrane Library, PubMed, Embase, Wanfang Data and ClinicalTrials.com were searched for studies from January,1990 to February, 2018. Randomized controlled trials (RCTs) comparing both two abovementioned fluid therapy protocols in colorectal surgery were included. The primary outcome was 30-day mortality after surgery. Secondary outcomes were length of hospital stay (LOS), complication rate, ICU admission and gastrointestinal indicators. RESULTS: Eleven studies were included, including a total of 1281 patients: the GDFT group included 624 patients and the control group included 657 patients. No significant differences were found between groups in 30-day mortality (relative risk, RR 0.86,0.28 to 2.63, P = 0.79), LOS (weighted mean difference, WMD 0.22,-0.1 to 0.55, P = 0.18), and ICU admission (RR 0.42, 0.17 to 1.04, P = 0.06). However, the GDFT group had a lower complication rate (RR 0.84,0.71 to 0.99, P = 0.04). In subgroup analyses, time to first flatus and time to tolerate an oral diet were shorter in GDFT group than the control group in studies who did not use the ERAS protocol. No publication bias was identified according to Begg's test. CONCLUSION: Compared with conventional fluid therapy, GDFT may not improve patients' postoperative outcome in colorectal surgery. However, the improvement of gastrointestinal function associated with GDFT over conventional fluid therapy was significant in the surgeries that did not use the ERAS protocol.

MicroRNA repertoire and comparative analysis of Andrias davidianus infected with ranavirus using deep sequencing.

Andrias davidianus is a large and economically important amphibian in China. Ranavirus infection causes serious losses in A. davidianus farming industry. MicroRNA mediated host-pathogen interactions are important in antiviral defense. In this study, five small-RNA libraries from ranavirus infected and non-infected A. davidianus spleens were sequenced using high throughput sequencing. The miRNA expression pattern, potential functions, and target genes were investigated. In total, 1356 known and 431 novel miRNAs were discovered. GO and KEGG analysis revealed that certain miRNA target genes are associated with apoptotic, signal pathway, and immune response categories. Analysis identified 82 downregulated and 9 upregulated differentially expressed miRNAs, whose putative target genes are involved in pattern-recognition receptor signaling pathways and immune response. These findings suggested miRNAs play key roles in A. davidianus's response to ranavirus and could provide a reference for further miRNA functional identification, leading to novel approaches to improve A. davidianus ranavirus resistance.

Comparative transcriptome reveal the potential adaptive evolutionary genes in Andrias davidianus.

To search the evidence of molecular evolution mechanism for aquatic and cave habitat in Andrias davidianus, the evolution analysis was carried out among several species transcriptome data. The transcriptome data of Notophthalmus viridescens, Xenopus tropicalis, Cynops pyrrhogaster, Hynobius chinensis and A. davidianus were obtained from the Genbank and reassembled except Xenopus tropicalis. The BLAST search of transcriptome data obtained 1244 single-copy orthologous genes among five species. A phylogenetic tree showed A. davidianus to have the closest relationship to H. chinensis. Fourteen positively selected genes were detected in A. davidianus and N. vridescens group and fifteen in A. davidianus and H. chinensis group. Five genes were shared in the both groups which involved in the immune system, suggesting that A. davidianus adaptation to an aquatic and cave environment required rapid evolution of the immune system compared to N. viridescens and H. chinensis.

Identification and expression of forkhead box genes in the Chinese giant salamander Andrias davidianus.

In the present study, 21 forkhead box (Fox) genes were identified in Andrias davidianus, including 13 full-length genes and eight partial sequences. Phylogenetic analysis showed that most were conserved in other investigated amphibians, whereas the Foxk1 gene was found exclusively in A. davidianus. Molecular evolution analysis indicated that most Fox genes underwent purifying selection, whereas two sites of the adFoxp4 gene showed positive selection and were located on the adFoxp4 protein surface. Expression profiles of all Fox genes identified were analysed in the hypothalamic-pituitary-gonad axis by reverse transcription-quantitative polymerase chain reaction. Eighteen genes exhibited sexually dimorphic expression (15 ovary-biased and three testis-biased genes), whereas two genes showed no difference between ovary and testis. Further investigation of 12 selected sexually dimorphic Fox genes showed changes in the expression profile of 11 genes in the ovary of larvae reared at high temperatures (28°C). The results of the present study provide information on Fox genes in an amphibian and suggest that they play key roles in sexual development and reproduction in A. davidianus.

Identification and expression of cytochrome P450 genes in the Chinese giant salamander Andrias davidianus.

Cytochrome P450 (cyp) genes play vital roles in biological processes, including in metabolism of endogenous and exogenous compounds. Exogenous hormone influences on gene expression that leads to disruption of gonad development have been studied in several species, but whether exogenous hormones affect cyp genes that show sexually dimorphic expression remains to be determined. Here, we identified and characterized cyp genes from the widely-cultured Chinese giant salamander Andrias davidianus. We obtained 20 cyp genes including 11 genes with complete sequences. Phylogenetic analyses supported the classification of cyp genes similar to other vertebrates. Expression profile of female and male salamanders showed multiple cyp genes to exhibit higher expression in ovary than in testis, including cyp26a, cyp19a, cyp1a1, cyp4v2, cyp3a24, cyp2c20, cyp2d14, cyp2d15, and cyp4b; while cyp11a, cyp2b11, cyp11b1, cyp4f22, cyp2j6, cyp2k1, cyp2k4, cyp1a5 was higher in testis than in ovary. Seven sex-biased genes were detected after aromatase inhibitor injection and with exposure to high water temperatures. Cyp2k1, cyp11b1, and cyp2j6 expression were increased, while cyp26a, cyp2c20, cyp4b genes were decreased with aromatase inhibition. In ovary, cyp26a and cyp2c20 expression were significantly reduced; whereas cyp2k1, cyp2j6, and cyp2k4 expression were significantly elevated with no changes in cyp11a and cyp11b1 expression after temperature treatment. These findings provide valuable information for further study of sex differentiation mechanisms and cyp gene evolution.

Characterization and tissue distribution of Lhx9 and Lhx9α in Chinese giant salamander Andrias davidianus.

Lhx9 is an LIM (named for the first three proteins in which the domain was found, Lin-11, Isl1 and Mec-3) homeodomain protein involved in development and differentiation of the gonad. In this study, we isolated the full-length Lhx9 and Lhx9α from Andrias davidianus, detected the tissue distribution and analysed the methylation of the promoters. We identified Lhx9 of 1411 bp and Lhx9α of 1153-bp length, differing in the 3'-flanking region, encoding 399 and 330 amino acids, respectively. The Lhx9 gene was detected primarily in liver, ovary and heart with moderate expression in brain, pituitary, intestine and spleen, and low expression in the remaining examined tissues, while Lhx9α expression was high in heart, pituitary and liver, and low in spleen and stomach. Significantly higher Lhx9 expression was observed in ovary than in testis, with no differences in Lhx9α expression between testis and ovary observed. Bisulphite sequencing revealed significantly higher methylation in testis compared to ovary. The methylation level of CpG sites -733, -673, -615 and -594 exhibited significantly higher levels in testis than in ovary, which was negatively correlated with Lhx9 expression. The methylation and expression patterns suggested that promoter methylation suppressed expression of Lhx9 in A. davidianus.

Sexually Dimorphic Expression of Foxl2 and Ftz-F1 in Chinese Giant Salamander Andrias Davidianus.

Foxl2 and FTZ-F1 play a crucial role in the regulation of gonad development in fish and mammals, but studies of their function in amphibians are scarce. We isolated the full length of Foxl2 (adFoxl2) and Ftz-F1 (adFtz-f1) cDNA from the Chinese giant salamander Andrias davidianus and quantified its expression in various tissues and developing gonads. The adFoxl2 gene encodes 301aa including a conserved forkhead box, and the adFtz-f1 gene encodes 467aa containing an Ftz-F1 box. The amino acid sequences showed high homology with other amphibians. adFoxl2 expression was high in ovary, whereas adFtz-f1 was higher in testis, moderate in pituitary, ovary, and kidney; and low in the remaining tested tissues. Expression of adFoxl2 gradually increased from 1Y to 5Y in ovary, whereas adFtz-f1 expression gradually decreased in testis. In addition, adFoxl2 and adFtz-f1 were detected in granulosa cell in ovary and in spermatocytes in testis. The adFoxl2 transcription was inhibited in brain and ovary after treatment with methyltestosterone and with letrozole, whereas adFtz-f1 expression was upregulated. High-temperature suppressed the expression of adFxl2 in ovary and enhanced the transcription of adFtz-f1. These results suggest that adFoxl2 functioned in ovary differentiation, whereas adFtz-f1 played a role in testis development, which lays a foundation for study of the sex differentiation mechanism in A. davidianus.

Characterization and expression of cyp19a gene in the Chinese giant salamander Andrias davidianus.

We cloned the full length cyp19a of Chinese giant salamander Andrias davidianus, determined its distribution in tissues and developing gonads, and analyzed the CpG methylation pattern of the cyp19a promoter. The results revealed isoforms of 1706 bp (G arom) and 1698 bp (B arom) in length, differing in the 5' flanking region, both encoding 502 amino acids. The G arom gene was observed mainly in the ovary and kidney, with little in other investigated tissues, while B arom expression was high in the brain, ovary, testis, and pituitary, with low or undetected expression in other examined tissues. Total aromatase expression was high in the ovary; moderate in the kidney, brain, testis, and pituitary; and low in the remaining tissues. G arom expression was significantly higher in the ovary than in the testis and gradually decreased with maturation of the salamander. A single injection of methyltestosterone or letrozole resulted in ovarian G arom expression decreasing over a 12-96 h period. A 1366 bp sequence of the cyp19a promoter was cloned and shown to be conserved in selected species. CpG methylation level was negatively correlated with cyp19a expression in the examined tissues and developing ovaries. Five and three CpG methylation sites positively correlated with DNA methylation levels in tissues and developing ovary, suggesting that they play an important role in regulating cyp19a expression. The aromatase gene showed two isoforms with distinct expression patterns, and the promoter methylation level at specific CpG sites was associated with variation in expression profiles of tissues and developing ovaries.

Cloning, expression of, and evidence of positive selection for, the prolactin receptor gene in Chinese giant salamander (Andrias davidianus).

Prolactin receptor (PRLR) is a protein associated with reproduction in mammals and with osmoregulation in fish. In this study, the complete length of Chinese giant salamander Andrias davidianus prolactin receptor (AD-prlr) was cloned. Andrias davidianus prlr expression was high in the kidney, pituitary, and ovary and low in other examined tissues. The AD-prlr levels were higher in ovary than in testis, and increased in ovaries with age from 1 to 6 years. To determine effect of exogenous androgen and aromatase inhibitor on AD-prlr expression, methyltestosterone (MT) and letrozole (LE) were injected, resulting in decreased AD-prlr in both brain and ovary, with MT repressing prlr transcription more rapidly than did LE. The molecular evolution of prlr was assessed, and found to have undergone a complex evolution process. The obranch-site test detected four positively selected sites in ancestral lineages prior to the separation of mammals and birds. Fourteen sites underwent positive selection in ancestral lineages of birds and six were positively selected in amphibians. The site model showed that 16, 7, and 30 sites underwent positive selection in extant mammals, amphibians, and birds, respectively. The positively selected sites in amphibians were located outside the transmembrane domain, with four in the extracellular and three in the intracellular domain, indicating that the transmembrane region might be conserved and essential for protein function. Our findings provide a basis for further studies of AD-prlr function and molecular evolution in Chinese giant salamander. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 707-719, 2015. © 2015 Wiley Periodicals, Inc.

Pathological and microbiological findings from mortality of the Chinese giant salamander (Andrias davidianus).

The Chinese giant salamander, Andrias davidianus, is a nationally protected and cultured species in China. Recently, a severe epizootic occurred in cultured Chinese giant salamanders in Hubei, Hunan, Sichuan, Shaanxi, and Zhejiang provinces of China, causing substantial economic losses. The typical clinical signs of diseased larval animals were jaw and abdominal swelling and subcutaneous hemorrhaging. Diseased adult animals exhibited skin hemorrhages, ulceration of the hind limbs, and multiple hemorrhagic spots in the visceral organs. Histopathological observation indicated tissue necrosis and cytoplasmic inclusions in the spleen, liver and kidney, suggestive of viral disease. A viral agent was isolated from affected tissues in cell culture. The virus was determined to be pathogenic after experimental infection. Electron microscopy revealed iridovirus-like virions with a size of 140-180 nm in diameter inside the kidney of naturally infected animals and in cell culture. The major capsid protein (MCP) of the virus exhibited 98-99 % sequence identity to ranaviruses. Additionally, phylogenetic analysis indicated that the virus belonged to the genus Ranavirus. Comparative analysis of the MCP gene sequence with those of other viruses previously isolated from Chinese giant salamanders revealed that these isolates were highly similar, although a few variations were observed. The virus was preliminarily named Chinese giant salamander iridovirus (GSIV).