Transcriptome profiling and digital gene expression analysis of the skin of Dybowski's frog (Rana dybowskii) exposed to Aeromonas hydrophila.

Recently, populations of Rana dybowskii, an important amphibian species in Northeast China, have decreased, mainly owing to the disease caused by Aeromonas hydrophila. However, effective control methods have not yet been developed. In order to explore the immune responses of R. dybowskii upon exposure to A. hydrophila infection, Illumina high-throughput transcriptome sequencing and digital gene expression (DGE) technology were employed to investigate transcriptomic changes in the skin of R. dybowskii exposed to A. hydrophila. In this work, a total of 26,244,446 transcriptome sequencing reads were obtained and assembled into 109,089 unique unigenes using de novo assembly, and a total of 37,105 unigenes (34.0%) were functionally annotated against the non-redundant (Nr), Swiss-Prot, Cluster of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Gene expression changes in the skin tissue of R. dybowskii exposed to A. hydrophila were investigated by a tag-based DGE system, and a total of 1435 significantly differentially expressed genes were identified, including 460 that were up-regulated and 975 that were down-regulated, indicating a large change in the host transcriptome profile exposed to A. hydrophila. Among these, 478 genes were associated with immune-relevant pathways, metabolic pathways, cellular components, growth, migration, and muscle and hormone signaling pathways. We confirmed the differential expression of 106 immune-relevant genes associated with innate and adaptive immune responses. Our data provide a fairly comprehensive molecular biology background for the deeper understanding of the amphibian immune system following A. hydrophila infection.

Analysis of skin and secretions of Dybowski's frogs (Rana dybowskii) exposed to Staphylococcus aureus or Escherichia coli identifies immune response proteins.

The aim of the present study was to investigate responses in Dybowski's frogs (Rana dybowskii) exposed to bacteria, using proteomic and transcriptomic approaches. Staphylococcus aureus and Escherichia coli were used as representative Gram-positive and Gram-negative bacteria, respectively, in an infectious challenge model. Frog skin and skin secretions were collected and protein expression in infected frogs compared to control frogs by two-dimensional gel electrophoresis, silver staining, and image analysis. Proteins that demonstrated differential expression were analysed by mass spectrometry and identified by searching protein databases. More than 180 protein spots demonstrated differential expression in E. coli- or S. aureus-challenged groups and, of these, more than 55 spots were up- or down-regulated at least sixfold, post-infection. Proteins with a potential function in the immune response were identified, such as stathmin 1a, annexin A1, superoxide dismutase A, C-type lectin, lysozyme, antimicrobial peptides, cofilin-1-B, mannose receptor, histone H4, prohormone convertase 1, carbonyl reductase 1 and some components of the Toll-like receptor (TLR) signalling pathway. These molecules are potential candidates for further investigation of immune mechanisms in R. dybowskii; in particular, TLR-mediated responses, which might be activated in frogs exposed to pathogenic bacteria as part of innate immune defence, but which might also impact on adaptive immunity to infection.

Induction of antimicrobial peptides from Rana dybowskii under Rana grylio virus stress, and bioactivity analysis.

The skin glands of Ranidae are a rich source of antimicrobial peptides. In this study, the genomic RNA of Rana dybowskii was extracted from its skin while under Rana grylio virus stress. Five new cDNA sequences encoding 5 mature peptides, Ranatuerin-2YJ (GLMDIFKVAVNKLLAAGMNKPRCKAAHC), Dybowskin-YJb (IIPLPLGYFAKKP), Dybowskin-YJa (IIPLPLGYFAKKKKKKDPVPLDQ), Temperin-YJa (VLPLLETCSMTCWENNQTFGK), and Temperin-YJb (VLPLVGNLLNDLLGK), were obtained by reverse transcription polymerase chain reaction with a pair of degenerate primers designed according to the conserved terminal sequences of cDNA encoding antimicrobial peptide precursors of genus Rana. The antimicrobial activities of the peptides were analyzed, and the results demonstrated that all these peptides showed a significant anti-Rana grylio virus activity, and the virus was gradually cleared with the increase in gene expression. Among the 5 peptides obtained in this work, Ranatuerin-2YJ also showed a broad-spectrum anti-Gram-positive bacteria and anti-Gram-negative bacteria activity with a minimal inhibitory concentration of 22.5 µg/mL and 7.64% hemolysis activity, both of which were significantly lower (p < 0.05) than that of the other peptides. Moreover, Ranatuerin-2YJ was widely distributed in the skin, liver, spleen, and blood of R. dybowskii, while the other 4 peptides could only be cloned from the skin, indicating that the Ranatuerin-2YJ in vivo plays an important role in the protection against pathogen invasion.

[Methane emission flux of Zhalong Phragmites australis wetlands in growth season].

Static chamber/gas chromatogram method was adopted to measure the methane emission flux of Zhalong Phragmites australis wetlands with different water levels in a growth season from May to October, 2009, aimed to understand the methane emission pattern in natural freshwater P. australis wetland in frigid region. During the observation period, the average methane emission flux of test wetlands ranged from -21.18 to 46.15 mg x m(-2) x h(-1), with a mean of 7.67 mg x m(-2) x h(-1). In deep water zone (average water level 100 cm) and shallow water zone (average water level 25 cm), the average methane emission flux was 5.81 and 9.52 mg x m(-2) x h(-1), with a peak in August and July, respectively, and the minimum in October. In summer (from June to July), the methane emission flux in deep water zone was significantly lower than that in shallow water zone; while in spring (May) and autumn (from August to October), a reversed trend was observed. The methane emission flux had a seasonal pattern of summer > autumn > spring, and a diurnal pattern of being the highest at 12:00 and 14:00 and the lowest at 0:00. Temperature and water level were the major factors affecting the methane emission flux in freshwater P. australis wetlands in frigid region.