Blunt snout bream (BSB), Megalobrama amblycephala, is a herbivorous freshwater fish native to China and a major aquaculture species in Chinese freshwater polyculture systems. Recently, the bacterium Aeromonas hydrophila has been reported to be its pathogen causing hemorrhagic septicemia clinical signs and great great ecomomic losses of farmed BSB. This study used next generation sequencing technology to better comprehensive the transcriptome profiles of response-related genes in the BSB which will facilitate further research into the resistance and susceptibility of this fish species to exogenously invasive pathogens. The data analysis of transcriptome profile and expression of immune-related genes from BSB upon A. hydrophila stimulation was also described. The main results are as follows:
1. As an initial step, the pathogenicity of A. hydrophila to BSB was carried out to confirm whether A. hydrophila is a main pathogen causing hemorrhagic septicemia in BSB. The fish was challenged with bacterial concentrations of 1.7×105, 1.7×106, 1.7×107 and 1.7×108 cfu/mL for 7 days post infection. The results showed that mortality was bacterial-dose dependent, with 100% mortality observed at day 1 at the highest dose (1.7×108 cfu/mL). Control fish exhibited cumulative mortalities of 0%. The median lethal dose (LD50) was 5×106 cfu/mL. A total of 15 Aeromonas strains were re-isolated from challenged fish and re-identified based on morphological characteristics, biochemical tests and genomic DNA gene sequencing. No bacteria were isolated from the control group. This study results indicated that A. hydrophila is capable of causing hemorrhagic septicemia in BSB. Moreover, antibiotic susceptibility test with two strains D4 and HU201301 was investigated; the results showed that both strains were sensitive to most of the tested antibiotic drugs.
2. To understand the immune response of the BSB to A. hydrophila infection, the RNA-Seq technology was utilized to analyze the transcriptomic profile after artificial bacterial infection. Two cDNA libraries synthesized from tissues collected from control BSB or those injected with A. hydrophila were sequenced by using Illumina HiSeq2000. After de novo assembly, 155,052 unigenes (average length 692.8 bp) were isolated. All unigenes were annotated using BLASTX relative to several public databases. The sequence similarity (86%) of the assembled unigenes was to zebrafish based on the Nr database. A number of unigenes (n = 30,482) were assigned to three GO categories: biological processes (25,242 unigenes), molecular functions (26,096 unigenes), and cellular components (22,778 unigenes). 20,909 unigenes were classified into 25 KOG categories and 28,744 unigenes were assigned to 315 specific signaling pathways. In total, 238 significantly differentially expressed unigenes (mapped to 125 genes) were identified: 101 upregulated genes and 24 downregulated genes. Another 303 unigenes were mapped to unknown or novel genes. Among the known expressed genes identified, 53 were immune-related genes and were distributed in 71 signaling pathways.
3. Microsatellites (n = 10,877), including di- to pentanucleotide repeat motifs, were also identified in the BSB transcriptome profiles. Dinucleotide repeats were most common (7,152, 65.8%), followed by tri- (3,172, 29.2%), tetra- (549, 5.05%), and pentanucleotide repeat motifs (4, 0.04%). The lengths of the SSRs ranged from 12 to 25 bp. PCR primers were successfully designed for 5077 (46.7%) of the identified microsatellites. Furthermore, a total of 36,326 putative SNPs were discovered from the transcripts. The frequency of SNP was one SNP in 29.2 bp sequence length. The transition and transversion mutation was 21,445 and 12,553 SNPs, respectively. A ratio of transition to transversion was 1.71. Within those yielded SNPs, 10,812 SNPs identified from 2,421 unigenes could be annotated to differential functionality in comparing to public database using BLASTX for GO, KOG and KEGG. A number of SNPs (n = 7,727, 71.5%) found from 1,628 unigenes were assigned to three main GO categories: ‘cellular components’, ‘molecular function’ and ‘biology process’. In total, 5,812 (53.8%) SNPs identified from 1,324 unigenes were classified into 25 KOG categories. 4,589 (42.4%) SNPs detected from 975 unigenes were assigned to 278 KEGG pathways. Furthermore, a number of 600 SNPs found from 111 unigenes were successfully annotated to the term ‘immune system’ via KEGG classification. The database could be useful for further genetic studies in BSB.
4. In silico characterization and homology modeling of encoded proteins, including MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6, MaC3, MaC7, MaCTSL, MaMMP-9, MaIL-8 and MaIL-10 in BSB were performed. Physicochemical and functional characteristics of proteins were analyzed. The secondary and tertiary of the protein’s domains were constructed applying the comparative modeling method.
5. Reverse transcription–quantitative PCR (RT–qPCR) was used to analyze the expression of immune-related genes (MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6, MaC3, MaC7, MaCTSL, MaMMP-9, MaIL-8, and MaIL-10) in response to A. hydrophila. The results showed that all investigated genes were highly up- or and downregulated in the liver, spleen and kidney during the challenge time from 4 to 120 hours post injection. This research provides the important roles of these genes in the BSB’s innate immune system.
The results provide significant valuable information of molecular data, which are useful for further study of the immunogenetics of BSB.