Pathogenicity of Aeromonas hydrophila causing mass mortalities of Procambarus clarkia and its induced host immune response.

Outbreaks of mass mortalities among cultured Procambarus clarkia occurred in a commercial hatchery during the spring of 2019 in Jiangsu province of China. Here, we exploit the pathogenicity and immune response of Aeromonas hydrophila (GPC1-2), which was isolated from diseased P. clarkia. Crayfish challenged showed similar pathological signs to the naturally diseased P. clarkia, lethal dose 50% (LD50) of the strain GPC1-2 to P. clarkia was 3.8 × 106 CFU/mL. Detection of virulence-associated genes by PCR indicated that the strain GPC1-2 carried hlyA, aerA, alt, ast, act, aha, ahp, ahpA, and ahpB. Histopathological analysis of hepatopancreas revealed that the hepatic tubule lumen and the gap between the hepatic tubules became larger, and the brush border disappeared in the P. clarkia infected by GPC1-2. Quantitive real-time PCR (qRT-PCR) was undertaken to measure mRNA expression levels for six immune-related genes in P. clarkia after A. hydrophila infection. The expression level of proPO, NOS, ALF1, TLR2, PX, and AST were detected in hemolymph, hepatopancreas, gill and intestine tissues, and clear transcriptional activation of these genes were observed in the infected individuals. These results revealed pathogenicity of A. hydrophila and its activation of host immune response, which will provide a scientific reference for the breeding and disease prevention in P. clarkia culture.

Transcriptome analysis and immune-related genes expression reveals the immune responses of Macrobrachium rosenbergii infected by Enterobacter cloacae.

Macrobrachium rosenbergii is an important cultural species in China and other Southeast Asian countries. However, Enterobacter cloacae infection has caused a great economic loss in M. rosenbergii culture industry. The immune responses of M. rosenbergii to the E. cloacae infection is not fully characterized. To investigate the immune response of M. rosenbergii against E. cloacae, we performed transcriptome analysis of the M. rosenbergii hepatopancreas with and without E. cloacae infection using RNA-seq. After assembly and annotation, 29,731 high quality unigenes were obtained from RNA-seq data. Differential expression analysis revealed the existence of 2498 significantly differently expressed genes (DEGs) at 12 h post infection, with 1365 up-regulated and 1133 down-regulated genes. Among these DEGs, some well-known immune-related genes were up-regulated significantly, including C-type lectin 1, lectin 3, anti-lipopolysaccharide factor 2, Cu/Zn superoxide dismutase and heat shock protein 70. GO analysis demonstrated 24 biological process subcategories, 14 cellular component subcategories, and 12 molecular function subcategories that were enriched among these DEGs, and some DEGs were clustered into immune related subcategories such as immune system process, response to stimulus, biological adhesion, and antioxidant activity. These DEGs were enriched into 216 KEGG pathways including a core set of immune correlated pathways notably in phagosome and lysosome. In addition, 5 up-regulated and 5 down-regulated immune-related DEGs were selected for further validation by quantitative real-time PCR and the results showed consistence with the RNA-seq data. Additionally, the expression level of six selected immune-related genes (ALF2, CLEC1, LEC3, hemocyanin1, HSP70 and SOD) based on the transcriptomic data were monitored at different point of time in hepatopancreas, gill, hemolymph and intestine. Results revealed these immune-related genes were significantly up-regulated in different tissues from 6 to 24 h after E. cloacae infection. Overall, these results provided valuable information for further studying the immune response of M. rosenbergii against E. cloacae infection.