Hemolymph exosomes inhibit Spiroplasma eriocheiris infection by promoting Tetraspanin-mediated hemocyte phagocytosis in crab.

Exosomes released from infected cells are thought to play an important role in the dissemination of pathogens, as well as in host-derived immune molecules during infection. As an intracellular pathogen, Spiroplasma eriocheiris is harmful to multiple crustaceans. However, the immune mechanism of exosomes during Spiroplasma infection has not been investigated. Here, we found exosomes derived from S. eriocheiris-infected crabs could facilitate phagocytosis and apoptosis of hemocytes, resulting in increased crab survival and suppression of Spiroplasma intracellular replication. Proteomic analysis revealed the altered abundance of EsTetraspanin may confer resistance to S. eriocheiris, possibly by mediating hemocyte phagocytosis in Eriocheir sinensis. Specifically, knockdown of EsTetraspanin in E. sinensis increased susceptibility to S. eriocheiris infection and displayed compromised phagocytic ability, whereas overexpression of EsTetraspanin in Drosophila S2 cells inhibited S. eriocheiris infection. Further, it was confirmed that intramuscular injection of recombinant LEL domain of EsTetraspanin reduced the mortality of S. eriocheiris-infected crabs. Blockade with anti-EsTetraspanin serum could exacerbate S. eriocheiris invasion of hemocytes and impair hemocyte phagocytic activity. Taken together, our findings prove for the first time that exosomes modulate phagocytosis to resist pathogenic infection in invertebrates, which is proposed to be mediated by exosomal Tetraspanin, supporting the development of preventative strategies against Spiroplasma infection.

iTRAQ-based differential proteomic analysis of high- and low-virulence strains of Spiroplasma eriocheiris.

Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

Development and application of the MIRA and MIRA-LFD detection methods of Spiroplasma eriocheiris.

The tremor disease (TD) caused by Spiroplasma eriocheiris is the most destructive disease of the Chinese mitten crab, Eriocheir sinensis. This study attempts to construct Multienzyme Isothermal Rapid Amplification (MIRA), a quick and simple nucleic acid amplification method that operates at room temperature. Based on the gene sequences of S. eriocheiris, appropriate amplification primers were constructed and screened in this investigation. Both the relevant specific probe and the chosen specific amplification primers were designed and labeled. The MIRA and MIRA-LFD reaction conditions were then optimized. The result showed MIRA and MIRA-FFD could identify S. eriocheiris at 37 °C in 30 min and 15 min, respectively. To investigate the specificity of MIRA and MIRA-LFD, three Gram-negative bacteria (Bacillus subtilis, Bacillus thuringiensis, and Staphylococcus aureus), three Gram-positive bacteria (Escherichia coli, Aeromonas hydrophila, and Salmonella typhimurium) and S. eriocheiris were selected. The result showed MIRA and MIRA-LFD were highly specific to S. eriocheiris and did not react with other six pathogens. The sensitivities of PCR, MIRA, and MIRA-LFD were then evaluated. The result showed the detection limit of PCR is 1 ng/L whereas the detection limit of MIRA and MIRA-LFD is 10 pg/L. Finally, the established MIRA and MIRA-LFD detection methods had the advantages of being quick, sensitive, and specific for S. eriocheiris detection, as well as not requiring any specialized equipment.

Integrated analysis of mRNA and microRNA transcriptome related to immunity and autophagy in shrimp hemocytes infected with Spiroplasma eriocheiris.

In recent years, the industry in charge of the cultivation of Macrobrachium nipponense (M.nipponense) has suffered significant economic losses due to an infectious pathogen called Spiroplasma eriocheiris (S.eriocheiris). There has therefore been a need to identify the key immune and autophagy genes that respond to M.nipponense's infection with S. eriocheiris to analyze its immune response mechanism and the regulation of related microRNAs (miRNAs). In this study, the mRNA and miRNA transcriptome of M.nipponense's hemocytes were analyzed at different stages of infection. This analysis employed the second and third-generation sequencing technologies. In the mRNA transcriptome, 1656 genes were expressed in healthy and susceptible M.nipponense. 892 of these were significantly up-regulated, while 764 were down-regulated. 118 genes with significant differences in autophagy, endocytosis, lysosome, Toll, IMD, and VEGF pathways were obtained from the transcriptome. In the miRNA transcriptome, 312 miRNAs (Conserved: 112, PN-type: 18, PC-type: 182) were sequenced. 74 were significantly up-regulated, and 57 were down-regulated. There were 25 miRNAs involved in regulating the Toll and IMD pathways, 41 in endocytosis, 30 in lysosome, and 12 in the VEGF pathway. An integrated analysis of immune-related miRNAs and mRNAs showed that miRNAs with significant differences (P < 0.05) such as ame-miR-29b-3p, dpu-miR-1and PC-3p-945_4074, had corresponding regulatory relationships with 118 important immune genes such as Relish, Dorsal, Caspase-3, and NF-κB. This study obtained the key immune and autophagy-related genes and corresponding regulatory miRNAs in M. nipponense's hemocytes in response to an infection by S.eriocheiris. The results can provide vital data that further reveals the defense mechanism of M.nipponense's immune system against S.eriocheiris. It can also help further comprehension and interpretation of M.nipponense's resistance mechanism to the invading S.eriocheiris, and provide molecular research information for the realization of host-directed therapies (HDT) for M.nipponense.

Integrated transcriptome analysis of immune-related mRNAs and microRNAs in Macrobrachium rosenbergii infected with Spiroplasma eriocheiris.

Macrobrachium rosenbergii (M. rosenbergii), is a major aquaculture species in China and Southeast Asia. However, infection with Spiroplasma eriocheiris (S. eriocheiris) has caused huge economic losses to the cultivation of M. rosenbergii. Currently, there are few reports on the immune response mechanism of M. rosenbergii that are infected with S. eriocheiris. To clarify the immune response mechanism of M. rosenbergii infected with S. eriocheiris, the key immune genes which respond to the infection with the pathogen and the regulation of related microRNAs (miRNAs) on them were identified. In this study, the mRNA and miRNA transcriptome of hepatopancreas of M. rosenbergii at different infection stages were analyzed using high-throughput sequencing and qRT-PCR. In the mRNA transcriptome, 27,703 and 33,402 genes were expressed in healthy and susceptible M. rosenbergii, respectively. By digital gene-expression profiling analysis, 23,929 and 24,325 genes were expressed, and 223 and 373 genes were significantly up-regulated and down-regulated, respectively. A total of 145 key genes related to Toll, IMD, JAK/STAT and MAPK were excavated from the transcriptome. In the miRNA transcriptome, 549 miRNAs (Conserved: 41, PN-type: 83, PC-type: 425) were sequenced, of which 87 were significantly up-regulated and 23 were significantly down-regulated. Among the related immune pathways, there are 259 miRNAs involved in the regulation of target genes in the Toll and IMD pathways, 231 JAK/STAT pathways and 122 MAPK pathways. qRT-PCR differential detection of immune-related miRNAs and mRNAs showed that 22 miRNAs with significant differences (P < 0.05) such as mro-miR-100, PC-mro-3p-27 and PN-mro-miR-316 had corresponding regulatory relationships with 22 important immune genes such as TLR2, TLR3, TLR4, TLR5, MyD88, Pelle and Relish in different stages after infection. In this study, the immune genes and related regulatory miRNAs of M. rosenbergii in response to S. eriocheiris infection were obtained. The results can provide basic data to further reveal the immune defense mechanism of M. rosenbergii against S. eriocheiris infection.