In recent years, studies on the septicemia of cultivated fish caused by Ea have been reported (Armwood et al., 2019; Shao et al., 2015), but the molecular mechanism of the anti-infection in eel is not clear. In a previous study, we found that the Ea mainly invaded the liver of eels (Zhai et al., 2021), and in this study the infected liver was found thrombosis, celluloid degeneration of vascular wall and hepatocyte atrophy in the control infected eels (Fig. 1E). Eels in the FCIA_Li group showed slight pathological changes in the liver post the challenge of Ea (Fig. 1C), and the RPS of FCIA_Li vs Con_Li was 50% due to the liver bacteria load of FCIA_Li group was much lower than that of the Con_Li group at 24 and 48 hpc (Fig. 1B). Based on the RNA-seq of mRNA and lncRNA, the lncRNAs of European eels in the compare of FCIA_Li vs Con_Li were preliminarily explored, and the anti-infection related DEGs targeted by DE-lncRNAs (padj < 0.05) were firstly analyzed to reveal the anti-infection effect of the FCA and FIA adjuvant post the challenge of Ea.
LncRNA has been found to play an important role in the pathogenesis of many diseases (Chen et al., 2013). From the functional point of view, lncRNA mainly acts on the transcription, post transcription, translation and apparent level of the target gene respectively through Cis and Trans regulation to regulate the expression of the target gene (Florian and Joshua, 2018), so as to find out the different GO term or KEGG pathways of different treatments through the regulation of lncRNA (Schmittand and Chang, 2016). Co-location refers to the fact that lncRNA may regulate the nearby protein coding genes through analyzed the genes within 100 kb upstream and downstream of lncRNA (Bao et al., 2019). In this study, the co-expression and co-location of lncRNA and mRNA were used to predict the target genes of all DE-lncRNAs. The results showed that there were 129 DE-lncRNAs between 2 infection groups of eels, and the target DEGs in co-expression and co-location were as high as 7742 and 942, respectively.
In terms of the RNA-seq in eel liver, the average number of total reads of the Li group was 9.5 G while it was more than 14 G in two infection groups, indicating Ea infection promoted the gene transcription of the infected eels (Houtz et al., 2017). In this study, we also analyzed the DEGs, DE-mRNAs and DE-lncRNAs. The results showed no distinctive difference between Li and FCIA_Li groups in DEGs and DE-mRNAs, but DE-lncRNAs were distinctively different (Fig. 2) and it was selected to investigate the cause of lower mortality in the FCIA _Li group (Fig. 1A). So far, there is no report on the molecular mechanism of the protection effect of Freund's adjuvant, and this study will preliminarily reveal the nonspecific protective mechanism of the adjuvant.
European eels were inoculated with the FCA and FIA at 0 d and 14 d respectively to boost the protective effect to the Ea infection, and the results showed the RPS was 50% in European eels, much higher than 11% RPS after Ea challenge post 28 d of the solo FIA inoculated (He et al., 2020). Reason for RNA-seq at 48 hpc was that eels start to die from the third day in the FCIA group resulting in unavailable sampling (Fig. 1A), and significant bacteria quantity and obvious pathological changes between the eel livers in two infection groups were observed at 48 hpc (Fig. 1B-E). Venn and volcano map of DE-lncRNAs showed, compared to the Li group, most of the DE-lncRNAs in two infected groups were downregulated (Fig. 3B, 44 and 34 significantly upregulated corresponding to 67 and 76 downregulated). However, volcano map showed 71 upregulated and 58 downregulated lncRNAs in the compare of FCIA_Li vs Con_Li group. Therefore, the DE-lncRNAs between two infected groups more likely reveal the anti-infection molecular mechanism of the Freund's adjuvant to the Ea challenge (Huang et al., 2015).
The results showed that 88 and 96 GO terms were enriched (p < 0.05) in the co-expressing and co-location target genes respectively, including cellular response to stimulus, membrane, protein binding and binding processes et al (Fig. 3C, 3D). Of which the process of cellular response to stimulus was directly correlate with the immune function of the host (Liu et al., 2017). Eight enriched KEGG pathways were found in the co-expression DEGs targeted by lncRNAs (Fig. 3E), among which the pathways of neuroactive ligand-receptor interaction (Wang et al., 2013), Calcium signaling pathway and Wnt signaling pathway (De, 2011), MAPK signaling pathway (Wu et al., 2019), CAMs (Krishnan et al., 2019), and Cytokine-cytokine receptor interaction (Han et al., 2019) were directly related to non-specific immune clearance function.
Cell adhesion molecules (CAMs) are glycol-proteins expressed on the cell surface and play a critical role in a wide array of biologic processes that include hemostasis, the immune response, inflammation, embryogenesis, and development of neuronal tissue (Khodabandehlou et al., 2017). There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins (Samanta & Almo, 2015). Membrane proteins that mediate immune cell–cell interactions fall into different categories, namely those involved in antigen recognition, costimulation and cellular adhesion (Gennarini & Furley, 2017). There is only the CAMs KEGG pathway was enriched in co-location (Fig. 3F) in this study, and 15 DEGs in CAMs targeted by 6 DE-lncRNAs mainly encodes Myelin-associated glycoprotein (Bornhöfft et al., 2018), B-cell receptor CD22 (Li et al., 2019), Class I histocompatibility antigen (He et al., 2020) and Hemicentin proteins (Schultz et al., 2005). These proteins played important role in the process of the host anti-Ea infection.
In this study, 23 immune-related DEGs were randomly selected for qRT-PCR verification. Although 20 DEGs were basically kept at the same level, there were some differences between the results of qPCR verification and RNA-seq (Fig. 4) and the fit linear result showed their correlative coefficient is relatively low, which probably due to the multiple transcripts of some gene in eukaryotic cells. Primers used in qPCR is actually designed based on the level of transcripts, but RNA-seq can be used for differential analysis at both gene and transcripts level. For the same gene, qPCR is the difference in the expression level of a single transcript between samples, while RNA-seq is the comprehensive result of the expression level of multiple transcripts due to alternative splicing of a gene. Compare to the Con_Li group, 9 genes of 23 immune related genes increased more than 10 times in the FCIA_Li group (Fig. 4C), indicating these genes involved in nonspecific immunity in eels (Zhao et al., 2020; He et al., 2020; Xiao et al., 2022).
The interaction between DE-lncRNAs of 2 infection groups and the target DEGs through co-expression and co-location showed 129 DE-lncRNAs targeted 8684 DEGs. To compare the possible anti-bacterial function of the target DEGs, we selected 16 DE-lncRNAs changed more than 20 times between 2 infected groups (Fig. 5 & Fig. 6; Table 3). The result showed most of DEGs targeted by upregulated DE-lncRNAs in the FCIA_Li group were directly or indirectly involved in the immune function of the host, but that of DEGs targeted by upregulated DE-lncRNAs in the Con_Li group were closely related to immunosuppression or apoptosis. Some of the upregulated DEGs were also verified by the qRT-PCR method (Fig. 4). Therefore, the Freund's adjuvant enhanced the non-specific immune function of European eel and prevented the apoptosis of hepatocytes after bacterial infection, but immune function of eels in the control group was suppressed after the Ea infection and hepatocytes were apoptotic due to pro-inflammatory factors, which eventually led to the increased mortality of infected eels.