3.1. Identification and nomenclature of HO genes
A total of 6 HO genes were identified in the common carp genome database, which was distributed on four chromosomes and more abundant than other vertebrate genomes. Among them, human, clawed frog, coelacanth, tilapia, medaka, channel catfish, stickleback and fugu have the same number of genes with two HO genes(Table 2). Significant amplification of HO genes was observed in common carp and zebrafish, with six and four HO genes, respectively. The mouse has the isoform HO-3 gene of the HO-2 gene compared to other species, which is consistent with previous studies. For gene annotation and nomenclature, six common carp HO genes were classified based on the similarity of HO genes to four known zebrafish HO homologs. Duplicated genes were assigned Latin numbers suffix (−1 and −2). So the HO genes in common carp were named HO-1a、HO-1b、HO-2a-1、HO-2a-2, etc. All HO gene sequences were deposited in the NCBI database with the continuous accession numbers MZ286949-MZ286954, and the detailed information about coding sequence, location, and accession number was summarized in Table 3.
3.2 Phylogenetic analysis of HO gene family
Cd2+ can be enriched in various species. In order to better study the distribution of HO genes in species and the phylogenetic relationship with common carp HO genes, we extracted the amino acid sequences of 29 HO genes from different representative species and used them to construct the phylogenetic topology(Fig 1). The ML topology is divided into 2 subfamilies, of which subfamily I (containing HO-1) and subfamily II (containing HO-2). In the teleost clade, common carp HO (blue pentagram) are all homologous to other scleractinians concentrated on another main branch and are particularly closed to the homologue of the model organism of bony fish, zebrafish (green pentagram), except for the clawed frog and coelacanth, which are on a branch with tetrapods. In the tetrapods clade, the mammals first come together (gray circles) and then in grouping with other tetrapods (red circles). Finally, the bony fish major clade and tetrapods major clade (blue part) converged into one major clade.
3.3 Tissue expression patterns of HO genes
Analysis of HO gene family expression in healthy tissues of carp based on qRT-PCR using gene-specific primers showed that HO genes were expressed in all selected tissues(Fig 2). HO-1a/b is mainly expressed in the liver, gills, blood, kidney and head kidney. The relative expression of HO-1 was significantly higher in the head kidney than in any other tissue (p < 0.05). The relative expression levels of HO-1 in the skin, heart and spleen were low and not significantly different. HO-2a-1/2 has high expression levels in the liver, blood, and muscle, but very low expression levels in the heart, spleen, and gonads. HO-2b-1/-2 has higher levels in more tissues, e.g., significantly higher expression levels in the liver, brain, gonads and intestine, and lower expression in the heart and blood.
3.4 Effects on HO gene expression levels under Cd2+ stress
To better understand the HO gene family, we chose to add the Cd2+ to the culture water and performed qRT-PCR to determine the expression levels of HO genes in the carp intestine(Fig 3) (Fig 4). In the foregut, when subjected to Cd2+ stress for 30d, HO-1b, HO-2a-1, and HO-2b-1 showed different levels of increase , in contrast to HO-1a, HO-2a-2, and HO-2b-2, which showed a decrease in expression levels. However, as the duration of Cd2+ stress increased, the expression levels of HO-1a and HO-2b-2 appeared to increase and were significantly higher than those of the control group at 60d. Overall, in the foregut, the expression of all HO genes increased in varying degrees after 30d / 60d of Cd2+ stress. In the midgut, compared with the control group, with the increase of Cd2+ stress time, almost all HO genes except HO-1b showed a significant decrease in expression level. The expression of HO-1b decreased on 60d compared with 30d, but still higher than that of the control group. In the hindgut, the opposite phenomenon was observed in the midgut, that is, all HO genes seem to have increased to varying degrees.
3.5 Effect of adding B. coagulans to feed under Cd2+ stress on the expression level of HO-1 gene
To better understand the effect of adding different concentrations of B. coagulans to the feed on HO-1 gene expression level under Cd2+ stress, we performed qRT-PCR assay to detect the dynamic changes of gene content(Fig 5). The results showed that after 30d and 60d of feeding, the expression of HO-1 gene was different by adding different concentrations of B. coagulans into the feed. In the 30d midgut, all other groups showed varying degrees of reduced expression levels compared to the control group and reached the lowest expression level in the L3 group. In the 30d hindgut, the L1 and L3 groups did not provoke an increase in HO-1a gene expression level compared with the control group, but L2 provoked HO-1a gene expression and reached the highest expression level. In the 60d foregut, the HO-1a gene showed a trend of increasing, then decreasing, then increasing and then decreasing again, and likewise the highest expression level of HO-1a gene was reached in the L2 group, however, the L1 and L3 groups did not provoke an increase in its expression level. In the 60d midgut, the L1, L2 and L3 groups all succeeded in provoking an increase in their expression levels, but differed in that the HO-1a gene reached its highest expression level in the L1 group. The HO-1a gene had the same expression trend in the 60d hindgut, compared with the 60 d foregut.
The HO-1b gene provoked an upregulation of its expression level in the 30d foregut in both the L2 and L3 groups, with the L2 group reaching the highest expression level at 30d. The L1 group suppressed HO-1b gene expression in the foregut at 30d, but significantly increased the gene expression level and reached the highest expression value at 30d in the midgut. The HO-1b gene had the same expression trend as the foregut in the 30d hindgut, and again reached the highest expression level in the L2 group. However, in the 60d foregut, the L1, L2 and L3 groups did not provoke an upregulation of HO-1b gene expression levels. In the 60d midgut, the expression level of the HO-1b gene was significantly increased in all kinds except the L3 group and reached the highest expression level in the L2 group. In the 60d hindgut, the Cd2+ stressed group significantly increased the expression level of HO-1b, but the L2 group still showed a better induction effect and reached the highest expression level at 60d.
In general, different concentrations of B. coagulans in the feed during Cd2+ stress caused effects on gene expression levels. For example, the addition of L2 concentration of B. coagulans provoked the expression of HO-1a/b genes in the foregut, midgut and hindgut for almost all periods, but L1 and L3 no significant effect. Thus, the L2 concentration of B. coagulans increased the expression level of the HO-1a/b gene and thus enhanced the resistance of the organism to oxidative stress compared with the control/stressed group.
3.6 Effect of adding B. coagulans to feed under Cd2+ stress on the expression levels of cytokines IL10 and TGF-β
In order to better understand the effect of Cd2+ stress on immune genes in the downstream pathway of HO gene, we selected two cytokines IL-10 and TGF-β for our study(Fig 6). We found that when subjected to Cd2+ stress for 30 or 60d, the expression levels of both cytokines were significantly inhibited compared with the control group. Each of the different experimental groups in the foregut, midgut and hindgut, the IL-10 gene showed the same expression trends of decreasing, then increasing and then decreasing, and reached the highest expression level at the L2 concentration group. Most of the L1, L2 and L3 groups almost provoked an increase in IL10 gene expression levels compared to the Cd2+ stressed group. However, compared with the control group, the expression level of the IL-10 gene remained lower in all experimental groups except in the midgut of the 60d L2 group, where the expression level was significantly higher than that of the control group. The TGF-β gene had a similar expression trend of decreasing, then increasing and then decreasing expression in the foregut, midgut and hindgut. In addition, compared with the stressed group, most of the L1, L2 and L3 groups almost always provoked an increase in TGF-β gene expression levels. However, compared with the control group, the expression levels of the TGF-β gene remained lower in all experimental groups except in the foregut of the 30d L2 group and the midgut of the 60d L2 group, which were significantly higher than the control group.
Interestingly, the expression levels of both IL10 and TGF-β genes were suppressed at significantly lower levels in Cd2+ stress compared to the control group. However, the addition of L1, L2 and L3 B. coagulans to the diet provoked an upregulation of IL10 and TGF-β gene expression in almost all periods and reached the highest expression level in the L2 group compared to the Cd2+ stressed group. However, the expression level remained below normal for most of the period compared to the control group.