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Additional file 1: Supplementary Figure 1. The alpha diversity of ARG Type in the intestinal microbiome of C. semilaevis. Except for Simpson, the diversity measured by ACE, Chao 1, good_coverage, observed species, Shannon, and was higher in the resistant family compared with susceptible family, but not significantly between two families. Supplementary Figure 2. The family-specific KEGG enrichment pathways of microbial genes. Circles and triangles represent the resistant family-specific and susceptible family-specific KEGG enrichment pathways, respectively. The color indicates the enrichment level (adjusted p < 0.05), the more enrichment of the pathway, the smaller the p value. The size of circles and triangles was dependent on the gene ratio. Supplementary Figure 3. The locations of Phaeobacter genes involved in lipid metabolism and immune pathways. Here we have used the assembled bins assigned to Phaeobacter for locating Phaeobacter genes. One bold arrow represents a KO gene, and its length indicates the length of the gene. The color of gene name represents the KO pathway of this gene involved. Supplementary Figure 4. Intestinal microbial, host functional, and combinatorial markers for discriminating the resistant family from the susceptible family in C. semilaevis. The accuracy, F1 score, and AUC were used for evaluating the performance of all microbes (yellow curve), top 4 important microbes (blue curve), top 3 important microbes (orange curve), 11 ko genes (the microbiome-associated DEGs significantly enriched pathways; green curve), top 5 important ko genes (pink curve) of 11 ko genes, top 5 important ko genes (wathet curve) of 11 ko genes, and the combination of top 4 important microbes and 11 ko genes (red curves). Supplementary Table 1. Detailed sample information of the Cynoglossus semilaevis samples collected from resistant and susceptible families. Supplementary Table 2. The detailed information of top 20 KEGG enrichment pathways (adjusted p < 0.05) for un- the down-expressed genes in the resistant family, respectively. Supplementary Table 3. The KEGG pathway of 8 differentially expressed genes between the resistant and susceptible families. Supplementary Table 4. The locations of Phaeobacter genes involved in lipid metabolism and immune pathways.

As an important farmed flatfish species, Chinese tongue sole (Cynoglossus semilaevis) is heavily threatened by vibriosis in recent years. Genetic selection for disease resistance is a sustainable and effective approach to reduce frequent outbreaks. To verify whether the resistance of vibriosis (caused by co-infections of different Vibrio spp. in natural outbreak) can be included in breeding programs, in this study, genetic analysis of resistance to vibriosis based on natural outbreak survival data was carried out by using four statistical models (three cross-sectional models and one longitudinal model). The magnitude of the genetic variation in the resistance of vibriosis was estimated through a 56-day natural outbreak test of 15,912 individuals from 78 full-sib families (the offspring of 77 sires and 78 dams). Variance components and heritabilities were estimated at two cut-off points respectively, i.e. day 35 with 49.2% cumulative mortality and day 56 with 71.6% finial cumulative mortality. Heritabilities of resistance to vibriosis were low to moderate, where values at day 56 (0.04–0.21) were significantly different from zero, while values at day 35 (0.03–0.10) were not significantly different from zero in each corresponding model. The Spearman rank correlations between family EBVs for different models were high (> 0.98), indicating a nearly identical ranking of families. Compare to three simpler cross-section models, the longitudinal model taken the time until death into account demonstrated the highest accuracy of family selection. These results confirmed the existence of genetic variation for resistance to vibriosis and provided novel insights into the selective breeding for disease resistance to vibriosis by using natural outbreak survival data in tongue sole.

The present study aimed at the development of an effective and efficient artificial reproduction technique for cultured female tongue sole (Cynoglossus semilaevis) in out-of-season. After about four-month enriching cultivation of broodstocks, a series of experiments were conducted to develop a protocol for the hormonal induction of ovulation and artificial fertilization. The results showed that single injection of combination of luteinizing hormone releasing hormone analogue (LHRH-A3), human chorionic gonadotropin (HCG) and domperidone (DOM) is effective to induce ovulation at different final maturation stages (i.e., stage IV and V). The optimal single injection dosages of combined hormones were (0.15 μg LHRH-A3 + 4 IU HCG + 2 mg DOM) / kg BW and (0.05 μg LHRH-A3 + 2 IU HCG + 1 mg DOM) / kg BW for females at stage IV and V respectively. At the optimized dose of hormones, the fertilization and hatching rates were 44.6% and 80.4%, 30.4% and 63.4% for stage IV and V respectively. The lapsed time to ovulation was very predictable and ranged from 35.5 to 39.5 h post injection. Ovulated eggs in ovarian cavity should be stripped within 15 min in order to obtain the highest fertilization and hatching rates, otherwise the values decreased significantly thereafter. These researches are expected to provide improved implementation of artificial reproduction techniques for the production of fertilized eggs in the commercial aquaculture of tongue sole.