Divergent selection was practiced for fillet yield to develop high (ARS-FY-H), and low (ARS-FY-L) yield genetic lines of rainbow trout. The two fish groups used in this study were collected after two generations of selection and were statistically different in their average muscle yield as indicated by a one-way Mann-Whitney U test (p<0.05; Fig. 1). The mean muscle yield of the high (ARS-FY-H) genetic was 0.53±0.01%, and that of the low (ARS-FY-L) genetic line was 0.51±0.02%.
Comparison of gut assemblages in high-(ARS-FY-H) and low-(ARS-FY-L) muscle yield genetic lines
Fish were reared and harvested under identical conditions, however, there was a significant difference in gut microbes between the two harvest days in the high- (ARS-FY-H; F1,15= 8.24, p<0.05, R2=37.06%) but not low-muscle yield genetic lines (ARS-FY-L; F1,17= 0.85, p>0.05). Therefore, harvest day was treated as a random effect in all models to test for the main effect of genetic line. Using a linear mixed model, we tested for differences in gut alpha diversity between fish genetic lines and found that diversity was higher in high (ARS-FY-H) genetic line (LMM, c2(1)=14.11, p<0.05, Fig. 2) when controlling for the harvest day effect. Both nMDS ordination and PERMANOVA results (F1,36= 4.7, p<0.05, R2=11.9%) indicated that the muscle-yield genetic line was predictive of gut microbial assemblages in rainbow trout (Fig. 3A). There were no significant differences in multivariate dispersion between gut assemblages of low (ARS-FY-L) and high (ARS-FY-H) genetic line samples. A total of 468 OTUs were shared between the two genetic lines (Fig. 3B). The high (ARS-FY-H) muscle-yield genetic line samples had almost double the number of unique OTUs compared to the low (ARS-FY-L) muscle-yield genetic line.
Together, these results indicate that the muscle-yield genetic lines are predictive of gut microbial assemblages and suggest that host genetic selective breeding might help curate a particular gut microbial assemblage. This notion is supported by recent studies in tilapia, showing host genetic selection for cold thermal tolerance has an effect on the microbiome [23]. Similarly, studies in stickleback fish identified an association between gut microbial differences and host genetic divergence [24]. Previous work from our lab group revealed significant variation in beta diversity of the bacterial communities of rainbow trout families showing variation in growth rate [25]. Together, these studies indicate a substantial impact of host selection or genetics in predicting host-associated microbial assemblages.
Taxonomy and functional diversity correlate with selection for fish muscle yield
A total of 8 phyla, 12 classes, 36 families, and 64 genera had significant differences in abundance between the two genetic lines (Kruskal-Wallis test; p<0.05, additional file 1). Phyla Bacteroidetes, Fusobacteria, Deniococcus, Acidobacteria, Patescibacteria, and Nitrospora had higher abundance in the high (ARS-FY-H) muscle yield genetic line whereas, the phylum Tenericutes had higher relative abundance in the low (ARS-FY-L) muscle yield genetic line (Fig. 4). Using a genus-level comparison, some unclassified genera belonging to family Burkholderiaceae and Gammaproteobacteria had higher abundance in high (ARS-FY-H) muscle yield genetic line. In contrast, genera Bacteroides, Deniococcus, Lutelibacter, Nitrosomonas, Pasteurella, and Negativibacillus were present only in the high (ARS-FY-H) muscle yield genetic line.
Higher abundances of the phyla Bacteroidetes, Fusobacteria, Deniococcus might be associated with higher muscle percentage, as these taxa are known symbionts and produce metabolites such as SCFAs that are beneficial to the host [26-29]. For example, genera in the phylum Bacteroidetes are associated with degradation of protein complex polymers and these are responsible for the formation of SCFAs like succinic acid, propionic acid, and acetic acid as the end products [28]. Similarly, genera in the phylum Fusobacteria, a phylum reported to be abundant in freshwater fish guts [30, 31], may produce butyrate, which supplies energy to gastrointestinal cells and inhibit pathogens in freshwater fish [32]. Fusobacteria are known to colonize the gut of zebrafish, synthesize vitamins, excrete butyrate, and metabolites associated with improving fish health [33]. Similarly, bacteria in the phylum Deinococcus can metabolize glucose [34]. Conversely, phylum Tenericutes had higher abundance in the low (ARS-FY-L) muscle yield genetic line samples. This phylum is found in Fathead minnow fish gut [35], however, the functional role of this phylum is not well studied in fish. A study on crabs showed that the Tenericutes phylum is correlated with Hepatopancreatic necrosis disease [36].
There are 64 genera with significant differential abundances between the two groups. Among them, 21 genera have >10 % and within which 4 genera have >15% differential abundances. These 21 genera include Arcicella, Clostridiaceae_1_unclassified, Burkholderiaceae_unclassified, Pedobacter, Absconditabacteriales, Arsenicibacter, Chiinophagaceae, Cloacibacterium, Hydrogenophaga, Rhizorhapis, Rhodoferax, Sphingomonadaceae_unclassified, Sphingorhabdus, Spirosomaceae_unclassified, Thermomonas, Thiothrix, Undibacterium, Veillonellaceae_unclassified, Chryseobacterium. All these significant genera have higher abundance in high (ARS-FY-H) muscle yield genetic line, whereas genera Mycoplasma (with differential abundance 16.82%), and Firmicutes_unclassified (with differential abundance 7.9%) have higher abundance in low (ARS-FY-L) muscle yield genetic line (additional file 1).
A study in rainbow trout showed that the genus Clostridium butyricum, family Clostridiaceae, enhances the disease resistance in host against pathogen Vibrio by increasing the phagocytic activity of leucocytes [37]. Inhibiting pathogenic bacteria from host might help to improve host health, including growth rate and metabolism. In addition, these genera have been used as a probiotic to improve immune response and survival in Paralichthys olivaceus fish [38]. Arcicella, belonging to phylum Bacteroidetes has been identified in freshwater environment and these bacteria can ferment carbohydrates [39]. Similarly, bacteria form genus Pedobacter is dominant in the healthy Atlantic Salmon gut [40].
Genus Bacteroides are very important bacteria colonizing the intestine of a wider variety of hosts, including humans [41, 42], mice [43], and tilapia fish [44]. These bacteria ferment carbohydrates and produce short-chain fatty acids (SCFAs) like acetate, propionate and butyrate. These SCFAs are key regulators of skeletal muscle metabolism and function [45]. A study showed that genus Cloacibacterium species isolated from the Abalone intestine can hydrolyze starch and ferment sugars like glucose, galactose, fructose, maltose and mannose and produce fatty acids [46]. The genera mentioned above in this section have higher abundance in high (ARS-FY-H) muscle yield genetic line and most of the genera are associated with digestion, fatty acid metabolism, pathogen inhibition, which are ultimately involved in improving host health and digestion.
Family Burkholderiaceae_unclassifed had higher abundance in high (ARS-FY-H), and genus Burkholderia belonging to this family were reported as the most abundant genus in fish gut [47]. However, their role in muscle yield and or host health in fish has not been reported before. In addition to the discussed genera in this section, there are other genera with significant differential abundance between groups; however, their functional role has not been previously reported in fish. Those genera include Absconditabacteriales, Arsenicibacter, Chiinophagaceae, Hydrogenophaga, Rhizorhapis, Rhodoferax, Sphingomonadaceae_unclassified, Sphingorhabdus, Spirosomaceae_unclassified, Thermomonas, Undibacterium, Veillonellaceae_unclassified, Chryseobacterium.
On the other hand, the taxa with significantly higher abundance in the low (ARS-FY-L) muscle yield genetic line were Mycoplasma (16.82%) and Firmicutes_unclassified (7.912 %). A previous study showed that genus Mycoplasma is the most abundant and consistently existing phylotypes in adult Atlantic salmon [48]. Bacteria belonging to this genus have also been described as pathogenic in gills of Tinca tinca [49]. The lower abundance of unclassified Firmicutes in low (ARS-FY-L) muscle yield genetic line might be associated with decreased body weight or correlated with decrease muscle percentage in fish. The ratio of Firmicutes to Bacteroides has been shown to correlate with weight gain in humans [50] and this trend might exist in fish as well. A previous study in our laboratory showed that body weight of rainbow trout is moderately correlated with muscle yield, regression coefficient (R2) values of 0.56 [51].
Tax4Fun analyses were used in this study to enumerate differential functional capabilities of microbial communities in high (ARS-FY-H) and low (ARS-FY-L) genetic lines (Fig. 5). Bacterial functional pathways related to calcium signaling, pentose and glucuronate interconversions, synthesis and degradation of ketone bodies, linoleic acid metabolism, lysine degradation, and arachidonic acid metabolism were enriched in most of the high (ARS-FY-H) genetic line samples. Microbial pathways involved in fatty acid metabolism are known to supply energy to muscle cells, which is essential for muscle growth [52], and was more abundant in the high (ARS-FY-H) genetic line. Genus Bacteroides belonging to phylum Bacteroidetes that showed higher abundance in the high (ARS-FY-H) genetic line is associated with fatty acid metabolism, producing SCFAs [53]. A study in mice revealed that SCFAs produced by microbiota in gut supports muscle function by preventing muscle atrophy and boost muscle strength [21].
Similarly, Biu et al. and Barkel et al reported that lysine degradation mediated production of SCFAs (butyrate and acetate) in human gut [54]. Genus Fusobacterium with higher abundance in high (ARS-FY-H) muscle yield genetic line is involved in lysine degradation and production of SCFAs [55]. Microbial synthesis and degradation of ketone bodies (KB), identified in the ARS-FY-H samples, were reported as associated with increased muscle mass in humans [56, 57]. Ketone bodies make an energy substrate that supplies energy to the brain and muscles, contributing to the maintenance of energy homeostasis through regulation of lipogenesis [56]. Arachidonic acid metabolism is essential for the functions of skeletal muscle and the immune system, which might be associated with increased muscle mass and health in the host [58, 59]. The Clostridiaceae family had higher abundance in high (ARS-FY-H) muscle yield genetic line fish and was reported as correlated with enriched Pentose and glucuronate interconversions [60]. Bacteria associated with this pathway are involved in the breakdown of complex substrates in pig gut microbiomes and improved carbon and energy uptake in the host [60]. Energy uptake is essential to achieve increase in muscle mass.
Fish with low (ARS-FY-L) muscle yield had unique functional profiles that differed from high (ARS-FY-H) muscle yield samples, Pyruvate metabolism, amino acid metabolism, folate biosynthesis, glycosphingolipid biosynthesis, glyoxylate and dicarboxylate metabolism, adipocytokine signaling pathway and two-complement system were enriched in most of the low (ARS-FY-L) genetic line samples (Fig. 5). Glycosphingolipids act as negative regulators of skeletal muscle differentiation and growth in rat [61, 62] [63]. However, association of glycosphingolipids in mediating low muscle yield in fish has not been studied so far. Similarly, Bile secretion is associated with lipid digestive functions [64, 65] and may reduce adiposity in host, which might result in lower muscle mass. In spite of the differential enrichment of pathways between the muscle yield groups, further investigation should be done to validate the role of these microbial pathways in the host.