Differences between the Microbiota of Fish pond water, Rhizosphere soil, and the Largemouth Bass Gut
A total of 899,370 high-quality sequences were obtained from 27 samples, including 9 gut samples, 9 rhizosphere soil samples, and 9 water samples. For subsequent analysis, 33,310 high-quality sequences were randomly selected from each sample. The aforementioned sequences were subsequently classified into 15,916 operational taxonomic units (OTUs) using a 97% sequence identity criterion. The alpha-diversity index values (Kruskal–Wallis H-test, p < 0.05) and compositions (PERMANOVA, F = 42.48, p < 0.001; Fig. 3a–h) differed significantly among the gut, fish pond water, and rhizosphere soil microbiota. The number of OTUs, shannon, ace, PD whole tree, chao1 and simpson index values of the rhizosphere soil microbiota were significantly higher than those of the fish pond water microbiota, followed by those of the gut microbiota (p < 0.05; Fig. 3a, b). These results indicated that the goods coverage of the gut microbiota was the highest, while that of the rhizosphere soil microbiota was the lowest (Fig. 3f), which consist with a prior study conducted by Wu et al. (Wu et al. 2012). Within the same habitat (the gut, water, or rhizosphere soil), the alpha-diversity index values of only a small number of samples differed significantly within the sampling times (Fig. 3c, e, f). Nevertheless, the microbiota compositions of water (PERMANOVA, F = 24.13, p < 0.001), rhizosphere soil (PERMANOVA, F = 5.11, p < 0.001), and the gut of largemouth bass (PERMANOVA, F = 4.07, p < 0.001) differed significantly among the different sampling times (Fig. 3h-k).
Except for a few operational taxonomic units (OTUs), the majority of OTUs were classified into 54 phyla, including 8 archaeal and 46 bacterial phyla. The most dominant phyla of the microbiota were Firmicutes, Fusobacteriota, Proteobacteria, Cyanobacteria, Actinobacteriota, Bacteroidota, Desulfobacterota, Euryarchaeota, Dependentiae, Verrucomicrobiota, Patescibacteria, Gemmatimonadota, Acidobacteriota, Chloroflexi, Deinococcota, Planctomycetota, Entotheonellaeota, Crenarchaeota, Nitrospirota, Myxococcota, Bdellovibrionota, Nanoarchaeota, Latescibacterota, Elusimicrobiota, Zixibacteria, Armatimonadota, MBNT15, Hydrogenedentes, NB1-j, Thermoplasmatota, Sumerlaeota, RCP2-54, SAR324_clade(Marine_group_B), Fibrobacterota, Methylomirabilota, WPS-2, Campylobacterota, Spirochaetota, FCPU426, Deferribacterota (Fig. 3h). These phyla comprised 99.53 ± 0.69% of the analyzed high-quality sequences. Significant variations in their relative abundances were observed among the different habitats (Fig. 3h).
At the genus level, 997 genera were detected, including 35 dominant genera. Notably, there were significant differences in the abundances of the most dominant genera among the fish pond water, rhizosphere soil, and fish gut microbiota (Fig. 4a). Abundances of Plesiomonas, Stenotrophomonas, Clostridium_sensu_stricto_1, Vibrio, Pseudoalteromonas, unidentified_Mitochondria, Ralstonia, Delftia belonging to Proteobacleria, Romboutsia, Paraclostridium, Cetobacterium, Lactococcus, Staphylococcus belonging to Firmicutes, and unidentified_Chloroplast belonging to Cyanobacteria were significantly enhanced in the largemouth bass gut microbiota; abundances of Vogesella, Rheinheimera, Pseudomonas belonging to Proteobacteria, Nitrosarchaeum belonging to Crenarchaeota, Nitrospira belonging to Nitrospirota, and Cyanobacterium_PCC-7202, Geitlerinema Cyanobacterium_PCC-10605 belonging to Cyanobacteria were significantly enhanced in the rhizosphere soil microbiota; and abundances of Luteolibacter belonging to Verrucomicrobiota, Bdellovibrio belonging to Bdellovibrionota, Aurantisolimonas, Flavobacterium, Haliscomenobacter, Runella belonging to Bacteroidota, Flectobacillus Alsobacter, Novosphingobium, Polynucleobacter, Limnohabitans belonging to Proteobacteria, and hgcl_clade belonging to Actinobacteriota were significantly enhanced in the water microbiota (Fig. 4a). In the gut microbiota, abundances of Macrococcus, Trichococcus, Lactiplantibacillus, Erysipelothrix, Lacticaseibacillus, Peptostreptococcus, Ligilactobacillus, Limosilactobacillus belonging to Firmicutes, Plesiomonas, Polynucleobacter, unidentified_Mitochondria belonging to Proteobacleria, Aurantimicrobium, Jonesia, Corynebacterium belonging to Actinobacteriota, unidentified_Chloroplast belonging to Cyanobacteria were significantly enhanced in the samples collected in May; abundances of Staphylococcus, Lactococcus and Romboutsia belonging to Firmicutes were significantly enhanced in the samples collected in July; and abundances of Brochothrix belonging to Firmicutes, Ralstonia, Alsobacter, Aeromonas, Shewanella, Pseudoalteromonas, Delftia, Stenotrophomonas belonging to Proteobacleria were significantly enhanced in the samples collected in September (Fig. 4b). In the rhizosphere soil microbiota, abundances of Sphingobium, Acidovorax, Vogesella, Methylophaga, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Pseudomonas, Arenimonas, Limnobacter belonging to Proteobacteria, Bacteroides belonging to Bacteroidota, were significantly enhanced in the samples collected in May; abundances of Tolypothrix, Nodosilinea_PCC-7104, Leptolyngbya_FYG, OscillatoriaPCC-6304, unidentified_Chloroplast belonging to Cyanobacteria, Nitrospira belonging to Nitrospirota, Azoarcus, Robertkochia belonging to Proteobacteria, Terrimonas, Rheinheimera, Lewinella, Fluviicola belonging to Bacteroidota, Nitrosarchaeum belonging to Crenarchaeota, were unidentified genera in the samples collected in July; abundances of Subgroup_10 belonging to Acidobacteriota, Candidatus_Nitrosotenuis belonging to Crenarchaeota, Cyanobacterium_PCC-7202, Geitlerinema CENA533, Cyanobacterium_PCC-10605 belonging to Cyanobacteria, MND1 belonging to Proteobacteria, Marivirga belonging to Bacteroidota, Halobacillus belonging to Firmicutes, were significantly enhanced in the samples collected in September (Fig. 4c). In the water microbiota, abundances of Aurantimicrobium belonging to Actinobacteriota, Flectobacillus, unidentified_NS11-12_marine_group, Flavobacterium belonging to Bacteroidota, Polaromonas, Limnohabitans, Aeromonas, Novosphingobium, Undibacterium, Vogesella, Pseudorhodobacter belonging to Proteobacteria were significantly enhanced in the samples collected in May; abundances of Alsobacter, GKS98_freshwater_group, Polynucleobacter, Plesiomonas, Bosea, Reyranella, Aurantisolimonas belonging to Proteobacteria, Dinghuibacter, Haliscomenobacter, Acidovorax belonging to Bacteroidota, Paraclostridium belonging to Firmicutes, IMCC26134, Cerasicoccus belonging to Verrucomicrobiota were significantly enhanced in the samples collected in July; and abundances of Rhodoluna, hgcl_clade belonging to Actinobacteriota, Acidovorax, unidentified_Paracaedibacteraceae, Caulobacter belonging to Proteobacteria, Cetobacterium belonging to Fusobacteriota, Runella, Niabella belonging to Bacteroidota, Peredibacter and Bdellovibrio belonging to Bdellovibrionota, Luteolibacter belonging to Verrucomicrobiota were significantly enhanced in the samples collected in September (Fig. 4d).
Connection between the Gut Microbiota of Largemouth Bass and Fish pond water and Rhizosphere soil Microbiota
The source tracking analysis was conducted to explore the relationship between the gut microbiota of largemouth bass and the environmental microbiota of the fish pond water and rhizosphere soil. The results based on all samples indicated that 11.99% and 62.01% of the bacterial composition in the gut microbiota could be attributed to the rhizosphere soil and water microbiota, respectively. Additionally, 0.17% of the bacterial composition in the rhizosphere soil microbiota and 3.59% of the bacterial composition in the water microbiota could be attributed to the gut microbiota. It was observed that approximately equal to or less than 1% of the bacterial composition in the rhizosphere soil microbiota could be attributed to both the gut and water microbiota (Fig. 5a).
The percentage of bacteria in the gut microbiota that originated from the fish pond water microbiota was 40.90%in May and 56.15% in September, but showed a significant rise to 88.97% in July (Fig. 5b–d). On the other hand, the percentage of bacteriain the pond water microbiota that originated from the rhizosphere soil microbiota was 0% in May and8.95% in September, but it increased significantly to 69.26% in July (Fig. 5b–d). It was also observed that the proportion of bacteria in the gut microbiota that originated from the tomato rhizosphere soilmicrobiota was 0.07% in May and 0% in September, which increased to 0.45% in July. In addition, the percentage of bacteria in the fish pond water microbiota that could be attributed to the gut microbiotawas 7.43%in May, 2.43%in July, and decreased to 0.62%in September (Fig. 5b–d). These findings provide insights into the complex interactions between the gut microbiota of largemouth bass and the surrounding environment, particularly the gut and fish pond water microbiota.