Evidence for a core gut microbiota in the zebrafish

Abstract

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.

Figure 1

Relationship between sampled zebrafish aquaculture facilities. The original University of Oregon (UO) facility was seeded by zebrafish acquired from commercial suppliers in the late 1960s (Grunwald and Eisen, 2002). Zebrafish from the UO facility was subsequently used to seed the Washington University (WU) facility in 1996 and the Zebrafish International Resource Center (ZIRC) in 2001. In 2000, zebrafish from the WU facility was used to seed a facility at the University of Texas at Austin, which was subsequently moved to the University of Washington (UW) facility in 2005. The University of North Carolina (UNC) facility was seeded in 2006 by zebrafish from the WU facility as well as from a facility at the University of California at San Francisco (UCSF) that has no historical connection to the UO facility.

Figure 2

The 16S rRNA gene sequencing reveals the relationship and membership of intestinal microbiotas of zebrafish from different locations. (a) Unweighted UniFrac tree comparing 5217 16S rRNA clone library sequences from the gut microbiotas of adult zebrafish, other teleost fish species, mouse and human (see Table 1). The distance P value for this entire UniFrac tree (the probability that there are more unique branches than expected by chance, using 1000 iterations) was found to be <0.002, assigning high condence to the overall structure of the tree. (b) Unweighted UniFrac tree of 17 763 16S rRNA pyrosequences spanning the V1–V2 hypervariable regions derived from the gut microbiotas of recently caught (India.1) and domesticated (UNC.1 and UW.1) zebrafish. Scale bars indicate distance between the samples in UniFrac units. The shape at the end of each branch indicates host superorder (triangles: freshwater Ostariophysi fish, circles: marine Acanthopterygii fish, squares: mammalian reference samples) with color indicating host order as shown in the key (Perciformes fish from Antarctica and Palmyra Atoll are labeled separately). (c) The relative abundance of bacterial classes observed in these data sets is represented in heatmaps below each tree. Bacterial classes are grouped by phylum: Proteobacteria (Pr), Firmicutes (Fi), Bacteroidetes (Ba), Chloroflexi (Ch), Fusobacteria (Fu), Actinobacteria (At), Spirochetes (Sp), Deferribacteres (De), Acidobacteria (Ai), Nitrospira (Ni), Planctomycetes (Pl), Verrucomicrobia (Ve), Lentisphaerae (Le), and Deinococcus-Thermus (DT)(see also Supplementary Table S3). Communities are clustered using PCoA of unweighted (d) and weighted (e) UniFrac distance matrices. The gray halos encircle the cluster of freshwater Ostariophysi fish. The percentage of the variation explained by the plotted principal coordinates is indicated on the axes.

Figure 3

The 16S rRNA gene sequence-based phylogenetic analysis of Fusobacteria within the intestinal microbiotas of zebrafish and other fishes. Maximum likelihood tree showing the phylogenetic relationship between Fusobacteria 16S rRNA gene sequences from uncultured and cultured bacteria derived from the intestinal contents of zebrafish (red text) and other teleost fishes (blue text), and those from other sources available in the public databases (black text). Branches representing multiple identical sequences are indicated with the number of sequences in parentheses. Bootstrap support (⩾50%) is shown as results from 1000 bootstrap replicates. The scale bar indicates 0.05% estimated sequence divergence.

Figure 4

Deep sequencing of 16S rRNA genes reveals a core intestinal microbiota shared among recently caught and domesticated zebrafish. (a) Rarefaction curves of 16S rRNA gene pyrosequences spanning the V1–V2 region from pooled intestinal samples collected from recently caught zebrafish (India; sample D.rerio.India.1; 5582 sequences) or domesticated zebrafish raised in aquaculture facilities at the University of North Carolina (UNC; sample D.rerio.UNC.1; 9357 sequences) or the University of Washington (UW; sample D.rerio.UW.1; 2824 sequences). Sequences are binned into OTUs using a pairwise sequence similarity threshold of 97%. (b) Venn diagram showing the distribution of all 525 OTUs (97%) identified in the combined 17 763 16S rRNA gene pyrosequences from India, UNC and UW, revealing a shared community of 21 OTUs found in all three locations. (c) Rank abundance plot showing the OTUs (97%) within each category of the Venn diagram in panel b ranked according to their abundance in the combined 17 763 sequence data set. The similarity between the rank abundance plots of all 525 OTUs (black line) and the 21 shared OTUs (green line) reveals that the OTUs found in all three locations include the most abundant OTUs in any location. (d) Pie charts showing the relative abundance of bacterial phyla in the intestinal microbiotas of zebrafish from India, UNC, and UW (see Supplementary Table S3), as well as the 21 OTUs shared between all three locations, which may comprise a ‘core' zebrafish gut microbiota (see Supplementary Table S4).