Long-term maintenance of species-specific bacterial microbiota in the basal metazoan Hydra

Abstract

Epithelia in animals are colonized by complex communities of microbes. Although a topic of long-standing interest, understanding the evolution of the microbial communities and their role in triggering innate immune responses has resisted analysis. Cnidaria are among the simplest animals at the tissue grade of organization. To obtain a better understanding of the microbiota associated with phylogenetically ancient epithelia, we have identified the epibiotic and endosymbiotic bacteria of two species of the cnidarian Hydra on the basis of rRNA comparisons. We analyzed individuals of Hydra oligactis and Hydra vulgaris from both laboratory cultures and the wild. We discovered that individuals from both species differ greatly in their bacterial microbiota. Although H. vulgaris polyps have a quite diverse microbiota, H. oligactis appears to be associated with only a limited number of microbes; some of them were found, unexpectedly, to be endosymbionts. Surprisingly, the microfauna showed similar characteristics in individuals of cultures maintained in the laboratory for >30 years and polyps directly isolated from the wild. The significant differences in the microbial communities between the two species and the maintenance of specific microbial communities over long periods of time strongly indicate distinct selective pressures imposed on and within the epithelium. Our analysis suggests that the Hydra epithelium actively selects and shapes its microbial community.

Fig. 1.

Analysis of Hydra-associated bacteria. (A) Phylogenetic position of the cnidarian Hydra. (B and C) Morphological characteristics of the two Hydra species analyzed. (D) Schematic representation of the approach. Bacterial microbiota were compared between H. vulgaris (blue) and H. oligactis (red) from laboratory culture (Right; drawn in plastic dishes) and the wild (Left; attached to water lily).

Fig. 2.

RFLP analysis of 16S rRNA genes of bacteria associated with two different Hydra species. (A) H. oligactis from the laboratory culture. (B) H. oligactis isolated from the wild (Lake Pohlsee). (C) H. vulgaris from the laboratory culture. (D) H. vulgaris isolated from the wild (Lake Pohlsee). Ho, H. oligactis; Hv, H. vulgaris.

Fig. 3.

Phylogenetic comparison of identified bacterial phylotypes from the different Hydra species. (A) Neighbor-joining tree (Olsen correction) with the 36 identified 16S rDNA phylotypes from five different samples. The number of RFLP patterns within each phylotype is shown in parentheses. Bootstrap values are shown at the corresponding nodes (n = 100). [Scale bar: evolutionary distance (0.1 substitution per nucleotide).] Specific bacterial lineages for the different Hydra species analyzed with UniFrac are indicated in blue (specific for H. vulgaris, P < 0.001) and red (specific for H. oligactis, P < 0.001). (B) Jackknife environment cluster tree (weighted UniFrac metric, based on the 36-sequence tree; ref. 10) of the analyzed bacterial communities. One hundred jackknife replicates were calculated, and each node was recovered with >99.9%. (Scale bar: distance between the environments in UniFrac units.) Hv, H. vulgaris; Ho, H. oligactis; lab, animals from laboratory culture; lake Pohlsee, animals taken from Lake Pohlsee; lake Ploen, animals taken from Lake Ploen.

Fig. 4.

Phylogenetic position (16S rRNA gene sequences, neighbor-joining tree) of identified phylotypes. (A) Phylogenetic position of identified bacterial phylotypes associated with H. vulgaris. (B) Phylogenetic position of identified bacterial phylotypes associated with H. oligactis. Light gray shadowed bacterial groups indicate species-specific bacterial guilds; dark gray shadowed bacterial phylotypes indicate species-specific bacterial species. The branch length indicator displays 0.1 substitution per site.

Fig. 5.

Microscopic analysis of endosymbiotic bacteria in H. oligactis. Macerated epithelial cell was stained with Hoechst and evaluated with phase-contrast microscopy (A) and epifluorescence microscopy (B). (C and D) Transmission electron micrographs of bacterial endosymbionts in the cytoplasm of ectodermal epithelial cell. Secondary membrane is indicated by yellow arrowheads. (E–M) In situ hybridization (FISH) reveals endosymbiont identity. (E–G) Trypsin-digested epithelial cells. (H–M) Macerated epithelial cells. (E, H, and K) Cells were stained with Hoechst. (F, I, and L) Bacteria cells were stained with the fluorescently labeled oligonucleotide probe EUB338. (G, J, and M) Bacterial cells were stained with the phylotype-specific probe HoSym1030. All cells were viewed with epifluorescence microscopy and appropriate filter sets.

Fig. 6.

Comparative RFLP analysis of bacterial 16S rRNA genes associated with H. oligactis isolated from Lake Ploen (A) and after cultivation in the laboratory for 2 months (B).