Microbial communities associated with holothurians: presence of unique bacteria in the coelomic fluid

Abstract

Marine invertebrates interact with various microorganisms ranging from pathogens to symbionts. One-to-one symbiosis between a single microbial species and a single host animal has served as a model for the study of host-microbe interactions. In addition, increasing attention has recently been focused on the complex symbiotic associations, e.g., associations between sponges and their symbionts, due to their biotechnological potential; however, relatively little is known about the microbial diversity associated with members of the phylum Echinodermata. Here, for the first time, we investigated microbial communities associated with a commercially important holothurian species, Apostichopus japonicus, using culture-dependent and -independent methods. Diverse and abundant heterotrophs, mostly Gammaproteobacteria members, were cultured semi-quantitatively. Using the cloning and sequencing technique, different microbial communities were found in different holothurian tissues. In the holothurian coelomic fluid, potentially metabolically active and phylogenetically unique members of Epsilonproteobacteria and Rickettsiales were discovered. This study suggests that coelomic fluids of marine invertebrates, at least those inhabiting intertidal areas where physical and chemical conditions fluctuate, provide microbes with unique and stable habitats.

Fig. 1

Composition of the culturable heterotrophs associated with holothurians. See Table 3 for sample codes.

Fig. 2

Similarity and composition of the bacterial population in holothurian tissues. The square distance (genetic similarity) was determined from the clonal frequency of each representative phylotype by the Ward method. Pie charts indicate the composition of bacterial population based on taxonomic grouping of 16S rRNA gene clone sequencing. See Table 3 for sample codes. 09: 24F-1509R primer set, 40: 24F-1540R primer set.

Fig. 3

Phylogenetic tree including representative holothurian clones as determined by neighbor-joining analysis. (A) and (B) were respectively constructed from 424 and 528 sites of the rRNA gene sequence that could be unambiguously aligned. Clones sequenced in this study are shown in red. The clonal frequency of each representative clone obtained in this study and DDBJ accession numbers are shown in parentheses. Branch points conserved with bootstrap value of >75% (solid circles) and with bootstrap values of 50 to 74% (open circles) are indicated. Some groups are represented by shaded trapezoids that indicate the numbers of sequences. Scale bars represent 0.05 substitutions per nucleotide position. (A) Tree indicating the phylogenetic relationship among members of the Alphaproteobacteria. (B) Tree indicating the phylogenetic relationship among members of the Epsilonproteobacteria.

Fig. 4

Epifluorescence micrograph of cells binding the Epsilonpro-teobacteria-specific probe (EP 402–423) in the holothurian coelomic fluid. Arrowheads indicate epsilonproteobacterial cells. Bar, 5 μm.