Microbes Environ. Vol. 21, No. 1, 16–22, 2006 http://wwwsoc.nii.ac.jp/jsme2/

Identification and in situ Detection of Two Lineages of Bacteroidales Ectosymbionts Associated with a Gut , Oxymonas sp.

SATOKO NODA1, MIHO KAWAI2,3, HIDEAKI NAKAJIMA2,4, TOSHIAKI KUDO2,3 and MORIYA OHKUMA1,2*

1 PRESTO, Japan Science and Technology Agency (JST), Wako, Saitama 351–0198, Japan 2 Environmental Molecular Biology Lab., RIKEN, Wako, Saitama 351–0198, Japan 3 Graduate School of Integrated Science, Yokohama City University, Tsurumi-ku, Yokohama 230–0045, Japan 4 Department of Applied Chemistry, Toyo University, Kawagoe, Saitama 350–8585, Japan

(Received August 9, 2005—Accepted October 8, 2005)

Bacterial attachments often cover the entire surface of flagellated in the guts of . Based on PCR-amplified 16S rRNA gene sequences, we investigated the phylogenetic positions of the rod-shaped (ectosymbionts) attached to the protist Oxymonas sp. in the termite Neotermes koshunensis. Two distinct and unique lineages of the ectosymbionts within the order Bacteroidales were identified, each belonging to a cluster exclusively comprised of the sequences from termite gut. We designed two oligonucleotide probes specific for the two lineages, and successfully detected the ectosymbionts, each of which distributed over the entire surface of Oxymonas sp. However, few cells of Oxymonas sp. simultaneously harbored both lineages of the ectosym- bionts.

Key words: ectosymbiont, protist, termite, Bacteroidales, FISH

Termites harbor a diverse community of microbes in their methanogens within protist cells3,22) and spirochetes at- gut, consisting of both flagellated protists (single-cell eu- tached to the cell surface of gut protists have been karyotes) and prokaryotes. The termite gut protists belong reported6,11,23). Recently, bacteria belonging to the order to either the phylum Parabasalia or the order Oxymonadida Bacteroidales were identified as ectosymbionts of a number but comprise diverse species, most of which are unique to of protist species in the gut of termites and a termites and wood-feeding cockroaches8,13). Recent culture- cockroach10,19,23). Those studies have disclosed the evolu- independent studies based on molecular sequences have en- tionary relationship of protist-bacteria associations and the abled one to classify the gut symbionts phylogenetically. distinct spatial distribution of the community members in These studies reveal that a great majority of prokaryotes in termite guts. Although the association between Bacte- the gut consist of yet-to-be cultivated novel organisms, re- roidales ectosymbionts and a number of parabasalid protists vealing our knowledge of termite gut symbionts to be limit- has been reported, only one species, Streblomastix sp., has ed. been studied with regard to the attachment of Bacteroidales The association of prokaryotes with gut protists is fre- within the order Oxymonadida11). Therefore, the phyloge- quently observed, and gut protists themselves are hosts of netic placement of the Bacteroidales ectosymbionts of an- prokaryotic symbionts12). Although there have been advanc- other protist species of Oxymonadida is important in order es in the molecular identification of the gut protists and their to understand the evolution of the Bacteroidales attach- associated prokaryotes, little is known about the nature of ments. these impressive symbiotic relationships. The presence of One of the large flagellates in the hindgut of Neotermes koshunensis is Oxymonas sp., a member of the order Oxy- * Corresponding author; E-mail: [email protected], Tel: +81–48– monadida. Multiple spirochete species attach to the cell-sur- 467–9648, Fax: +81–48–462–4672. face of Oxymonas sp.11). Many protists species in the termite Ectosymbiotic Bacteroidales Bacteria 17 gut harbor both spirochetes and other bacterial species as sequencer (PE Biosystems, Foster City, CA, USA). The ectosymbionts. With the exception of spirochetes, however, DNA sequences determined in this study will appear in the the associated prokaryote of Oxymonas sp. has never been nucleotide sequence databases under the accession numbers investigated. In this study, we analyzed 16S rRNA gene AB231289-AB231292. sequences of ectosymbiotic bacteria of Oxymonas sp. to Sequence data used to infer a phylogenetic tree were re- clarify the Bacteroidales attachment. Their localization and trieved from the public DNA sequence databases. Their ac- distribution were investigated by fluorescent in situ hybrid- cession numbers are shown in Fig. 1. Sequences were ization (FISH) with specific oligonucleotide probes for the aligned with the CLUSTAL X package21) and corrected identified 16S rRNA gene sequences. manually. Phylogenetic analyses were restricted to unam- biguously aligned positions. To infer the 16S rRNA gene Materials and Methods phylogeny, we used a general time-reversible model with gamma-distributed rate variation and a proportion of invari- Termites and protist able sites that was selected as the appropriate model of se- The termite Neotermes koshunensis was collected in quence evolution with Modeltest 3.0617). The phylogenetic Okinawa prefecture, Japan. The protist Oxymonas sp. in N. tree was constructed by the maximum likelihood (ML) koshunensis was identified by morphological characteris- method using the program PHYML v2.4.42). The robustness tics. The protistan small subunit rRNA gene has been ana- of the branching pattern was confirmed by a bootstrap anal- lyzed, and from pooled Oxymonas sp. cells, clones showing ysis of 1000 replicates. Bayesian inference was performed an almost identical sequence were obtained, suggesting using MrBayes v3.0b45), which was run for 100,000 genera- that the Oxymonas sp. in this termite comprises a single tions, and the posterior probabilities of each node were mea- genetically homogeneous species8). Hindgut material of N. sured after discarding the first 10,000 generations. koshunensis was extracted by removing the hindgut from PAUP*4.10b20) was used for the maximum parsimony (MP) live specimens. The protist cell was physically isolated with method and a bootstrap analysis with 200 replicates was a micromanipulator (TransferMan, Eppendorf, Hamburg, conducted using a heuristic search with TBR branch-swap- Germany) as described previously11). The isolation step was ping and with 10 random additions of sequences. repeated three times to remove contaminated protist cells and free-swimming bacteria. Five to ten cells of the isolated FISH protist were put in acetone, and after evaporating, used for FISH was performed according to the methods reported the PCR amplification. by Noda et al.11). The specimen was observed under an Olympus model BX-60 epifluorescence microscope (To- 16S rRNA gene analysis kyo, Japan). The previously reported probe EUBAC6), The Bacterial 16S rRNA gene was amplified by PCR us- which binds to most eubacterial cells and was labeled at the ing ExTaq DNA polymerase (Takara, Otsu, Japan). The 5' end with Texas-Red, was used as a control for the perme- PCR conditions were 22 or 25 cycles at 94°C for 30 s, 50°C ability of the cells. Also used was the previously reported for 45 s, and 72°C for 2 min. The universal PCR primers for probe CFBV-69010) which binds to cluster V of Bacteroi- the eubacterial 16S rRNA gene used were Eub27F and dales and was labeled at the 5' end with Texas-Red or 6- 1392R10). We cloned the PCR products into the pCR2.1- FAM. The specificity of this probe has already been TOPO vector (Invitrogen, Carlsbad, CA, USA), and con- established10). The number of protist cells that harbored bac- structed three clone-libraries from three independent PCRs. teria detected by this probe was measured in six indepen- Clones containing inserts of the expected size were picked dent hybridization specimens. The probes for the identified and sequenced. The primer used for the partial sequencing sequences of the ectosymbionts of Oxymonas sp. were de- of the 16S rRNA gene in all the clones was Eub750R10) and signed for this study. These probes are NkOxy16S-B157 the primers, T7, Sp6 (Promega, Madison, WI, USA) and (5'-GAAAGCCTATCCCGGTGTA-3') and NkOxy16S- Univ5 (5'-CAGCMGCCGCGGTAA), were used for the 9B439 (5'-CGCAGGGTACTTACAACAC-3'), each of complete sequencing of the 16S rRNA gene in representa- which was labeled at the 5' end with 6-FAM or Texas-Red. tive clones. Based on a comparison of the partial sequences, clones with more than 97% nucleotide identity were sorted into phylotypes. Nucleotide sequences were determined us- ing ABI dye-terminator chemistry with an ABI3700 DNA 18 NODA et al.

Fig. 1. Phylogenetic positions of the ectosymbiotic bacteria of the protist Oxymonas sp. The tree was inferred by the maximum likelihood meth- od based on comparisons of 16S rRNA gene sequences. Numbers at nodes indicate (from left to right divided by slashes) bootstrap values for maximum likelihood, Bayesian inference and parsimony methods. The scale bar indicates 0.1 nucleotide substitutions per position. Clusters IV and V of the sequences from termite guts are specified by the vertical bar on the right-side of the tree. Sequences from termite guts are tagged by Rs (Reticulitermes speratus), BCf (Coptotermes formosanus) and COB (Cubitermes orthognathus). The sequences obtained in this work and those reported as ectosymbionts of protists are indicated in bold. The database accession numbers are shown after the names of taxa. Genus abbreviations: B., Bacteroides; Po., Porphyromonas; Pr., Prevotella; D., Dysgonomonas. The sequences of Sphingobacterium spiritivorum and Rikenella microfusus were used as outgroups.

the major phylotypes obtained from the protist. Nk-OxyU1- Results 3 and Nk-OxyU1-9 were obtained from two of the three in- dependent libraries. These two phylotypes, showing 89.7% 16S rRNA genes and their phylogeny identity to each other, were phylogenetically affiliated to In order to address the phylogenetic position of the ecto- Bacteroidales. Two phylotypes (Nk-OxyU1-14 and Nk- symbionts of the protist Oxymonas sp., the protist cells were OxyU2-15) that were moderately abundant in only one of isolated with careful physical manipulation, and the bacteri- the three libraries, respectively, were affiliated to Trepone- al 16S rRNA gene was PCR-amplified. The PCR products ma or Clostridiales (see Table 1). It is reported that the Oxy- were cloned and sorted into phylotypes using the criterion monas sp. harbors many ectosymbiotic spirochetes10), but of 97% sequence identity. In order to exclude phylotyes the clones assigned to spirochetes were not abundant. This from bacteria contaminating the isolated protists and/or bac- was probably due to a difference of amplification efficiency teria engulfed by the protists, we investigated only major in the applied PCR or of effectiveness of DNA release from and reproducibly obtained phylotypes which were presumed bacterial cells used directly for the amplification. to be abundant and to be associated stably. Table 1 shows Ectosymbiotic Bacteroidales Bacteria 19

Table 1. Major phylotypes abundant in clones of the 16S rRNA gene identified from the Oxymonas sp. cells

a Library Number of Number of Representative clone Affiliation Acc. No. clones analyzed phylotypes (Number of clonesb) 1 23 7 Nk-OxyU1-3 (12) Bacteroidales AB231289 Nk-OxyU1-9 (4) Bacteroidales AB231290 2 20 5 Nk-OxyU1-3 (13) Bacteroidales Nk-OxyU1-14c (3) Treponema AB231291 3 38 13 Nk-OxyU1-9 (17) Bacteroidales Nk-OxyU2-15c (6) Clostridiales AB231292 a Minor phylotypes representing less than 10% of the total clone number are not shown. They were affiliated to Bacteroidales, Spirochaetaceae, Mycoplasmatales, TM 7 phylum, alpha-Proteobacteria, beta-Proteobacteria, Termite group 1 and Sphingobacteriales. b Number of clones that were grouped into the same phylotype as the representative clone is indicated. c The sequences most similar to Nk-OxyU1-14 and Nk-OxyU2-15 in the databases are the clone NkS15 (AB084958) obtained from the gut of the termite N. koshunensis (93.0% identity) and the environmental clone LKB99 (AJ746500) obtained from a landfill leachate (85.9% identity in ca. 600 bp), respectively.

Our previous study of termite gut symbionts showed the detected ectosymbiont cells in various numbers, ranging presence of five phylogenetic clusters in Bacteroidales, des- from approximately 10 to several hundred per protist cell. ignated as clusters I to V15). In the constructed phylogenetic Due to the strong background autofluorescence of the pro- tree (Fig. 1), each of the two major phylotypes branched out tist cells, we failed to estimate the average number of ecto- within cluster V, which was exclusively comprised of the symbionts per protist cell. The ectosymbionts attached to sequences identified from termite guts. Cluster V including the rostellum (a nose-like structure for attachment to the ter- these phylotypes was supported robustly by a bootstrap mite gut wall) through the posterior part of the protist cells. analysis of the Bayesian inference, ML and MP methods. No difference in distribution was observed between the two However, the two phylotypes formed distinct lineages from lineages of the ectosymbionts. Neither of the two probes de- each other, and one of them (Nk-OxyU1-3) was monophyl- tected free-swimming bacteria in the gut fluid. etic with the ectosymbionts of the protists, Devescovina The probe specific for the phylotype Nk-OxyU1-3 re- spp. (NkD2-1 and CdD3-1)10), showing 92.4% and 89.8% vealed that 20.7% (198 cells were examined) of Oxymonas identity, respectively. sp. cells, and 14.0% of Oxymonas sp. cells (50 cells were examined) were associated with the ectosymbiont corre- Detection of the ectosymbionts by FISH sponding to the phylotype Nk-OxyU1-9. When both probes The cluster V-specific probe gave positive signals in rod- labeled with different fluorescence were simultaneously shaped bacteria on not all but a considerable number of the used for the detection, the ectosymbionts of the two distinct Oxymonas sp. cells. The fraction of Oxymonas sp. cells that lineages were rarely detected on the same single protist cell harbored bacteria detected by this probe was 31.6±18.9%. (less than 1% of Oxymonas cells that harbored symbiotic Transmission electron microscopic observation revealed no Bacteroidales). The results indicated that the single Oxy- bacterial cell in the cytoplasm of Oxymonas sp., while bac- monas sp. cells harbored the ectosymbionts of only one of teria attached to the protist cell surface were observed (data the two lineages. not shown). Thus, the bacteria detected by the FISH were ectosymbionts of Oxymonas sp. The ectosymbionts were Discussion distributed over the entire surface of Oxymonas sp. We designed two oligonucleotide probes targeting the In this study, we identified two distinct lineages of ecto- phylotypes Nk-OxyU1-3 and Nk-OxyU1-9, respectively, symbiotic bacteria of the protist Oxymonas sp., each be- and successfully used them for the FISH-based identifica- longing to cluster V of Bacteroidales. The attachment of tion of the ectosymbiotic bacteria of Oxymonas sp. (Fig. 2). Bacteroidales members has so far been reported for the pro- The two specific probes did not detect the ectosymbionts as- tist species in the orders Oxymonadida, Cristamonadida, sociated with Devescovina sp., indicating that these probes and Trichonymphida10,19,23) (the latter two belong to the phy- were specific for each phylotype. Each of the two probes lum Parabasalia). Both of the ectosymbiont phylotypes of 20 NODA et al.

Fig. 2. FISH detection of ectosymbiotic bacteria. Specific probes NkOxy16S-B157 (panels A1 and D1) and NkOxy16S-9B439 (panels A2 and D2) were labeled with 6-FAM and used simultaneously with the eubacterial consensus probe labeled with Texas Red (panels B1 and B2). Panels C1 and C2 are phase-contrast micrographs of the same samples. Panels D1 and D2 show magnified views of ectosymbiotic bacteria on Oxymonas sp. The protist species are labeled with O (Oxymonas) and D (Devescovina). Arrowheads in panels D1 and D2 show the ectosym- biotic bacteria. Scale bars in panels C1 and C2 are 50 µm. The green fluorescence derived from the 6-FAM-labeled probes enables us to dis- tinguish the positive signals from the amorphous yellow background of auto-fluorescence or wood particles in the gut.

Oxymonas sp. are distantly related to those of Streblomastix group in cluster IV (Fig. 1). The host-ectosymbiont evolu- sp., which formed a distinct lineage of Bacteroidales10) (see tionary relationships are not simple. Fig. 1), although both of the genera Oxymonas and Streblo- One of the salient findings in this study is that, though the mastix belong to the order Oxymonadida. One of the two ec- two lineages of the Bacteroidales ectosymbionts were iden- tosymbiont phylotypes from Oxymonas sp., Nk-OxyU1-3, tified in Oxymonas sp., these two ectosymbiont lineages did was grouped with the sequences of ectosymbionts from the not occur simultaneously on individual cells of Oxymonas protists Devescovina spp. (Cristamonadida), but the protist sp. Very few, if any, cells of Oxymonas sp. harbored both genus Oxymonas (Oxymonadida) is distantly related to the lineages. In previous studies10,19,23), a single Bacteroidales genus Devescovina8,13). Thus, the phylogenetic relationships phylotype has been identified from each of the gut protist of the Bacteroidales ectosymbionts described above are un- species examined so far, except for one case (Streblomastix related to their hosts’ classifications. The phylogenetic in- sp.; see below). Thus, it seems that a single protist cell usu- congruence implies that termite gut protists have acquired ally harbors only one species of Bacteroidales as an abun- their Bacteroidales ectosymbionts multiple times during dant ectosymbiont. A Bacteroidales ectosymbiont species their evolution. On the other hand, the associations between might have competitively excluded another from the protist gut protists and their ectosymbionts in some cases seem to cell, and as a result, different Bacteroidales species could be coevolved10), since the ectosymbionts of the protists in not coexist on the same protist cell. Alternatively, the physi- the order Trichonymphida (Hoplonympha, Urinympha, Bar- ological state of the protist cells or variation in their genetic bulanympha and Staurojoenina) formed a monophyletic background, though probably small because of the homoge- Ectosymbiotic Bacteroidales Bacteria 21 neity of the rRNA gene, might affect the selection of the Acknowledgements species of Bacteroidales ectosymbionts. In the case of Stre- blomastix sp. (Oxymonadida), three major phylotypes have This work was partially supported by grants for the Bio- been identified from pools of the cells of this protist architect Research Program and the Eco Molecular Science species10). However, it is not yet clear whether each of these Research Program from RIKEN, and by a Grant-in-Aid for phylotypes is attached to individual protist cells separately Scientific Research from JSPS (No. 16380065). One of us as in the case of the ectosymbionts of Oxymonas sp., or they (H. N.) is a recipient of the Junior Research associate Pro- attach to the same protist cell simultaneously. gram from RIKEN. We thank A. Yamada for providing the In contrast, in the case of spirochetes attached to protist termites. cells in the termite gut, a single protist cell often harbors 6,11) multiple spirochete species . Moreover, some of the ecto- References symbiotic spirochetes are common to different protist species6,11). The Bacteroidales ectosymbionts investigated 1) Dyer, B.D. and O. Khalsa. 1993. Surface bacteria of Streblomas- tix strix are sensory symbionts. BioSystems 31: 169–180. so fardo not share different protist species, as shown in this 2) Guindon, S. and O. Gascuel. 2003. A simple, fast, and accurate and previous studies10). In any case, many gut protists simul- algorithm to estimate large phylogenies by maximum likelihood. taneously harbor spirochetes and Bacteroidales members as Syst. Biol. 52: 696–704. their ectosymbionts10,11,23). 3) Hara, K., N. Shinzato, T. 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