Bell-shaped ciliates of the subclass Peritrichia, such as Vorticella, Carchesium and Epistylis, are commonly found in freshwater and other aquatic environments, either solitary or colonial. Peritrichs attach to a substratum via a contractile or non-motile stalk, and collect food particles by water current using ciliary rows around the edge of the bell, called the peristome. Some peritrichs are epibiotic and ectocommensalistic associates of aquatic insects and other animals, settling on the surface of their specific hosts. Only a few peritrichs are known to establish a more internal association with their hosts, locating within the preoral cavity or esophagus of water beetles and presumably subsisting on food materials chewed and ingested by the insects. To date, no endoparasitic or endocommensalistic peritrichs have been reported from insects. Host insects reported to date have all been aquatic, and given the aquatic lifestyle of peritrichs, terrestrial hosts have been considered unlikely. In the present study, we report a dense population of bizarre microbes within the gut of a terrestrial insect, and histological, ultrastructural and molecular phylogenetic analyses identified it as a peritrich ciliate. The highly-developed hindgut of the stag beetle Aegus currani contained oval colonial peritrichs connected by branched stalks resembling grape clusters. Each zooid exhibited a reduced peristome without disc, a vestibulum with active ciliary movement inside, and an elongated macronucleus. These features are morphologically reminiscent of but distinct in some respects from those in Operculariella parasitica, known from the esophagus of dysticid diving beetles. Taxonomic, ecological and functional aspects of this gut-dwelling peritrich warrant future study.
Members of the subclass Peritrichia (Ciliophora: Oligohymenophorea) comprise a large group of morphologically peculiar ciliates. Well-known representatives of the Peritrichia include Vorticella, Carchesium, and Epistylis. These and other peritrich ciliates are commonly found in aquatic environments, either solitary or colonial. Peritrichs attach to a substratum via a contractile or non-motile stalk, and possess a bell-shaped structure with ciliary rows along the edge of the bell (the peristome or collar), the movement of which drives food particles into a cellular opening (known as the vestibule, infundibulum, or funnel) that leads to the cytopharynx where food particles are endocytosed to form food vacuoles (Fenchel, 1987; Lynn, 2008; Foissner and Hawksworth, 2009).
In European freshwater environments, some extensive surveys of peritrich ciliates have been conducted (Stammer, 1948; Nenninger, 1948; Lust, 1950; Matthes, 1950; Biegel, 1954), and revealed that (i) approximately 30% of sessile peritrichs settle on diverse abiotic, plant and animal substrata; (ii) another 30% of the sessile peritrichs exhibit some specificity, for example, settling only on animals, for which the host species span various classes and orders; and (iii) the remaining 40% of sessile peritrichs are highly specific, each species settling on only a few or a single animal host species (Nenninger, 1948; Lust, 1950; Steffan, 1967). While most of the highly host-specific peritrich ciliates are epibionts/ectocommensals that attach themselves to the outer surfaces of host animals, previous studies reported two notable epibiotic peritrich taxa that are ecologically closer to endoparasites/endocommensals: Orbopercularia ominosa and O. gnathophila, which inhabits the preoral cavity of whirligig beetles (Coleoptera: Gyrinidae) (Lust, 1950; Matthes and Scheubel, 1971); and Operculariella parasitica, which inhabits the esophagus of diving beetles (Coleoptera: Dytiscidae) (Stammer, 1948a). These peritrich ciliates exhibit several peculiar morphological traits, including reduced size of the peristome. It has been argued that the smaller peristome is related to their special ecological niche in which they subsist on food materials chewed and ingested by host insects (Steffan, 1967). There have been no reports to date on true endoparasitic/endocommensalistic peritrichs associated with insects.
Stag beetles (Coleoptera: Lucanidae) comprise a large group of morphologically spectacular insects that includes some 1500 described species in the world (Fujita, 2010). Stag beetle larvae generally feed on decaying wood, although it is the fungal bodies decomposing the wood, not the wood materials themselves, which are nutritionally important for sustaining larval growth (Tanahashi et al., 2009; Tanahashi and Kubota, 2013). All stag beetles examined thus far possess a membranous pouch specialized for harboring symbiotic yeasts, called the mycangium, in association with the ovipositor of adult females (Tanahashi et al., 2010; Tanahashi and Fremlin, 2013; Tanahashi and Howes, 2016). Yeast symbionts in the mycangia of Lucanus, Dorcus and Platycerus species are closely related to the xylosefermenting yeast Scheffersomyces stipitis (formerly called Pichia stipitis) (Tanahashi et al., 2010; Hawes, 2013; Tanahashi and Fremlin, 2013), which is suggestive of involvement of mycangial yeasts in the xylophagous nutritional physiology of their lucanid hosts.
Here we report the discovery of a novel microbial associate in the Lucanidae. During a global survey of fungus—lucanid mycangial symbiotic associations, we encountered a stag beetle species harboring bizarre microorganisms in its highly-developed hindgut, which we identify here as peritrich ciliates. The intra-intestinal association of a peritrich with a non-aquatic insect is to our knowledge unprecedented.
MATERIALS AND METHODS
An adult male (Fig. 1A) and four adult females (Fig. 1B) of Aegus currani, originating from General Nakar, Quezon, Luzon Island, Philippines (Table 1), were purchased at an insect shop (Mushi-sha, Tokyo, Japan; http://www.mushi-sha.com). A. currani is endemic to Luzon island, Philippine, and the insects were field-collected and imported to Japan. Stag beetles of the genus Aegus are distributed across southwestern Asia and include over 200 described species, of which A. currani is the largest in size; adults males sometimes exceed 70 mm in body length (Fujita, 2010).
Light and electron microscopy
The insects were dissected in a phosphate-buffered saline (PBS; 8 g NaCl, 2.9 g NaHPO4·12H2O, 0.2 g KCl and 0.2 g KH2PO4 in 1 L distilled water) in Petri dishes using fine forceps and scissors under a dissection microscope (S8APO, Leica Microsystems) connected to a digital camera. The dissected alimentary tracts were observed under a light microscope (Axiophot, Carl Zeiss Microscopy) with differential interference and epifluorescence optics, and subjected to histological preparations. For tissue sectioning and staining, the samples were fixed in 4% paraformaldehyde in PBS, dehydrated and cleared through a xylene-ethanol series, embedded in paraffin, processes into serial tissue sections (10–15 µm thick), mounted on glass slides, dewaxed through a xylene-ethanol-water series, stained with hematoxylin and eosin, dehydrated, and cleared through a water-ethanol-xylene series, mounted in balsam with coverslips, and observed under a light microscope. For confocal fluorescence imaging, the samples were fixed in Carnoy's solution (60% ethanol, 30% chloroform and 10% glacial acetic acid), stained with Sytox Orange for visualizing DNA, with a mouse anti-ß-tubulin antibody (primary antibody) and a rabbit anti-mouse lgG antibody (secondary antibody) labeled with Alexa Fluor 488 or 567 for visualizing ß-tubulin, and with an oligonucleotide probe EUB338 (5′-GCT GCC TCC CGT AGG AGT-3′) targeting eubacterial 16S rRNA for visualizing bacteria, and observed under a laser confocal microscope (Pascal 5, Carl Zeiss Microscopy). For inspecting fine structure, the samples were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C overnight, and subsequently with 2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) at 4°C for 60 min. After dehydration through a water-ethanol series, the samples were embedded in Spurr resin, processed into ultrathin sections (80 nm thick) using an ultramicrotome (Ultracut-N, Leichert-Nissei), mounted on copper meshes, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (model H-7000, Hitachi).
Samples of A.currani examined in this study.
Molecular phylogenetic analysis
The dissected hindgut samples were crushed in liquid nitrogen, suspended in a lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton-X, 20 mg/ml lysozyme) and subjected to DNA extraction using QIAmp DNA Mini Kit (Qiagen). A 1.5 kb region of eukaryotic 18S rRNA gene was amplified by PCR from the DNA samples using the primers NS1 (5′-GTA GTC ATA TGC TTG TCT C-3′) and FS2 (5′-TAG GNA TTC CTC GTT GAA GA-3′) under a temperature profile of 95°C for 5 min followed by 28 cycles consisting of 95°C for 1 min, 53°C for 1 min and 72°C for 1.5 min. The PCR products were subcloned into pT7Blue plasmid vector (Novagen). Competent cells of Escherichia coli DH5α (TaKaRa) were transformed with the plasmids, and white colonies (8-16 colonies per plate) were subjected to PCR amplification using pT7Blue-specific primers U-19 and R-20 (Novagen). The PCR products were sequenced using a DNA sequencer (3130×1 Genetic Analyser, Applied Biosystems) with the sequencing primers U-19, R-20 and NS3 (5′-GCA AGT CTG GTG CCA GCA GCC-3′) (White et al., 1990). These short reads (˜600 bases) were then assembled into contigs using the software ContigExpress (Invitrogen). The contig sequences were inspected by BLAST searches ( http://blast.ncbi.nlm.nih.gov/Blast.cgi) to exclude non-ciliate contigs.
The ciliate 18S rRNA gene sequences were subjected to multiple alignment using the program ClustalW (Larkin et al., 2007). The alignments were inspected and corrected manually, from which ambiguously aligned sites were removed. Phylogenetic analyses were conducted by the maximum-likelihood (ML) method using the program Mega 6 (Tamura et al., 2013). We selected the Tamura + G + I model on the basis of the Akaike criterion. Bootstrap tests were performed with 1000 replications.
RESULTS AND DISCUSSION
In order to examine the yeast-harboring mycangium, an adult female of A. currani was dissected. The mycangium is an invaginated pouch-like organ made of inter-segmental membrane located at female's dorsal abdominal tip (Fig. 1C). During the dissection, we noticed that, when the hindgut was torn in the saline, numerous cyst-like particles spilt out (Fig. 1D). The particles were connected to each other by branching filaments, forming clusters resembling bunches of grapes (Fig. 1E–G). Careful dissection of the hindgut revealed that the particles filled the hindgut cavity as huge clusters, being attached to the inner wall of the hindgut via the filaments (Fig. 1F).
Light microscopic observations showed that the particles exhibited active ciliary movements inside, but not outside (Fig. 1H and I; Supplementary Movies S1 (AegusPeritrichMovieS1.mp4) and S2 (AegusPeritrichMovieS2.mp4)), suggesting the possibility that the particles are ciliate cells of a particular type. Given their characteristic traits, we conjectured that the particles might represent a ciliate of the subclass Peritrichia. Members of the Peritrichia embrace Vorticella, Carchesium, Epistylis and other bell-shaped sessile filter feeders commonly found in aquatic environments, which generally lack surface cilia except for ciliary rows around the cytostome, namely the peristome and the vestibule, for feeding (Fenchel, 1987; Lynn, 2008; Foissner and Hawksworth, 2009). Each particle was regarded as a peritrich cell or a zooid (Fig. 1D and E). The particle cluster looked like a peritrich colony consisting of zooids and branching stalks (Fig. 1E). The colonies were often so large and intermingled in the hindgut cavity that it was difficult to count the number of zooids in a colony (Fig. 1F and G). Distinct from free-living colonial peritrichs, such as Carchesium and Epistylis, whose zooids are bell-shaped with a ciliated peristome extending outward for efficiently collecting food particles, the zooids in the hindgut of A. currani were egg- or bud-shaped, their peristome was reduced and encased internally, and consequently their cytostome opening was very small, which probably reflects adaptation to the nutrition-rich intrahost environment (Fig. 1G-I). Morphometry of the zooids yielded ciliate cell dimensions of 127 ± 22 µm in length and 94 ± 22 µm in width (mean ± SD; n = 20).
By gently pressing the zooids on glass slides using a coverslip, we were able to observe ciliary movements and cellular activities within the ciliate cells (Fig. 1H and I; Supplementary Movies S1 (AegusPeritrichMovieS1.mp4) and S2 (AegusPeritrichMovieS2.mp4)). In addition to the movement of shorter cilia around the reduced peristome, very active movement of longer cilia was seen within the vestibule, which appears to contribute to food intake from the surrounding milieu. At the bottom of the vestibule was located the cytopharynx, in which food vacuoles form one after another (Fig. 1H; Supplementary Movie S1 (AegusPeritrichMovieS1.mp4)). We also observed, in association with the vestibule, a contractile vacuole, the contraction of which forces excess water into the vestibule cavity (Fig. 1I; Supplementary Movie S2 (AegusPeritrichMovieS2.mp4)).
Tissue sectioning and histological staining of the ciliateharboring hindgut clarified the structural and cytological configuration of the host and the microbe. Hematoxylin and eosin staining visualized the zooids distributed throughout the hindgut cavity, each containing a densely-stained intracellular body (Fig. 1J). Fluorescent DNA binding dye intensely stained a slender C-shaped structure in each zooid, which sometimes exceeded 150 µm in length (Fig. 1K and L; Supplementary Movie S3 (AegusPeritrichMovieS3.mp4)). The structure is evidently the macronucleus of the ciliate. Note that this structure was also distinguishable in light microscopic images of the live ciliate cells (Fig. 1H and I). Transmission electron microscopy revealed fine structures of the macronucleus, mitochondria, vestibular cilia, food vacuoles, etc. (Fig. 2A-C).
Fluorescence in situ hybridization using an oligonucleotide probe targeting bacterial 16S rRNA detected some signals within the ciliate cells (Fig. 1L). Transmission electron microscopy consistently detected bacterial cells within the cytoplasm (Fig. 2D), which probably represent endosymbiotic bacteria of the ciliate; their characterization will require future study. Note that endosymbiotic bacteria are commonly found in diverse ciliates and other protozoans (Heckmann and Görtz, 1992; Fujishima, 2009).
On a review of the literature, we noted that the ciliate in the hindgut of A. currani is morphologically and cytologically similar to Operculariella parasitica known from the esophagus of dysticid diving beetles (Stammer, 1948). The cell shape and size, the reduced peristome, the basic configuration of stalks, the structure of vestibule, etc., were quite similar between the ciliate and O. parasitica (Table 2). On the other hand, the ciliate exhibited much larger colony size, different morphology of macronucleus, and above all, distinct ecology and host specificity in comparison with O. parasitica (Table 2). Molecular phylogenetic analysis based on 18S rRNA gene sequence confirmed the placement of the ciliate within the Peritrichia (Fig. 3), but because no genetic information is available for O. parasitica, it is currently unknown whether the ciliate is phylogenetically related to O. parasitica. The morphological resemblance between them seems to support their phylogenetic affinity, but the possibility that their hostassociated lifestyle has led to convergent evolution cannot be excluded. Hence, while we suggest that the ciliate in the hindgut of A. currani may be a new member of the genus Operculariella, we withhold its taxonomic treatise at this stage until more samples from other host populations are examined and its molecular phylogenetic relationship to O. parasitica is established.
In the present study, we inspected five adult insects of A. currani in total, and the peritrich ciliates were found in the hindgut of all individuals. In four insects inspected in 2013, the ciliate density was very high, whereas the infection density was low in an insect additionally inspected in 2014 (Table 1). All these insects were derived from the same locality in Philippines; however, as A. currani is a relatively rare lucanid species, we were able to examine only a limited number of the insects.
In conclusion, we report a peritrich ciliate symbiotically inhabiting the hindgut of the stag beetle A. currani. Our finding is unprecedented in that (i) this is the first peritrich ciliate living deep in a host alimentary tract and isolated from the ambient environment, and (ii) this is the first peritrich ciliate symbiotically associated with a terrestrial insect. All peritrich ciliates identified to date are aquatic, and all known peritrich ciliates associated with animals are epibiotic/ectocommensalistic (Nenninger, 1948; Lust, 1950; Steffan, 1967). It thus seems reasonable that all host insects of peritrich ciliates reported to date have been aquatic, such as water beetles and water bugs, enabling the ciliates to swim in, settle on, and swim away in the aquatic environment unrestricted. However, the stag beetle studied here is a terrestrial insect which lives in and feeds on decaying wood as a larva, and consumes tree sap as an adult (Fujita, 2010). This poses an enigma regarding how this ciliate is able establish and maintain its association with the hindgut of the host insect. Where does the ciliate come from? How does it infect the hindgut of the host insect? Is the ciliate acquired from the environment or vertically transmitted across host generations? Is the ciliate also present in the larval alimentary tract? If so, how does it persist through the host metamorphosis? Is the ciliate infection parasitic, commensalistic, or mutualistic for the host insect? These questions remain to be addressed in future studies on both natural populations and laboratory experimental colonies of A. currani.
Comparison between Operculariella parasitica and the ciliate in the hindgut of Aegus currani.
MT designed and conducted the research. XYM performed transmission electron microscopy. TF and MT wrote the paper.
Supplementary materials for this article are available online (URL: http://www.bioone.org/doi/suppl/10.2108/zs170012).
Supplementary Movie S1 (AegusPeritrichMovieS1.mp4). The peritrich ciliate cells dissected from the hindgut of A. currani. Ciliary movements in the vestibule and the peristome, and formation of a food vacuole at the cytopharynx are highlighted. Also see Fig. 1H.
Supplementary Movie S2 (AegusPeritrichMovieS2.mp4). The peritrich ciliate cells dissected from the hindgut of A. currani. Ciliary movements in the vestibule and the peristome, and contraction of the contractile vacuole are highlighted. Also see Fig. 11.
Supplementary Movie S3 (AegusPeritrichMovieS3.mp4). Serial optical cross-sectioning of the ciliate-harboring hindgut of A. currani obtained by confocal fluorescence microscopy. DNA and tubulin are visualized in red and blue, respectively. The 3D structures of C-shaped macronuclei and branching stalks are highlighted in the movie. See also Fig. 1K and L.
We thank Dr. Yu Matsuura for technical advice and suggestion in the histological experiments, and Dr. Wilhelm Foissner for providing the information and literature on the peritrichs of the genera Orbopercularia and Operculariella associated with water beetles.