Chilacis Typhae

Characterization of an Obligate Intracellular Bacterium in the Midgut Epithelium of the Bulrush Bug Chilacis typhae (Heteroptera, Lygaeidae, Artheneinae)ᰔ Stefan Martin Kuechler,* Konrad Dettner, and Siegfried Kehl Department of Animal Ecology II, University of Bayreuth, Universita¨tsstraße 30, 95440 Bayreuth, Germany

Received 21 December 2010/Accepted 21 February 2011

Many members of the suborder Heteroptera have symbiotic bacteria, which are usually found extracellularly in specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describe the second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug of the family Lygaeidae (sensu stricto). Chilacis typhae possesses at the end of the first section of the midgut a structure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled with an intracellular endosymbiont. This “mycetocytic belt” might represent an evolutionarily intermediate stage of the usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the groEL genes showed that the bacterium belongs to the Gammaproteobacteria, and it revealed a phylogenetic relationship with a secondary bacterial endosymbiont of Cimex lectularius and free-living plant pathogens such as Pectobacterium and Dickeya. The distribution and ultrastructure of the rod-shaped Chilacis endosymbiont were studied in adults and nymph stages using fluorescence in situ hybridization (FISH) and electron microscopy. The detection of symbionts at the anterior poles of developing eggs indicates that endosymbionts are transmitted vertically. A new genus and species name, “Candidatus Rohrkolberia cinguli,” is proposed for this newly characterized clade of symbiotic bacteria.

The large number of species as well as individuals make sacs or tubular outgrowths are still connected with the midgut insects the most successful animal group in the terrestrial eco- lumen, but a complete separation from gut lumen has also system. This great diversity would hardly be possible without been reported for Acanthosomatidae (36). The importance of the help of symbiosis with microorganisms, in particular, bac- these specific symbionts is apparent in aposymbiotic nymphs of teria. It is estimated that 20 to 50% of all insects are associated several heteropterans, where the absence of symbionts results with symbiotic microorganisms (8, 16, 32). They reside extra- in retarded growth, mortality, and/or sterility (1, 17, 24, 32, 36, or intracellularly in the gut or body cavity or in specific host 44, 47, 48). Typically, symbionts of midgut crypts will be trans- cells called bacteriocytes or mycetocytes, which form complex mitted vertically by three postnatal transmission mechanisms: organs called bacteriomes or mycetomes (9). In particular, (i) direct superficial bacterial contamination of egg surfaces insects that feed exclusively on nutritionally restricted diets (egg smearing; Pentatomidae, Acanthosomatidae) (1, 36, 46, such as cellulose (woody material), plant sap, seeds, vertebrate 50), (ii) deposition of bacteria-containing capsules with eggs blood, or keratin materials possess obligate mutualistic symbi- (capsule transmission; Plataspidae) (17, 22, 44), and (iii) feed- onts. They aid in the degradation of the diet or supply essential ing on parental bacteria-containing excrement (coprophagy; nutrients (amino acids and vitamins) to the host that the insect Cydnidae, Coreidae, Reduviidae) (6, 14, 27, 54). itself can neither synthesize nor obtain from its diet in sufficient Other intestinal tract symbionts are described from different quantities (5). families of superfamilies Lygaeoidea and Coreoidea (Blissidae, A variety of symbiotic associations is found in the large Rhyparochromidae, Pachygronthidae, Coreidae, and Alydi- group of the true bugs (suborder Heteroptera), which consists dae), as well as reduviid and pyrrhocorid stink bugs, which are of 42,300 described species (20). Bacterial symbionts are asso- associated with betaproteobacterial symbionts (Burkholderia ciated with the intestinal tract, especially in plant-sucking stink spp.) and actinobacterial symbionts, respectively (30, 35, 37). bugs of the infraorder Pentatomorpha. Species of the families In contrast to symbionts in midgut crypts of stink bugs of the Acanthosomatidae, Cydnidae, Plataspidae, Scutelleridae, and superfamily Pentatomoidea, Burkholderia symbionts of lygae- Pentatomidae as well as the small family of Parastrachiidae oid and coreoid stink bugs, which do not form a monophyletic harbor their bacterial symbionts, which belong to a distinct group, are not transmitted vertically by eggs, but instead their lineage of Gammaproteobacteria, extracellularly in well-sepa- symbionts must be acquired by every new generation from the rated sections of the posterior midgut, so-called crypts or ceca environment again (34). (21, 26, 29, 36, 44, 46, 50, 54). Normally, the lumina of these Stink bugs of the families Cimicidae and Lygaeidae (sensu stricto) harbor their symbionts in specific mycetomes, which are well separated from the gut system (23, 38, 51, 53). As a * Corresponding author. Mailing address: Department of Animal consequence of living intracellularly in the body cavity, these Ecology II, University of Bayreuth, Universita¨tsstrasse 30, 95440 Bay- reuth, Germany. Phone: 49 0921 55 2733. Fax: 49 0921 55 2743. E-mail: symbionts are transmitted vertically by transovarial mecha- [email protected]. nisms, which is typical for intracellular symbiosis (43). The ᰔ Published ahead of print on 4 March 2011. occurrence of facultative, secondary symbiotic strains of

2869 2870 KUECHLER ET AL. APPL.ENVIRON.MICROBIOL.

Wolbachia sp. and Rickettsia sp. in the midgut epithelium, fat protocol. Slides were hybridized in hybridization buffer (20 mM Tris-HCl [pH 8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate [SDS], 20% formamide) con- body, and hemolymph of different stink bugs appears to be Ϫ1 widespread, as in other insects (11, 14, 33, 38). Interestingly, taining 10 pmol of fluorescent probes ml , incubated at 46°C for 90 min, rinsed in washing buffer (20 mM Tris-HCl [pH 8.0], 450 mM NaCl, 0.01% SDS), the Wolbachia endosymbiont of the bedbug Cimex lectularius mounted with antibleaching solution (Vectashields Mounting Medium; Vector occurs in mycetomes and has an obligate, nutritional, mutual- Laboratories, Peterborough, United Kingdom), and viewed under a fluorescent istic function (23). microscope. In this study, we report the first finding of a rod-shaped, Electron microscopy. The midgut of C. typhae was fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h, embedded in 2% agarose, and endosymbiotic bacterium in epithelial cells of the first midgut fixed again in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) overnight. section in the bulrush bug Chilacis typhae (Heteroptera: The midgut was washed in 0.1 M cacodylate three times for 20 min. Following Lygaeidae). The palearctic lygaeoid C. typhae, present in North fixation in 2% osmium tetroxide for 2 h, the sample was washed and stained en America since 1987 (59), spends most of its time on bulrush bloc in 2% uranyl acetate for 90 min. After fixation, the midgut was dehydrated (Typha latifolia and T. angustifolia) and feeds on seeds at dif- serially in ethanol (30%, 50%, 70%, 95%, and three times at 100%), transferred to propylene oxide, and embedded in Epon. Ultrathin sections (70 nm) were cut ferent stages of maturation (58). Both fresh and old seed heads using a diamond knife (Micro-Star, Huntsville, TX) on a Leica Ultracut UCT are used. Up to 1,000 animals were once found in one seed microtome (Leica Microsystems, Vienna, Austria). Ultrathin sections were head. We investigated the localization as well as the transmis- mounted on polyvinyl butyral (Pioloform)-coated copper grids and stained with sion route of the C. typhae symbiont using molecular tech- saturated uranyl acetate, followed by lead citrate. The sections were viewed using a Zeiss CEM 902 A transmission electron microscope (Carl Zeiss, Oberkochen, niques (fluorescence in situ hybridization [FISH]). The phylo- Germany) at 80 kV. genetic position of the bacterial symbiont was elucidated by Phylogenetic analysis. High-quality sequences of the 16S rRNA and the groEL analysis of the 16S rRNA and groEL genes. Finally, the mor- genes were aligned using the ClustalW software in BioEdit (19) and edited phological characteristics of the gammaproteobacterium were manually. A likelihood ratio test was performed using MrModeltest, version 2.3 analyzed by electron microscopic (EM) observations. (45), to find the best-fitting models for the underlying molecular data. The Akaike criterion selected the GTRϩIϩG (general time-reversible model of nucleotide substitution with a proportion of invariant sites and gamma-distrib- uted rate heterogeneity) model for 16S rRNA and groEL gene data. Using this MATERIALS AND METHODS assumption of sequence evolution, a Bayesian analysis with MrBayes (version Sampling. Adults and larval stages of C. typhae were collected from bulrushes 3.1.2) (28) was performed with four simultaneous Markov chains for each data of the species Typha latifolia. Altogether, we investigated four populations from set. For the 16S rRNA gene data, 10,000,000 generations were used; in total, ϭ different areas around Bayreuth (shortest distance between habitats: 25 km 10,000 trees were obtained (samplefreq 1,000), and the first 2,500 of these linear distance) in the spring of 2010. Live bugs were brought into the laboratory were considered the burn-in and were discarded. For the groEL gene data, ϭ and were embedded for histology/FISH or dissected for bacteria characteriza- 5,000,000 generations were run; 10,000 trees were obtained (samplefreq 500), tion. We used 52 individuals for PCR, from which 10 were taken for sequencing. and the first 2,500 of these were discarded as burn-in. A maximum parsimony Seventeen individuals were required for FISH, and five individuals were used for analysis was performed in PAUP*, version 4.0b10 (56). To verify faster evolution EM. Altogether, we prepared 158 Chilacis individuals for our investigations. rates in endosymbionts, relative-rate tests were carried out using Kimura’s two- Symbiont cultivation. Only insects that had been surface sterilized by ethanol parameter model in the program RRTree (49). For groEL gene sequences, only with a subsequent external flame treatment were used. The possible cultivability nucleotide sites at the first- and second-codon positions were analyzed. Nucle- of the symbiotic, midgut bacteria was verified by plating the extracted midgut otide sites at the third-codon position could not be taken into account for tissue on two standard microbiological media: brain heart infusion (BHI) and analysis due to saturated nucleotide substitution. lysogeny broth (LB) medium. Nucleotide sequence accession numbers. The DNA sequences of the 16S Histology. Before bugs were fixed in 4% paraformaldehyde overnight, the rRNA gene and the groEL gene determined in this study were deposited in the hemelytra were removed. The fixed bugs were washed in 1ϫ phosphate-buffered DDBJ/EMBL/GenBank nucleotide sequence databases under the accession saline and 96% ethanol (1:1), dehydrated serially in ethanol (70%, 90%, and numbers FR729476 and FR729479. twice in 100%), and embedded in Unicryl (Plano GmbH, Germany). Serial sections (2 mm) were cut using a Leica Jung RM2035 rotary microtome (Leica Instruments GmbH, Wetzlar, Germany), mounted on epoxy-coated glass slides, RESULTS and subjected to FISH. DNA extraction, cloning, and sequencing. DNA was extracted using a Qiagen General observation of mycetocytes. Upon dissection of C. DNeasy Tissue Kit (Qiagen GmbH, Hilden, Germany) following the protocol for typhae, 3.8 to 4.7 mm in size (Fig. 1A), a red, narrow “belt,” the animal tissue. The eubacterial 16S rRNA gene was PCR amplified using the symbiotic organ, can be found at the end of enlarged first Ј Ј universal primer set 07F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1507R midgut section (Fig. 1B and C, m1) (nomenclature according (5Ј-TACCTTGTTACGACTTCAC-3Ј) (39). A 1.65-kb segment of the bacterial groEL gene was amplified with the primers Gro-F2 (5Ј-ATGGCAGCTAAAG to reference 55). It consists of a number of enlarged epithelial AMGTAAAATTYGG-3Ј) and Gro-R2 (5Ј-TTACATCATRCCRCCCAT-3Ј) cells (ϳ100 ␮m wide), arranged in a circle, which harbor in- (38). All PCRs were performed in a Biometra thermal cycler with the following tracellular symbiotic bacteria and are richly supplied with tra- program: an initial denaturing step at 94°C for 3 min, followed by 34 cycles of cheae. All analyzed individuals were positive for the symbiotic 94°C for 30 s, 50°C for 2 min, and 72°C for 1 min. A final extension step of 72°C bacterium and presented the same localization pattern and for 10 min was included. PCR products of the expected sizes were cloned using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Suitable clones for se- morphological characteristics in all adult and larval stages (Fig. quencing were selected by restriction fragment length polymorphism (RFLP). 1D). The mycetocytic belt was generally much larger in females Inserts were digested by restriction endonucleases RsaI and HhaI. Plasmids than in males (data not shown). containing the DNA inserts of the expected sizes were sequenced with M13 Cultivation experiments of the Chilacis symbiont resulted forward and M13 reverse sequencing primers (Invitrogen). FISH. The following probes were used for FISH targeted to the 16S rRNA in a negative outcome. No microbial colonies could be ob- gene: eubacterial probe EUB338, 5Ј-Cy3-GCTGCCTCCCGTAGGAGT-3Ј (2); tained. EUB388 II, 5Ј-Cy3-GCAGCCACCCGTAGGTGT-3Ј; EUB338 III, 5Ј-Cy3-GC Identification of a bacterial symbiont. A 1.5-kb segment of TGCCACCCGTAGGTGT-3Ј (13); and the symbiont-specific probe Chila500, the eubacterial 16S rRNA gene was amplified by PCR from Ј Ј 5 -Cy3-TTGCTGCCTTCTTCCTCGCT-3 (this study). A single mismatch dis- DNA samples of the midgut epithelium of C. typhae and sub- tinguishes the specific probe Chila500 from Ewingella americana. In addition, a nonsense probe complementary to EUB338, NON338 (5Ј-Cy3-ACTCCTACGG jected to cloning and RFLP typing. All RFLP types of 40 GAGGCAGC-3Ј) (41), was used as a negative control of the hybridization clones were identical. Five clones were sequenced and com- VOL. 77, 2011 MIDGUT ENDOSYMBIONT OF THE BULRUSH BUG CHILACIS TYPHAE 2871

FIG. 1. Intracellular endosymbiont of Chilacis typhae. (A) Adult female. (B) Dissected digestive system showing the three midgut sections (m1 to m3), pylorus (p), rectum (r), and a red-colored midgut belt with endosymbionts (s). This section is also well supplied with trachea (arrow). (C) Schematic illustration of the dissected digestive system. (D) Fourth larval stage of C. typhae. The red midgut cells with symbiotic bacteria (s) are already visible through the transparent cuticle. (E to G, I, and J) Fluorescence in situ hybridization of the endosymbiont in midgut sections stained with the specific probe Chila500 (Cy3; yellow) and DAPI (blue). (E) Cross-section of the symbiotic midgut tissue. The epithelial cells, which are circularly arranged around the intestinal lumen (il), are completely filled with endosymbionts. (F) Longitudinal section of the symbiotic midgut tissue. (G) Enlarged image of the transition from normal midgut tissue (asterisk) to midgut epithelial cells with endosymbionts. There are no other cells between the intestinal lumen (il), which is filled with nutrient, and the symbiotic organ. (H) Schematic illustration of a longitudinal section through an ovariole. (Adapted from reference 53 with kind permission of Springer ScienceϩBusiness Media.) (I) Infection zone of the ovariole. Endosymbionts were transferred from the germarium to a developing oocyte. (J) Agglomeration of the Chilacis endosymbionts at the anterior pole end of the oocyte (o), surrounded by follicle cells (f), indicating transovarial transmission. pared with other sequences in the GenBank database. All didae) (unpublished data). Furthermore, a 1.65-kb segment of nucleotide sequences were identical and exhibited 98% simi- the gammaproteobacteria groEL gene was amplified and was larity to the sequence of Pectobacterium sp. (DQ418491.1), an subjected to cloning and sequencing. It is one of the most isolate from the woolly apple aphid Eriosoma lanigerum (Aphi- highly expressed proteins in endosymbiotic bacteria of insects 2872 KUECHLER ET AL. APPL.ENVIRON.MICROBIOL. and is well suited for phylogenetic analysis because of its vari- DISCUSSION ability. RFLP genotyping and sequencing of the clones identi- fied only a single sequence type, which showed the highest The molecular characterization of the intracellular endo- agreement of 84% with the free-living, plant-pathogenic bac- symbiont of Chilacis typhae presents the second description of terium Dickeya dadantii (CP001836.1). The symbiont genes a symbiotic bacterium in the family of Lygaeidae (sensu exhibited AT contents comparable to levels in free-living gam- stricto), after “Candidatus Kleidoceria schneideri,” isolated from the birch catkin bug Kleidocerys resedae (38). Although maproteobacteria, with 46.36% for the 16S rRNA gene and both lygaeids feed exclusively on seeds, two completely differ- 48.51% for the groEL gene. ent forms of endosymbiosis have developed in these two insect Phylogenetic position of the Chilacis symbiont. Phylogenetic species. While K. resedae as well as Kleidocerys ericae (unpub- analysis of 16S rRNA gene sequences showed that the endo- lished data) exhibits a red, raspberry-shaped mycetome, which symbiont of C. typhae had a phylogenetic relationship to the is completely separate from the intestine, the endosymbiont of secondary endosymbiont (S-endosymbiont) of Cimex lectu- C. typhae is intracellularly localized in the epithelial cells of the larius and plant-pathogenic bacteria Pectobacterium, Dickeya, midgut. This might be a link between the occurrence of extra- and Edwardsiella (95% posterior probability) (Fig. 2) This phy- cellular symbionts in intestinal crypts in the Pentatomoidea logenetic placement was also seen in a phylogenetic analysis and Coreoidea (1, 22, 26, 29, 35, 36, 37, 44, 46, 50, 54) and the using the groEL gene (Fig. 3). The Chilacis symbiont clustered occurrence of symbiotic bacteria in mycetomes, as in Kleido- together with Dickeya and Edwardsiella as near relatives. Fur- cerys and other Lygaeidae (38, 53), as well as in bed bugs (23). thermore, phylogenetic classification revealed that the endo- Additionally, phylogenetic analysis reveals that the symbionts symbiont of the Chilacis cluster was distinct from other sym- of Chilacis and Kleidocerys cluster completely separately from biotic bacteria isolated from midguts of Pentatomoidea each other. This indicates that “Candidatus Kleidoceria species, as well as from “Candidatus Kleidoceria schneideri” schneideri” and the Chilacis endosymbiont were probably from the lygaeid bug Kleidocerys resedae. taken up independently from each other and might represent The evolutionary substitution rates of the 16S rRNA and in their current conditions different stages of development of groEL gene sequences of the Chilacis endosymbiont were host-symbiont coevolution. slightly higher than those of free-living, culturable gammapro- Furthermore, relative-rate tests of the 16S rRNA and groEL teobacteria but far from the significantly high rates of the gene sequences of the Chilacis and Kleidocerys endosymbionts intracellular Kleidocerys symbiont (Table 1). indicate that the evolutionary rates of the gammaproteobacte- In situ hybridization of endosymbiotic gammaproteobacterium of Chilacis were slightly higher than in related free-living rium. Cross-sections of the body were subjected to in situ bacteria and can be compared, for example, with the rate ratio hybridization with specific oligonucleotide probes. Specific sig- of the secondary, Sodalis-allied symbiont from the scutellerid nals could be detected only in a belt-shaped zone at the end of bug Cantoa ocellatus (29). In contrast, the rates of substitution the first midgut section (Fig. 1E and F). This structure con- for “Candidatus Kleidoceria schneideri” are significantly sisted of epithelial cells, which were distinctly enhanced and higher than for other obligate endosymbiotic bacteria, as in completely filled with endosymbionts (Fig. 1G). Thus, the my- Acanthosomatidae (36), Parastrachiidae (26), or Scutelleridae cetocytic epithelial cells are located in direct contact with the (29). This ratio is also reflected in the nucleotide composition intestinal lumen. The distribution of the Chilacis endosymbiont of the two studied genes. The 16S rRNA and groEL gene was also investigated in ovarioles and in all five larval stages of sequences of the Chilacis endosymbiont exhibit no remarkable C. typhae. It could be demonstrated that symbionts, typical for AT-biased nucleotide composition and are comparable to Chilacis vertical transmission of endosymbiotic bacteria, were trans- those of free-living bacteria. This indicates that the endosymbiont is at an early stage of a symbiotic relationship or ferred to the eggs via an “infection zone” of the ovarioles. has only recently diverged from a free-living ancestor. The Symbionts were circularly arranged in follicle cells of the ger- identification of the symbiont genome size will show whether marium surrounding the developing oocytes (Fig. 1H and I). the genome is comparable to genomes of free-living bacteria or Subsequently, they were integrated central to the proximal was already strongly reduced, as in other intracellular bacteria pole end of the oocytes (Fig. 1H and J). As in adults, the (43). mycetocytes occurred with the midgut tract in all larval stages. However, the form of the transmission of the Chilacis sym- In the last larval stage (larval stage 5 [L5]) and in emerging biont is comparable with that of “Candidatus Kleidoceria.” As females, the mycetocytic midgut section was most strongly de- typical for intracellular bacteria, the Chilacis and Kleidocerys veloped (data not shown). symbionts are transmitted transovarially to offspring via a belt- Electron microscopy of midgut cells. Ultrastructural exam- shaped infection zone in each ovariole. This is in contrast to ination of midgut epithelial cells of C. typhae, which were the posthatching symbiont transmission mechanism, which is enclosed by a thin epithelium layer, showed that the cells were found in representatives of Pentatomoidea (1, 6, 17, 22, 27, 36, completely filled with a rod-shaped bacteria (Fig. 4A). With 44, 46, 50, 54). the exception of the nucleus, no further cell organelles could Most endosymbionts cannot be cultured outside their hosts be observed. Mitochondria, which are usually frequent in my- (43). This may also be true for extracellular gut bacteria of cetocytes (e.g., in Kleidocerys), were uncommon or were miss- pentatomomorphan species (25, 36). Therefore, little informa- ing completely. Due to their small diameter of only 0.5 ␮m and tion is available regarding the interaction between the stink a length up to 10 to 15 ␮m, the bacteria offered a very long, bug hosts and their gut and mycetomic symbionts. For exam- filamentary structure (Fig. 4A and B). ple, it is assumed that the cecum-associated bacteria of the VOL. 77, 2011 MIDGUT ENDOSYMBIONT OF THE BULRUSH BUG CHILACIS TYPHAE 2873

FIG. 2. Phylogenetic position of the endosymbiont of C. typhae. Consensus tree of the Bayesian interference with 47 sequences of the 16S rRNA gene (MrBayes; 1,353 bp [554 variable sites, 373 parsimony-informative], 10,000,000 generations, 10,000 trees; samplefreq ϭ 1,000; burn-in, 2,500). The tree has been rooted with Vibrio cholerae as an outgroup. Support values of Ͼ0.5 are indicated at the nodes.

shield bug Parastrachia japonensis (Parastrachiidae) are in- substances (29). A recent study describes the possible involve- volved in recycling of uric acid (26, 31). Moreover, it is spec- ment of the intracellular Wolbachia sp. endosymbiont and vi- ulated that the gut symbiont of the shield bug Cantoa ocellatus tamin biosynthesis in the bedbug Cimex lectularius (23), an (Scutelleridae) is involved in detoxiﬁcation of plant defense interrelationship which is already proved for Rhodococcus in 2874 KUECHLER ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 3. Phylogenetic position of the endosymbiont of C. typhae. Consensus tree of the Bayesian interference with 29 sequences of the groEL gene (MrBayes; 1,574 bp [824 variable sites, 690 parsimony-informative], 5,000,000 generations, 10,000 trees; samplefreq ϭ 500; burn-in, 2,500). The tree has been rooted with Vibrio cholerae as an outgroup. Support values of Ͼ0.5 are indicated at the nodes.

Triatominae (Reduviidae) (3) and other blood-sucking insects detected in diverse cells and tissues, particularly in Malpighian (9). Interestingly, a secondary endosymbiont which is closely tubules and ovariole pedicels (23). Moreover, these tubular, related to the Chilacis symbiont could be observed in the same gammaproteobacterial cells were not found in all studied bed- studied Cimex specimens. But in contrast, the secondary sym- bugs. Thus, it was assumed that this endosymbiont is not es- biont was not concentrated in speciﬁc cells but, rather, was sential for bedbugs.

TABLE 1. Relative-rate tests for the 16S rRNA and groEL gene sequences between the lineages of Chilacis and Kleidocerys endosymbionts and Dickeya zeae and Pectobacterium carotovorum as free-living relatives, as well as Vibrio cholerae as an outgroup

Rate Host and Lineage 1 Lineage 2 Outgroup K a K b K Ϫ K (ϮSD) ratio P valuec gene (accession no.) (accession no.) (accession no.) 1 2 1 2 (K1/K2) Chilacis 16S rRNA Gut symbiont of D. zeae (CP001655) and V. cholerae 0.111 0.092 0.019 Ϯ 0.0048 1.2 5.6eϪ05 C. typhae (FR729479) P. carotovorum (AF373185) (X74694) groEL Gut symbiont of D. zeae (CP001655) and V. cholerae 0.104 0.087 0.017 Ϯ 0.007 1.2 0.017 C. typhae (FR729476) P. carotovorum (CP001657) (CP001235)

Kleidocerys 16S rRNA “Candidatus Kleidoceria D. zeae (CP001655) and V. cholerae 0.149 0.09 0.06 Ϯ 0.0098 1.7 1eϪ07 schneideri” (FN555107) P. carotovorum (AF373185) (X74694) groEL “Candidatus Kleidoceria D. zeae (CP001655) and V. cholerae 0.16 0.087 0.073 Ϯ 0.0115 1.8 1eϪ07 schneideri” (FN555108) P. carotovorum (CP001657) (CP001235)

a Estimated mean distance between lineage 1 and the last common ancestor of lineages 1 and 2. b Estimated mean distance between lineage 2 and the last common ancestor of lineages 1 and 2. c P values were generated using the program RRTree. VOL. 77, 2011 MIDGUT ENDOSYMBIONT OF THE BULRUSH BUG CHILACIS TYPHAE 2875

FIG. 4. Transmission electron microscopy of midgut epithelial cells, including the gammaproteobacterial endosymbiont of C. typhae. (A) Rod- shaped symbiotic bacteria, which lie closely packed in the cytoplasm of midgut epithelial cells, directly adjacent to the intestinal lumen (il). Endosymbionts appear as ﬁlamentous structures (asterisk), with a length up to 10 ␮m, before extending out of the plane of the section. (B) High magniﬁcation of longitudinal sections of a Chilacis symbiont showing typical Gram-negative morphology.

The consistent occurrence of the Chilacis symbiont in the epithelium in adults (15, 42). In the olive fly Bactrocera oleae midgut epithelium in all larval stages and adult females sug- the symbionts were additionally localized in a special cephalic gests a mutualistic relationship, which could be important es- organ (esophageal bulb or pharyngeal bulb) connected to the pecially in development. In particular, the close proximity of pharynx (9, 10). A recent study indicates that these bacteria the endosymbiont to the gut lumen of the first midgut section have an effect on olive fly fitness and are involved in the (m1) suggests a potential participation in nutrient digestion. nitrogen cycle and/or with supplementation of essential amino This idea is conceivable when one considers that the next acids (7). However, Erwinia and related species are not re- phylogenetically related bacteria are plant-pathogenic entero- stricted to the intestine as mutualistic bacteria. In the pea bacteria such as Dickeya or Pectobacterium (52), which can aphid Acyrthosiphon pisum and the fruit fly Drosophila mela- break down plant cell wall material by the production of dif- nogaster, Erwinia (alternatively, Dickeya) is also pathogenic (4, ferent kinds of pectolytic enzymes, cellulases, and proteases, 18). The potential virulence factors produced by most species causing necrosis, blight, and soft rot, a form of progressive of the Erwinia group to infect plant tissue (12) or insect tissue tissue degradation (12, 57). A similar decomposition process of (18) might have originally played a role for the penetration of carbohydrates with the help of symbionts may take place in the intestinal cells in Chilacis. In this context, further research is anterior midgut of Chilacis. The fact that symbiotic gut bacte- required to investigate in more detail the function and evolu- ria contribute to seed digestion is also assumed for granivorous tionary pathways of the Erwinia-related endosymbiont in beetles (40). Analysis of freshly ingested food in the first gut Chilacis. region (m1) of Oncopeltus fasciatus, which is also a seed-suck- On the basis of the distinct genetic, phylogenetic, and mi- ing lygaeid bug, revealed that it consisted of 50% water, 46% crobiological traits described above, we propose the name lipid, 5% protein, and only 1% soluble carbohydrates (60). If “Candidatus Rohrkolberia cinguli” for this new lineage. The these proportions of the composition of the ingested nutrient intracellular life cycle in an animal organism particularly sep- also occurred in the seeds of Typha sp., then the use of pecto- arates the Chilacis endosymbiont from the next related and lytic enzymes and/or cellulase would be rather insignificant described strains of Dickeya and Pectobacterium. The generic because of the low ratio of carbohydrates. name refers to the German name of Typha species, whereas Interestingly, the midgut ceca of stink bugs of the family the specific name refers to the belt-shaped structure of midgut Pentatomidae also contained symbiotic bacteria which were mycetocytes. also placed in a clade with Erwinia and Pantoea species (46). The gut symbionts of Platia stali are found in a structure (1) ACKNOWLEDGMENTS which is quite comparable to the mycetocytic belt of Chilacis. We thank S. Geimer and R. Grotjahn for assistance in electron In addition, long bacilliform microorganisms are located intra- microscopy analysis, as well as D. Scholz and B. Westermann for the cellularly in the cytoplasm of epithelial cells of the gastric ceca. opportunity to use the fluorescence microscope and for providing help. Furthermore, E. Helldo¨rfer is acknowledged for drawing the schematic But in contrast to Chilacis, these cells are on the hemolymph illustrations. We also thank A. Kirpal for technical assistance. We are side of the posterior midgut and are connected to the midgut grateful to J. Woodring for reading and correcting the manuscript. lumen via a fine pore. It was speculated that symbionts in these REFERENCES mycetocytes produce vitamin A or carotene and vitamin E 1 1. Abe, Y., K. Mishiro, and M. Takanashi. 1995. Symbiont of brown winged because crystals of vitamin A1 or carotene could be detected in green bug, Plautia stali Scott. Jpn. J. Appl. Entomol. Z. 39:109–115. the ceca. 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