Characterization of an Obligate Intracellular Bacterium in the Midgut ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2011, p. 2869–2876 Vol. 77, No. 90099-2240/11/$12.00 doi:10.1128/AEM.02983-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Characterization of an Obligate Intracellular Bacterium in the MidgutEpithelium of the Bulrush Bug Chilacis typhae

(Heteroptera, Lygaeidae, Artheneinae)�

Stefan Martin Kuechler,* Konrad Dettner, and Siegfried KehlDepartment of Animal Ecology II, University of Bayreuth, Universitatsstraße 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 extracellularlyin specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describethe second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug ofthe family Lygaeidae (sensu stricto). Chilacis typhae possesses at the end of the first section of the midgut astructure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled withan intracellular endosymbiont. This “mycetocytic belt” might represent an evolutionarily intermediate stage ofthe usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the groELgenes showed that the bacterium belongs to the Gammaproteobacteria, and it revealed a phylogenetic relation-ship with a secondary bacterial endosymbiont of Cimex lectularius and free-living plant pathogens such asPectobacterium and Dickeya. The distribution and ultrastructure of the rod-shaped Chilacis endosymbiont werestudied 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 trans-mitted vertically. A new genus and species name, “Candidatus Rohrkolberia cinguli,” is proposed for this newlycharacterized clade of symbiotic bacteria.

The large number of species as well as individuals makeinsects the most successful animal group in the terrestrial eco-system. This great diversity would hardly be possible withoutthe help of symbiosis with microorganisms, in particular, bac-teria. It is estimated that 20 to 50% of all insects are associatedwith symbiotic microorganisms (8, 16, 32). They reside extra-or intracellularly in the gut or body cavity or in specific hostcells called bacteriocytes or mycetocytes, which form complexorgans called bacteriomes or mycetomes (9). In particular,insects that feed exclusively on nutritionally restricted dietssuch as cellulose (woody material), plant sap, seeds, vertebrateblood, or keratin materials possess obligate mutualistic symbi-onts. They aid in the degradation of the diet or supply essentialnutrients (amino acids and vitamins) to the host that the insectitself can neither synthesize nor obtain from its diet in sufficientquantities (5).

A variety of symbiotic associations is found in the largegroup of the true bugs (suborder Heteroptera), which consistsof 42,300 described species (20). Bacterial symbionts are asso-ciated with the intestinal tract, especially in plant-sucking stinkbugs of the infraorder Pentatomorpha. Species of the familiesAcanthosomatidae, Cydnidae, Plataspidae, Scutelleridae, andPentatomidae as well as the small family of Parastrachiidaeharbor their bacterial symbionts, which belong to a distinctlineage of Gammaproteobacteria, extracellularly in well-sepa-rated sections of the posterior midgut, so-called crypts or ceca(21, 26, 29, 36, 44, 46, 50, 54). Normally, the lumina of these

sacs or tubular outgrowths are still connected with the midgutlumen, but a complete separation from gut lumen has alsobeen reported for Acanthosomatidae (36). The importance ofthese specific symbionts is apparent in aposymbiotic nymphs ofseveral heteropterans, where the absence of symbionts resultsin retarded growth, mortality, and/or sterility (1, 17, 24, 32, 36,44, 47, 48). Typically, symbionts of midgut crypts will be trans-mitted vertically by three postnatal transmission mechanisms:(i) direct superficial bacterial contamination of egg surfaces(egg smearing; Pentatomidae, Acanthosomatidae) (1, 36, 46,50), (ii) deposition of bacteria-containing capsules with eggs(capsule transmission; Plataspidae) (17, 22, 44), and (iii) feed-ing on parental bacteria-containing excrement (coprophagy;Cydnidae, Coreidae, Reduviidae) (6, 14, 27, 54).

Other intestinal tract symbionts are described from differentfamilies of superfamilies Lygaeoidea and Coreoidea (Blissidae,Rhyparochromidae, Pachygronthidae, Coreidae, and Alydi-dae), as well as reduviid and pyrrhocorid stink bugs, which areassociated with betaproteobacterial symbionts (Burkholderiaspp.) and actinobacterial symbionts, respectively (30, 35, 37).In contrast to symbionts in midgut crypts of stink bugs of thesuperfamily Pentatomoidea, Burkholderia symbionts of lygae-oid and coreoid stink bugs, which do not form a monophyleticgroup, are not transmitted vertically by eggs, but instead theirsymbionts must be acquired by every new generation from theenvironment again (34).

Stink bugs of the families Cimicidae and Lygaeidae (sensustricto) harbor their symbionts in specific mycetomes, whichare well separated from the gut system (23, 38, 51, 53). As aconsequence of living intracellularly in the body cavity, thesesymbionts are transmitted vertically by transovarial mecha-nisms, which is typical for intracellular symbiosis (43). Theoccurrence of facultative, secondary symbiotic strains of

* Corresponding author. Mailing address: Department of AnimalEcology II, University of Bayreuth, Universitatsstrasse 30, 95440 Bay-reuth, Germany. Phone: 49 0921 55 2733. Fax: 49 0921 55 2743. E-mail:[email protected].

� Published ahead of print on 4 March 2011.

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Wolbachia sp. and Rickettsia sp. in the midgut epithelium, fatbody, and hemolymph of different stink bugs appears to bewidespread, as in other insects (11, 14, 33, 38). Interestingly,the Wolbachia endosymbiont of the bedbug Cimex lectulariusoccurs in mycetomes and has an obligate, nutritional, mutual-istic function (23).

In this study, we report the first finding of a rod-shaped,endosymbiotic bacterium in epithelial cells of the first midgutsection in the bulrush bug Chilacis typhae (Heteroptera:Lygaeidae). The palearctic lygaeoid C. typhae, present in NorthAmerica since 1987 (59), spends most of its time on bulrush(Typha latifolia and T. angustifolia) and feeds on seeds at dif-ferent stages of maturation (58). Both fresh and old seed headsare used. Up to 1,000 animals were once found in one seedhead. We investigated the localization as well as the transmis-sion route of the C. typhae symbiont using molecular tech-niques (fluorescence in situ hybridization [FISH]). The phylo-genetic position of the bacterial symbiont was elucidated byanalysis of the 16S rRNA and groEL genes. Finally, the mor-phological characteristics of the gammaproteobacterium wereanalyzed by electron microscopic (EM) observations.

MATERIALS AND METHODS

Sampling. Adults and larval stages of C. typhae were collected from bulrushesof the species Typha latifolia. Altogether, we investigated four populations fromdifferent areas around Bayreuth (shortest distance between habitats: 25 kmlinear distance) in the spring of 2010. Live bugs were brought into the laboratoryand were embedded for histology/FISH or dissected for bacteria characteriza-tion. We used 52 individuals for PCR, from which 10 were taken for sequencing.Seventeen individuals were required for FISH, and five individuals were used forEM. Altogether, we prepared 158 Chilacis individuals for our investigations.

Symbiont cultivation. Only insects that had been surface sterilized by ethanolwith a subsequent external flame treatment were used. The possible cultivabilityof the symbiotic, midgut bacteria was verified by plating the extracted midguttissue on two standard microbiological media: brain heart infusion (BHI) andlysogeny broth (LB) medium.

Histology. Before bugs were fixed in 4% paraformaldehyde overnight, thehemelytra were removed. The fixed bugs were washed in 1� phosphate-bufferedsaline and 96% ethanol (1:1), dehydrated serially in ethanol (70%, 90%, andtwice in 100%), and embedded in Unicryl (Plano GmbH, Germany). Serialsections (2 mm) were cut using a Leica Jung RM2035 rotary microtome (LeicaInstruments GmbH, Wetzlar, Germany), mounted on epoxy-coated glass slides,and subjected to FISH.

DNA extraction, cloning, and sequencing. DNA was extracted using a QiagenDNeasy Tissue Kit (Qiagen GmbH, Hilden, Germany) following the protocol foranimal tissue. The eubacterial 16S rRNA gene was PCR amplified using theuniversal primer set 07F (5�-AGAGTTTGATCMTGGCTCAG-3�) and 1507R(5�-TACCTTGTTACGACTTCAC-3�) (39). A 1.65-kb segment of the bacterialgroEL gene was amplified with the primers Gro-F2 (5�-ATGGCAGCTAAAGAMGTAAAATTYGG-3�) and Gro-R2 (5�-TTACATCATRCCRCCCAT-3�)(38). All PCRs were performed in a Biometra thermal cycler with the followingprogram: an initial denaturing step at 94°C for 3 min, followed by 34 cycles of94°C for 30 s, 50°C for 2 min, and 72°C for 1 min. A final extension step of 72°Cfor 10 min was included. PCR products of the expected sizes were cloned usinga TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Suitable clones for se-quencing were selected by restriction fragment length polymorphism (RFLP).Inserts were digested by restriction endonucleases RsaI and HhaI. Plasmidscontaining the DNA inserts of the expected sizes were sequenced with M13forward and M13 reverse sequencing primers (Invitrogen).

FISH. The following probes were used for FISH targeted to the 16S rRNAgene: eubacterial probe EUB338, 5�-Cy3-GCTGCCTCCCGTAGGAGT-3� (2);EUB388 II, 5�-Cy3-GCAGCCACCCGTAGGTGT-3�; EUB338 III, 5�-Cy3-GCTGCCACCCGTAGGTGT-3� (13); and the symbiont-specific probe Chila500,5�-Cy3-TTGCTGCCTTCTTCCTCGCT-3� (this study). A single mismatch dis-tinguishes the specific probe Chila500 from Ewingella americana. In addition, anonsense probe complementary to EUB338, NON338 (5�-Cy3-ACTCCTACGGGAGGCAGC-3�) (41), was used as a negative control of the hybridization

protocol. Slides were hybridized in hybridization buffer (20 mM Tris-HCl [pH8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate [SDS], 20% formamide) con-taining 10 pmol of fluorescent probes ml�1, incubated at 46°C for 90 min, rinsedin washing buffer (20 mM Tris-HCl [pH 8.0], 450 mM NaCl, 0.01% SDS),mounted with antibleaching solution (Vectashields Mounting Medium; VectorLaboratories, Peterborough, United Kingdom), and viewed under a fluorescentmicroscope.

Electron microscopy. The midgut of C. typhae was fixed in 2.5% glutaralde-hyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h, embedded in 2% agarose, andfixed again in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) overnight.The midgut was washed in 0.1 M cacodylate three times for 20 min. Followingfixation in 2% osmium tetroxide for 2 h, the sample was washed and stained enbloc in 2% uranyl acetate for 90 min. After fixation, the midgut was dehydratedserially in ethanol (30%, 50%, 70%, 95%, and three times at 100%), transferredto propylene oxide, and embedded in Epon. Ultrathin sections (70 nm) were cutusing a diamond knife (Micro-Star, Huntsville, TX) on a Leica Ultracut UCTmicrotome (Leica Microsystems, Vienna, Austria). Ultrathin sections weremounted on polyvinyl butyral (Pioloform)-coated copper grids and stained withsaturated uranyl acetate, followed by lead citrate. The sections were viewed usinga Zeiss CEM 902 A transmission electron microscope (Carl Zeiss, Oberkochen,Germany) at 80 kV.

Phylogenetic analysis. High-quality sequences of the 16S rRNA and the groELgenes were aligned using the ClustalW software in BioEdit (19) and editedmanually. A likelihood ratio test was performed using MrModeltest, version 2.3(45), to find the best-fitting models for the underlying molecular data. TheAkaike criterion selected the GTR�I�G (general time-reversible model ofnucleotide substitution with a proportion of invariant sites and gamma-distrib-uted rate heterogeneity) model for 16S rRNA and groEL gene data. Using thisassumption of sequence evolution, a Bayesian analysis with MrBayes (version3.1.2) (28) was performed with four simultaneous Markov chains for each dataset. For the 16S rRNA gene data, 10,000,000 generations were used; in total,10,000 trees were obtained (samplefreq � 1,000), and the first 2,500 of thesewere considered the burn-in and were discarded. For the groEL gene data,5,000,000 generations were run; 10,000 trees were obtained (samplefreq � 500),and the first 2,500 of these were discarded as burn-in. A maximum parsimonyanalysis was performed in PAUP*, version 4.0b10 (56). To verify faster evolutionrates in endosymbionts, relative-rate tests were carried out using Kimura’s two-parameter model in the program RRTree (49). For groEL gene sequences, onlynucleotide sites at the first- and second-codon positions were analyzed. Nucle-otide sites at the third-codon position could not be taken into account foranalysis due to saturated nucleotide substitution.

Nucleotide sequence accession numbers. The DNA sequences of the 16SrRNA gene and the groEL gene determined in this study were deposited in theDDBJ/EMBL/GenBank nucleotide sequence databases under the accessionnumbers FR729476 and FR729479.

RESULTS

General observation of mycetocytes. Upon dissection of C.typhae, 3.8 to 4.7 mm in size (Fig. 1A), a red, narrow “belt,” thesymbiotic organ, can be found at the end of enlarged firstmidgut section (Fig. 1B and C, m1) (nomenclature accordingto reference 55). It consists of a number of enlarged epithelialcells (�100 �m wide), arranged in a circle, which harbor in-tracellular symbiotic bacteria and are richly supplied with tra-cheae. All analyzed individuals were positive for the symbioticbacterium and presented the same localization pattern andmorphological characteristics in all adult and larval stages (Fig.1D). The mycetocytic belt was generally much larger in femalesthan in males (data not shown).

Cultivation experiments of the Chilacis symbiont resultedin a negative outcome. No microbial colonies could be ob-tained.

Identification of a bacterial symbiont. A 1.5-kb segment ofthe eubacterial 16S rRNA gene was amplified by PCR fromDNA samples of the midgut epithelium of C. typhae and sub-jected to cloning and RFLP typing. All RFLP types of 40clones were identical. Five clones were sequenced and com-

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pared with other sequences in the GenBank database. Allnucleotide sequences were identical and exhibited 98% simi-larity to the sequence of Pectobacterium sp. (DQ418491.1), anisolate from the woolly apple aphid Eriosoma lanigerum (Aphi-

didae) (unpublished data). Furthermore, a 1.65-kb segment ofthe gammaproteobacteria groEL gene was amplified and wassubjected to cloning and sequencing. It is one of the mosthighly expressed proteins in endosymbiotic bacteria of insects

FIG. 1. Intracellular endosymbiont of Chilacis typhae. (A) Adult female. (B) Dissected digestive system showing the three midgut sections (m1to 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 sectionsstained with the specific probe Chila500 (Cy3; yellow) and DAPI (blue). (E) Cross-section of the symbiotic midgut tissue. The epithelial cells, whichare circularly arranged around the intestinal lumen (il), are completely filled with endosymbionts. (F) Longitudinal section of the symbiotic midguttissue. (G) Enlarged image of the transition from normal midgut tissue (asterisk) to midgut epithelial cells with endosymbionts. There are no othercells between the intestinal lumen (il), which is filled with nutrient, and the symbiotic organ. (H) Schematic illustration of a longitudinal sectionthrough an ovariole. (Adapted from reference 53 with kind permission of Springer Science�Business 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 poleend of the oocyte (o), surrounded by follicle cells (f), indicating transovarial transmission.

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and is well suited for phylogenetic analysis because of its vari-ability. RFLP genotyping and sequencing of the clones identi-fied only a single sequence type, which showed the highestagreement of 84% with the free-living, plant-pathogenic bac-terium Dickeya dadantii (CP001836.1). The symbiont genesexhibited AT contents comparable to levels in free-living gam-maproteobacteria, with 46.36% for the 16S rRNA gene and48.51% for the groEL gene.

Phylogenetic position of the Chilacis symbiont. Phylogeneticanalysis of 16S rRNA gene sequences showed that the endo-symbiont of C. typhae had a phylogenetic relationship to thesecondary endosymbiont (S-endosymbiont) of Cimex lectu-larius and plant-pathogenic bacteria Pectobacterium, Dickeya,and Edwardsiella (95% posterior probability) (Fig. 2) This phy-logenetic placement was also seen in a phylogenetic analysisusing the groEL gene (Fig. 3). The Chilacis symbiont clusteredtogether with Dickeya and Edwardsiella as near relatives. Fur-thermore, phylogenetic classification revealed that the endo-symbiont of the Chilacis cluster was distinct from other sym-biotic bacteria isolated from midguts of Pentatomoideaspecies, as well as from “Candidatus Kleidoceria schneideri”from the lygaeid bug Kleidocerys resedae.

The evolutionary substitution rates of the 16S rRNA andgroEL gene sequences of the Chilacis endosymbiont wereslightly higher than those of free-living, culturable gammapro-teobacteria but far from the significantly high rates of theintracellular Kleidocerys symbiont (Table 1).

In situ hybridization of endosymbiotic gammaproteobacte-rium. Cross-sections of the body were subjected to in situhybridization with specific oligonucleotide probes. Specific sig-nals could be detected only in a belt-shaped zone at the end ofthe first midgut section (Fig. 1E and F). This structure con-sisted of epithelial cells, which were distinctly enhanced andcompletely filled with endosymbionts (Fig. 1G). Thus, the my-cetocytic epithelial cells are located in direct contact with theintestinal lumen. The distribution of the Chilacis endosymbiontwas also investigated in ovarioles and in all five larval stages ofC. typhae. It could be demonstrated that symbionts, typical forvertical transmission of endosymbiotic bacteria, were trans-ferred to the eggs via an “infection zone” of the ovarioles.Symbionts were circularly arranged in follicle cells of the ger-marium surrounding the developing oocytes (Fig. 1H and I).Subsequently, they were integrated central to the proximalpole end of the oocytes (Fig. 1H and J). As in adults, themycetocytes occurred with the midgut tract in all larval stages.In the last larval stage (larval stage 5 [L5]) and in emergingfemales, the mycetocytic midgut section was most strongly de-veloped (data not shown).

Electron microscopy of midgut cells. Ultrastructural exam-ination of midgut epithelial cells of C. typhae, which wereenclosed by a thin epithelium layer, showed that the cells werecompletely filled with a rod-shaped bacteria (Fig. 4A). Withthe exception of the nucleus, no further cell organelles couldbe observed. Mitochondria, which are usually frequent in my-cetocytes (e.g., in Kleidocerys), were uncommon or were miss-ing completely. Due to their small diameter of only 0.5 �m anda length up to 10 to 15 �m, the bacteria offered a very long,filamentary structure (Fig. 4A and B).

DISCUSSION

The molecular characterization of the intracellular endo-symbiont of Chilacis typhae presents the second description ofa symbiotic bacterium in the family of Lygaeidae (sensustricto), after “Candidatus Kleidoceria schneideri,” isolatedfrom the birch catkin bug Kleidocerys resedae (38). Althoughboth lygaeids feed exclusively on seeds, two completely differ-ent forms of endosymbiosis have developed in these two insectspecies. While K. resedae as well as Kleidocerys ericae (unpub-lished data) exhibits a red, raspberry-shaped mycetome, whichis completely separate from the intestine, the endosymbiont ofC. typhae is intracellularly localized in the epithelial cells of themidgut. This might be a link between the occurrence of extra-cellular symbionts in intestinal crypts in the Pentatomoideaand Coreoidea (1, 22, 26, 29, 35, 36, 37, 44, 46, 50, 54) and theoccurrence of symbiotic bacteria in mycetomes, as in Kleido-cerys and other Lygaeidae (38, 53), as well as in bed bugs (23).Additionally, phylogenetic analysis reveals that the symbiontsof Chilacis and Kleidocerys cluster completely separately fromeach other. This indicates that “Candidatus Kleidoceriaschneideri” and the Chilacis endosymbiont were probablytaken up independently from each other and might representin their current conditions different stages of development ofhost-symbiont coevolution.

Furthermore, relative-rate tests of the 16S rRNA and groELgene sequences of the Chilacis and Kleidocerys endosymbiontsindicate that the evolutionary rates of the gammaproteobacte-rium of Chilacis were slightly higher than in related free-livingbacteria and can be compared, for example, with the rate ratioof the secondary, Sodalis-allied symbiont from the scutelleridbug Cantoa ocellatus (29). In contrast, the rates of substitutionfor “Candidatus Kleidoceria schneideri” are significantlyhigher than for other obligate endosymbiotic bacteria, as inAcanthosomatidae (36), Parastrachiidae (26), or Scutelleridae(29). This ratio is also reflected in the nucleotide compositionof the two studied genes. The 16S rRNA and groEL genesequences of the Chilacis endosymbiont exhibit no remarkableAT-biased nucleotide composition and are comparable tothose of free-living bacteria. This indicates that the Chilacisendosymbiont is at an early stage of a symbiotic relationship orhas only recently diverged from a free-living ancestor. Theidentification of the symbiont genome size will show whetherthe genome is comparable to genomes of free-living bacteria orwas already strongly reduced, as in other intracellular bacteria(43).

However, the form of the transmission of the Chilacis sym-biont is comparable with that of “Candidatus Kleidoceria.” Astypical for intracellular bacteria, the Chilacis and Kleidoceryssymbionts are transmitted transovarially to offspring via a belt-shaped infection zone in each ovariole. This is in contrast tothe posthatching symbiont transmission mechanism, which isfound in representatives of Pentatomoidea (1, 6, 17, 22, 27, 36,44, 46, 50, 54).

Most endosymbionts cannot be cultured outside their hosts(43). This may also be true for extracellular gut bacteria ofpentatomomorphan species (25, 36). Therefore, little informa-tion is available regarding the interaction between the stinkbug hosts and their gut and mycetomic symbionts. For exam-ple, it is assumed that the cecum-associated bacteria of the



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shield bug Parastrachia japonensis (Parastrachiidae) are in-volved in recycling of uric acid (26, 31). Moreover, it is spec-ulated that the gut symbiont of the shield bug Cantoa ocellatus(Scutelleridae) is involved in detoxification of plant defense

substances (29). A recent study describes the possible involve-ment of the intracellular Wolbachia sp. endosymbiont and vi-tamin biosynthesis in the bedbug Cimex lectularius (23), aninterrelationship which is already proved for Rhodococcus in

FIG. 2. Phylogenetic position of the endosymbiont of C. typhae. Consensus tree of the Bayesian interference with 47 sequences of the 16S rRNAgene (MrBayes; 1,353 bp [554 variable sites, 373 parsimony-informative], 10,000,000 generations, 10,000 trees; samplefreq � 1,000; burn-in, 2,500).The tree has been rooted with Vibrio cholerae as an outgroup. Support values of �0.5 are indicated at the nodes.



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Triatominae (Reduviidae) (3) and other blood-sucking insects(9). Interestingly, a secondary endosymbiont which is closelyrelated to the Chilacis symbiont could be observed in the samestudied Cimex specimens. But in contrast, the secondary sym-biont was not concentrated in specific cells but, rather, was

detected in diverse cells and tissues, particularly in Malpighiantubules and ovariole pedicels (23). Moreover, these tubular,gammaproteobacterial cells were not found in all studied bed-bugs. Thus, it was assumed that this endosymbiont is not es-sential for bedbugs.

FIG. 3. Phylogenetic position of the endosymbiont of C. typhae. Consensus tree of the Bayesian interference with 29 sequences of the groELgene (MrBayes; 1,574 bp [824 variable sites, 690 parsimony-informative], 5,000,000 generations, 10,000 trees; samplefreq � 500; burn-in, 2,500).The tree has been rooted with Vibrio cholerae as an outgroup. Support values of �0.5 are indicated at the nodes.

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

Host andgene

Lineage 1(accession no.)

Lineage 2(accession no.)

Outgroup(accession no.) K1

a K2b K1 � K2 (SD)

Rateratio

(K1/K2)P valuec

Chilacis16S rRNA Gut symbiont of

C. typhae (FR729479)D. zeae (CP001655) and

P. carotovorum (AF373185)V. cholerae

(X74694)0.111 0.092 0.019 0.0048 1.2 5.6e�05

groEL Gut symbiont ofC. typhae (FR729476)

D. zeae (CP001655) andP. carotovorum (CP001657)

V. cholerae(CP001235)

0.104 0.087 0.017 0.007 1.2 0.017

Kleidocerys16S rRNA “Candidatus Kleidoceria

schneideri” (FN555107)D. zeae (CP001655) and

P. carotovorum (AF373185)V. cholerae

(X74694)0.149 0.09 0.06 0.0098 1.7 1e�07

groEL “Candidatus Kleidoceriaschneideri” (FN555108)

D. zeae (CP001655) andP. carotovorum (CP001657)

V. cholerae(CP001235)

0.16 0.087 0.073 0.0115 1.8 1e�07

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.



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The consistent occurrence of the Chilacis symbiont in themidgut epithelium in all larval stages and adult females sug-gests a mutualistic relationship, which could be important es-pecially in development. In particular, the close proximity ofthe endosymbiont to the gut lumen of the first midgut section(m1) suggests a potential participation in nutrient digestion.This idea is conceivable when one considers that the nextphylogenetically related bacteria are plant-pathogenic entero-bacteria such as Dickeya or Pectobacterium (52), which canbreak down plant cell wall material by the production of dif-ferent kinds of pectolytic enzymes, cellulases, and proteases,causing necrosis, blight, and soft rot, a form of progressivetissue degradation (12, 57). A similar decomposition process ofcarbohydrates with the help of symbionts may take place in theanterior midgut of Chilacis. The fact that symbiotic gut bacte-ria contribute to seed digestion is also assumed for granivorousbeetles (40). Analysis of freshly ingested food in the first gutregion (m1) of Oncopeltus fasciatus, which is also a seed-suck-ing lygaeid bug, revealed that it consisted of 50% water, 46%lipid, 5% protein, and only 1% soluble carbohydrates (60). Ifthese proportions of the composition of the ingested nutrientalso occurred in the seeds of Typha sp., then the use of pecto-lytic enzymes and/or cellulase would be rather insignificantbecause of the low ratio of carbohydrates.

Interestingly, the midgut ceca of stink bugs of the familyPentatomidae also contained symbiotic bacteria which werealso placed in a clade with Erwinia and Pantoea species (46).The gut symbionts of Platia stali are found in a structure (1)which is quite comparable to the mycetocytic belt of Chilacis.In addition, long bacilliform microorganisms are located intra-cellularly in the cytoplasm of epithelial cells of the gastric ceca.But in contrast to Chilacis, these cells are on the hemolymphside of the posterior midgut and are connected to the midgutlumen via a fine pore. It was speculated that symbionts in thesemycetocytes produce vitamin A1 or carotene and vitamin Ebecause crystals of vitamin A1 or carotene could be detected inthe ceca. Such crystals could not be observed in Chilacis. An-other relationship of Erwinia-related symbiotic bacteria wasdescribed in fruit flies (Diptera: Tephritidae). Here, the bac-teria reside intracellularly in the epithelial cells of digestivetissue in larval stages, as in Chilacis, but also extracellularly inthe midgut lumen between the peritrophic tube and the midgut

epithelium in adults (15, 42). In the olive fly Bactrocera oleaethe symbionts were additionally localized in a special cephalicorgan (esophageal bulb or pharyngeal bulb) connected to thepharynx (9, 10). A recent study indicates that these bacteriahave an effect on olive fly fitness and are involved in thenitrogen cycle and/or with supplementation of essential aminoacids (7). However, Erwinia and related species are not re-stricted to the intestine as mutualistic bacteria. In the peaaphid Acyrthosiphon pisum and the fruit fly Drosophila mela-nogaster, Erwinia (alternatively, Dickeya) is also pathogenic (4,18). The potential virulence factors produced by most speciesof the Erwinia group to infect plant tissue (12) or insect tissue(18) might have originally played a role for the penetration ofintestinal cells in Chilacis. In this context, further research isrequired to investigate in more detail the function and evolu-tionary pathways of the Erwinia-related endosymbiont inChilacis.

On the basis of the distinct genetic, phylogenetic, and mi-crobiological traits described above, we propose the name“Candidatus Rohrkolberia cinguli” for this new lineage. Theintracellular life cycle in an animal organism particularly sep-arates the Chilacis endosymbiont from the next related anddescribed strains of Dickeya and Pectobacterium. The genericname refers to the German name of Typha species, whereasthe specific name refers to the belt-shaped structure of midgutmycetocytes.

ACKNOWLEDGMENTS

We thank S. Geimer and R. Grotjahn for assistance in electronmicroscopy analysis, as well as D. Scholz and B. Westermann for theopportunity to use the fluorescence microscope and for providing help.Furthermore, E. Helldorfer is acknowledged for drawing the schematicillustrations. We also thank A. Kirpal for technical assistance. We aregrateful to J. Woodring for reading and correcting the manuscript.

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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) Highmagnification of longitudinal sections of a Chilacis symbiont showing typical Gram-negative morphology.



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