APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2011, p. 2869–2876 Vol. 77, No. 9 0099-2240/11/$12.00 doi:10.1128/AEM.02983-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved. 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 relation- ship 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 trans- mitted 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% glutaralde- hyde 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