Resedae (Heteroptera: Lygaeidae, Ischnorhynchinae) Stefan Martin Kuchler,¨ Konrad Dettner & Siegfried Kehl

RESEARCH ARTICLE Molecular characterization and localization of the obligate endosymbiotic bacterium in the birch catkin bug Kleidocerys resedae (Heteroptera: Lygaeidae, Ischnorhynchinae) Stefan Martin Kuchler,¨ Konrad Dettner & Siegfried Kehl

Department of Animal Ecology II, University of Bayreuth, Bayreuth, Germany

Correspondence: Stefan Martin Kuchler, ¨ Abstract Department of Animal Ecology II, University of Bayreuth, Universitatsstraße¨ 30, 95440 In contrast to speciﬁc bacterial symbionts of many stinkbugs, which are harboured Bayreuth, Germany. Tel.: 149 921 552733; extracellularly in the lumina of midgut sacs or tubular outgrowths, the obligate fax: 149 921 552743; e-mail: endosymbiont of birch catkin bug Kleidocerys resedae (Heteroptera: Lygaeidae) [email protected] resides in a red-coloured, raspberry-shaped mycetome, localized abdominally, close to the midgut section. Phylogenetic analysis, based on the 16S rRNA gene and Received 21 December 2009; revised 23 March the groEL (chaperonin) gene, showed that the bacteria belong to the g-subdivision 2010; accepted 8 April 2010. of the Proteobacteria and revealed a phylogenetic relationship with bacterial Final version published online 25 May 2010. endosymbionts of Wigglesworthia glossinidia, the primary symbiont of tse-tse ﬂy Glossina brevipalpis. Furthermore, RFLP analysis and sequencing revealed that DOI:10.1111/j.1574-6941.2010.00890.x K. resedae was also infected by Alphaproteobacteria of the genera Wolbachia and

Editor: Michael Wagner Rickettsia. The distribution and transmission of Kleidocerys endosymbiont in adults and all nymph stages were studied using FISH. The detection of symbionts Keywords at the anterior poles of developing eggs indicated that endosymbionts are Gammaproteobacteria; Kleidocerys; Lygaeidae; transmitted vertically to offspring. Ultrastructural examinations by electron groEL; electron microscopy. microscopy revealed the packed accommodation of rod-shaped bacteria in the cytoplasm of mycetocytes. A new genus and species name, ‘Candidatus Kleidoceria schneideri’, is proposed for this newly characterized clade of symbiotic bacteria.

(Buchner, 1965). On the other hand, facultative symbiotic Introduction bacteria (secondary symbionts or S-symbionts) are errati- Symbiotic bacteria have diverse ecological and evolutionary cally distributed and are not essential for host survival and effects on hosts, influencing aspects of ecological interac- reproduction. Nevertheless, facultative mutualists also exert tions from nutrition to defence and affecting reproductive fitness benefits, such as protection against natural enemies, systems, with consequences for population structure, repro- and resistance to pathogenic fungi, heat and other mortality ductive isolation and speciation (Buchner, 1965; Moran factors (Kellner, 2002; Russell & Moran, 2006; Kaltenpoth & et al., 2008). In particular, the most intimate mutualistic Strohm, 2007; Scott et al., 2008), allowing their carriers to associations are found in obligate insect symbionts (called live longer and reproduce more offspring. In contrast, primary symbionts or P-symbionts), which are required for facultative symbionts such as Rickettsia or Wolbachia can successful host development and reproduction of their also appear as reproductive manipulators that cause repro- hosts, exemplified by provisioning of essential nutrients in ductive aberrations such as cytoplasmatic incompatibility, aphids (Douglas, 1998, 2006), tse-tse flies (Akman et al., male-killing, feminization of genetic males and partheno- 2002) and carpenter ants (Sauer et al., 2000). In general, they genesis. The maternally inherited manipulators increase the are inherited, usually maternally, and lack a replicative or a host reproduction through daughters, often at the expense

MICROBIOLOGY ECOLOGY MICROBIOLOGY dormant phase outside their hosts. Typically, primary sym- of reproduction through sons, and the host ﬁtness in general bionts are restricted to special cells, called mycetocytes or (Stouthamer et al., 1999; Perlman et al., 2006). bacteriocytes, which are summarized in a compact symbio- Members of the Hemiptera, such as cicadas, aphids, tic organ – the mycetome or the bacteriome, respectively mealybugs and whiteﬂies, exhibit a strong phytophageous

c 2010 Federation of European Microbiological Societies FEMS Microbiol Ecol 73 (2010) 408–418 Published by Blackwell Publishing Ltd. All rights reserved Primary endosymbiont of birch catkin bug Kleidocerys resedae 409 living, by sucking extremely unbalanced plant sap. Feeders endocellular symbionts of other insects, show remarkable of plant sap are well provided with carbohydrates available evolutionary patterns, including AT-biased nucleotide com- in the form of sucrose, but are not adequately supplied with position, accelerated molecular evolution and reduced gen- lipids, proteins, amino acids or vitamins. These shortfalls ome size (Hosokawa et al., 2006; Kikuchi et al., 2009). The will be compensated by the metabolic and biosynthetic important roles of these specific symbionts are especially capabilities of the primary symbionts of hemipteran insects, apparent in aposymbiotic nymphs of several heteropterans, best known in aphids, where the endosymbiont Buchnera where the absence of symbionts results in retarded growth aphidicola provides the host with essential amino acids and and/or nymphal mortality (Muller,¨ 1956; Abe et al., 1995; some vitamins (Douglas, 1998, 2003, 2006; Akman Gund¨ uz¨ Fukatsu & Hosokawa, 2002; Kikuchi et al., 2009). & Douglas, 2009). In the present study, we report the first molecular In addition to the ‘homopteran’ insects, most members of description of the endosymbiotic bacteria in the birch catkin Heteroptera, which feed on restricted diets – such as blood- bug Kleidocerys resedae (Heteroptera: Lygaeidae). Kleidocerys sucking bed bugs (Cimicidae), assassin bugs (Reduviidae)or resedae live on birch and alder trees (Betula spp. and Alnus plant-sapping bugs in different taxonomic groups – are spp.), feeding and breeding on the seed catkins (Wachmann dependent on a wide variety of symbiotic bacterial associa- et al., 2007) and are sometimes considered a ‘nuisance pest’ tions (Buchner, 1965; Dasch et al., 1984; Kikuchi et al., species because they invade homes in the autumn in large 2008). Both symbionts in the midgut epithelium and numbers (Sweet, 2000). As described in early histological abdominal mycetomes have been described for different studies, K. resedae exhibit a red-coloured, raspberry-shaped heteropteran bug species (Schneider, 1940; Chang & Mus- mycetome that overlies the midgut and runs in the long- grave, 1970; Dasch et al., 1984). itudinal direction of the body (Schneider, 1940). The grape- However, in many stinkbugs, specific bacterial symbionts shaped accumulation of mycetocytes, which are completely are harboured extracellularly in the lumina of midgut sacs or filled with Gammaproteobacteria, could be detected in all tubular outgrowths (Glasgow, 1914; Rosenkranz, 1939; larval stages and adults. Phylogenetic analysis inferred by Kikuchi & Fukatsu, 2008). These sac-like appendages of the 16S rRNA gene and particularly the groEL gene (chapero- posterior end of the midgut are called caeca or crypts and nin) revealed the phylogenetic position of the endosymbiont vary in number and arrangement in different taxonomic in the existing phylogenetic system of heritable endosym- groups. In most plant-sucking stinkbugs, the lumen of the bionts of insects. FISH was used to investigate bacterial midgut crypts is connected to the midgut main tract. distribution and vertical transmission. The morphological However, a complete separation of the lumen crypts from characteristics of the Gammaproteobacteria were analysed by the gut lumen has also been reported (Dasch et al., 1984; electron microscopic observations. Kikuchi et al., 2009). As a consequence of their extracellular associations in the gut cavity, the vertical transmission mechanisms of sym- Materials and methods bionts in heteropteran insects are more multifaceted than the transovarial mechanisms typical of intracellular symbio- Samples sis (Fukatsu & Nikoh, 2000; Braendle et al., 2003; Kikuchi Adults and larval stages of K. resedae were collected by heavy et al., 2007). Besides the direct superficial bacterial contam- shaking of birch branches (Betula spp.) on the university ination of egg surfaces (egg smearing; Pentatomidae, campus of Bayreuth in 2008 and 2009. Bugs were brought to Acanthosomatidae) (Rosenkranz, 1939; Abe et al., 1995), the laboratory alive and were embedded for histology/FISH two other impressive forms of transmission have been or dissected for bacteria characterization. reported: probing of parental bacteria-containing excrement (coprophagy; Cydnidae, Coreidae, Reduviidae) (Huber- Schneider, 1957; Schorr, 1957; Dasch et al., 1984; Beard Histology et al., 2002) and deposition of bacteria-containing capsules with eggs (capsule transmission; Plataspidae) (Schneider, Before all the bugs were fixed in 4% paraformaldehyde 1940; Muller,¨ 1956; Fukatsu & Hosokawa, 2002; Hosokawa overnight, the hemelytra were carefully removed. The fixed et al., 2005). Although such extracellular associations appear bugs were washed in 1 phosphate-buffered saline and 96% to be evolutionarily more casual than the endocellular ethanol (1 : 1), dehydrated serially in ethanol (70%, 90%, symbiosis, strict phylogenetic relationships of the gut sym- 2 100%) and embedded in UnicrylTM (Plano GmbH, biont, except in alydid stinkbugs (Kikuchi et al., 2007), are Germany). Serial sections (2 mm) were cut using a Leica mostly reflected in those of their hosts (Hosokawa et al., Jung RM2035 rotary microtome (Leica Instruments GmbH, 2006; Kikuchi et al., 2009). In contrast to free-living Wetzlar, Germany), mounted on epoxy-coated glass slides bacteria, gut symbionts of true bugs, just like obligate and subjected to FISH.

DNA extraction, cloning and sequencing washed in 0.1 M cacodylate three times for 20 min. Follow- s ing postfixation in 2% osmium tetroxide for 2 h, the sample DNA was extracted using a Qiagen DNeasy Tissue Kit was washed and stained en bloc in 2% uranyl acetate for (Qiagen GmbH, Hilden, Germany) following the protocol 90 min. After fixation, mycetome was dehydrated serially in for animal tissue. Eubacterial 16S rRNA gene was PCR ethanol (30%, 50%, 70%, 95% and 3 100%), transferred amplified using the universal primer set 07F (50-AGAGTTT to propylene oxide and embedded in Epon. Ultrathin GATCMTGGCTCAG-30) and 1507R (50- TACCTTGTTAC sections (70 nm) were cut using a diamond knife (Micro- GACTTCAC-30) (Lane, 1991). A 1.65-kb segment of the Star, Huntsville, TX) on a Leica Ultracut UCT microtome bacterial groEL gene was amplified with the primers Gro-F2 (Leica Microsystems, Vienna, Austria). Ultrathin sections (50-ATGGCAGCTAAAGAMGTAAAATTYGG-30) and Gro- were mounted on pioloform-coated copper grids and R2 (50-TTACATCATRCCRCCCAT-30). All PCR reactions stained with saturated uranyl acetate, followed by lead were performed in a Biometra thermal cycler with the citrate. The sections were viewed using a Zeiss CEM 902 A following program: an initial denaturating step at 94 1C for transmission electron microscope (Carl Zeiss, Oberkochen, 3 min, followed by 34 cycles of 94 1C for 30 s, 50 1C for 2 min Germany) at 80 kV. Micrographs were taken using an SO- and 72 1C for 1 min. A final extension step of 72 1C for 163 EM film (Eastman Kodak, Rochester, NY). 10 min was included. PCR products of the expected sizes were cloned using the TOPO TA Clonings Kit (Invitrogen, Carlsbad, CA). Suitable clones for sequencing were selected Phylogenetic analysis by restriction fragment length polymorphism (RFLP). High-quality sequences of the 16S rRNA gene and the groEL Inserts were digested by restriction endonucleases RsaI and gene were aligned using the CLUSTALW software in BIOEDIT HhaI. Plasmids containing the DNA inserts of the expected (Hall, 1999) and edited manually. A likelihood ratio test was sizes were sequenced at the DNA analytics core facility of the performed using MRMODELTEST V.2.3 (Nylander, 2004) to find University of Bayreuth with M13 forward and M13 reverse the best-fitting models for the underlying molecular data. sequencing primers (Invitrogen). The Akaike criterion selected the GTR1I1G model for groEL and 16S rRNA gene data. Using this assumption on FISH sequence evolution, a Bayesian analysis with MRBAYES (v.3.1.2) (Huelsenbeck & Ronquist, 2001) was performed The following probes were used for FISH targeted to the 16S with four simultaneous Markov chains for each dataset. For rRNA gene: eubacterial probe EUB338 [50-(Cy5)-GCTGC the 16S rRNA gene data, 60 000 000 generations were used; CTCCCGTAGGAGT-30](Amannet al., 1995), EUB388 II in total, 600 000 trees were obtained (samplefreq = 1000) [50-(Cy3)-GCAGCCACCCGTAGGTGT-30], EUB338 III and the first 15 000 of these were considered the ‘burnin’ and [50-(Cy3)-GCTGCCACCCGTAGGTGT-30](Daimset al., discarded. For the groEL gene data, 1 000 000 generations 1999) and symbiont-specific probe Kleido100 [50-(Cy3)-GAATC were run; 10 000 trees were obtained (samplefreq = 100) and TACCTAATGGAGA-3 0]. A single mismatch distinguishes the the first 2500 of these were discarded as ‘burnin’. A maxi- specific probe Kleido100 from Candidatus Baumannia cicadelli- Ã mum parsimony analysis was performed in PAUP nicola and the endosymbiont of Haematomyzus elephantis.In v. 4.0b10 (Swofford, 2000). addition, a nonsense probe complementary to EUB338, NON338 [50-(Cy3)-ACTCCTACGGGAGGCAGC-30](Manz et al., 1992), was used as a negative control of the hybridization Sequence data protocol. Slides were hybridized in hybridization buffer [20 mM The DNA sequences of 16S rRNA gene and the groEL gene Tris-HCl (pH 8.0), 0.9 M NaCl, 0.01% sodium dodecyl sulphate determined in this study were deposited in the DDBJ/ (SDS), 10% formamide] containing 10 pmol fluorescent pro- EMBL/GenBank nucleotide sequence databases under the bes mL–1, incubated at 46 1C for 90 min, rinsed in washing accession numbers FN555107 and FN555108, respectively. buffer [20 mM Tris-HCl (pH 8.0), 450 mM NaCl, 0.01% SDS], s mounted with antibleaching solution (Vectashield Mounting Results Medium; Vector Laboratories, Peterborough, UK) and viewed under a fluorescent microscope. General observation of mycetome In the process of dissection of the animals of K. resedae Electron microscopy 4.5–6.0 mm in size (Fig. 1a), a red-coloured, raspberry- Mycetome of K. resedae was fixed in 2.5% glutaraldehyde in shaped organ – the mycetome – could be found adhering 0.1 M cacodylate buffer (pH 7.3) for 1 h, embedded in 2% dorsally at the posterior part of the midgut section M2 agarose and fixed again in 2.5% glutaraldehyde in 0.1 M (according to Schuh & Slater, 1995) running in the long- cacodylate buffer (pH 7.3) overnight. The mycetome was itudinal direction of the body (Fig. 1b). It consisted of a

Fig. 1. Intracellular harbouring of the Kleido- cerys resedae endosymbiont in the abdominal mycetome. (a) Habitus of an adult female. Scale bar = 1 mm. (b) Dissected digestive system with three midgut sections (M1–3) and adhesive mycetome (yellow circle). Scale bar = 0.5 mm. (c) Enlarged image of the red, raspberry-shaped mycetome, which is composed of numerous mycetocytes (grapes) and well supplied with tracheae (arrows). Scale bar = 75 mm. (d,e) FISH double hybridization of mycetome cross-sections stained with specific probe Kleido100 (Cy3) and DAPI. (d) Mycetocytes (My) completely filled with endosymbionts (orange) with centrally located double nuclei (blue). Scale bar = 20 mm. (e) Agglomeration of the Kleidocerys endosymbiont (S; light yellow) at the anterior pole end of the oocyte (O), surrounded by follicle cells (F; blue, DAPI) – indicating transovarial transmission. Scale bar = 250 mm. (f–h) FISH double hybridization of mycetome cross-sections. Scale bar = 100 mm. Left panel (red): stained with the specific probe Kleido100 (Cy3). Middle panel (green): stained with the universal probe EUB338 (Cy5). Right panel (yellow): overlay. With the exception of a few other bacterial signals, which could be allocated to Rickettsia and Wolbachia (data not shown), all bacteria in the mycetome belong to the Kleidocerys symbiont. multiplicity of large mycetocytes (50–80-mm wide), which of mealybug Pseudococcus kraunhiae (AB374417.1). The were highly supplied by tracheae and surrounded by a thin validity of the sequences was confirmed by FISH performed epithelium layer (Fig. 1c). In some specimens, the relative using probe Kleido100. The sequences of the other two types position of the abdominal mycetome to the gut was not exhibited the highest similarities to 16S rRNA gene se- compelling and an intimate contact with the intestine was quences of the Wolbachia endosymbiont of parasitoid wasp missing completely. Additionally, no connection was found Nasonia vitripennis (Pteromalidae; M84686.1) and the se- between the mycetome and the midgut main tract. The quence of the Rickettsia endosymbiont of stone beetle mycetome was generally much larger in females than in Coccotrypes dactyliperda (Scolytidae; AY961085.1). males (data not shown). Furthermore, a 1.65-kb segment of the Gammaproteobac- teria groEL gene (chaperonin) was amplified by PCR from DNA samples of K. resedae and was subjected to cloning and Identification of a bacterial symbiont sequencing. It is applicable for phylogenetic analysis within A 1.5-kb segment of the eubacterial 16S rRNA gene was the endosymbiotic bacteria of insects because of its varia- amplified by PCR from DNA samples of K. resedae and bility. In addition, it was applied in current studies and used subjected to cloning and RFLP typing. Three RFLP types especially for the description of symbiotic bacteria in true were identified among 40 clones examined, of which two or bugs (e.g. Kikuchi et al., 2009), which enables a good more clones from each of the types were sequenced and comparability within the bugs. RFLP genotyping and se- compared with other sequences found in GenBank. The quencing of the clones identified only a single sequence type. sequence of one type belongs to the Gammaproteobacteria BLAST searches revealed that the groEL gene sequences showed and showed a 91% sequence similarity to the endosymbiont the highest agreement of 82% with the g-endosymbiont

B. aphidicola of aphid Thelaxes suberi (AJ439085.1). The biont. In addition, a small number of signals belong to symbiont genes exhibited high AT contents of 51.96% for other bacteria, which could be identified as Rickettsia and the 16S rRNA gene and 64.36% for the groEL gene. Wolbachia (data not shown). Besides the mycetome, scat- tered positive signals with Rickettsia- and Wolbachia-specific probes could also be detected in other tissues as well as Phylogenetic analysis of the Kleidocerys oocytes. Tissues with a high metabolism, for example body symbiont fat, were predominantly infected, but always in small Phylogenetic analyses on the basis of the 16S rRNA gene numbers. sequence revealed that the endosymbiont from Kleidocerys was placed in the phylogenetic relationship of Candidatus Electron microscopy of mycetocytes Blochmannia floridanus (from ant), Wigglesworthia glossinidia (from tse-tse fly) and other gammaproteobacterial Ultrastructural examinations of mycetocytes of K. resedae, representatives of mealybugs (97% posterior probability) which were enclosed by a thin epithelium layer, revealed a (Fig. 2). To resolve this group, the phylogenetic placement tight aggregation of rod-shaped bacteria with a thin cell wall was additionally determined with the help of the groEL gene that resides free in the cytoplasm (Fig. 4a and b). Besides (Fig. 3). Those results, which were generally concordant packed bacteria, which sometimes exhibited a very long and with results of the 16S rRNA gene analyses, showed that the filamentary structure of up to 15 mm (Fig. 4a), the mycetocytes gammaproteobacterial strain from Kleidocerys clustered included only the nucleus and a number of mitochondria again in a well-supported clade with endosymbionts of (Fig. 4b). W. glossinidia (100% posterior probability). With its close relationship with W. glossinidia, the endosymbiont of seed Discussion bug Kleidocerys is much more separated from the well- supported monophyletic groups of other bug symbionts In 1940, Schneider first described symbiotic associations in (P-symbionts) such as Candidatus Ishikawaella capsulatus detail in several species of the Lygaeidae using light micro- (plataspid bugs) and Candidatus Rosenkranzia clausaccus scopy. Here, we provide the first molecular identification of (acanthosomatid stinkbugs). The phylogenetic position of symbiotic bacteria in birch catkin bug K. resedae and Kleidocerys endosymbiont was comparable in the 16S rRNA generally for seed bugs of the family Lygaeidae. Only a few gene and groEL gene trees when the maximum-parsimony molecular descriptions of symbiotic bacteria in heteropteran method was used (data not shown). insects have been documented to date. However, in contrast to the intracellular living gammaproteobacterial symbionts of K. resedae, which are housed in specialized mycetocytes, In situ hybridization of c-bacteria hitherto existing microbiological characterizations have For the localization of the Kleidocerys endosymbiont, a 16S often described extracellular symbiotic bacteria that were rRNA gene-targeted in situ hybridization was accomplished harboured in the gut or in the lumen of midgut caeca/ with specific oligonucleotide probes for cross-sections of crypts: for example betaproteobacterial Burkholderia spp. the entire body of the bug. Specific signals could be detected from broad-headed bugs of the family Alydidae (Kikuchi only in cells of the mycetome (Fig. 1d). The numerous et al., 2005), Rhodococcus actinomycete from assassin bugs mycetocytes, which contain two nuclei, are completely filled Tritoma of the family Reduviidae (Beard et al., 2002; Yassin, with endosymbionts. There is no connection that would 2005), actinobacterial Coriobacterium glomerans from red show a transition from the midgut main tract to the firebugs Pyrrhocoris apterus of the family Pyrrhocoridae mycetome; therefore, no signals could be observed in the (Haas & Konig,¨ 1987, 1988; Kaltenpoth et al., 2009), caeca- intestine. The distribution of the Kleidocerys endosym- associated symbionts from the southern green stinkbug bionts was also investigated in ovarioles and all five larval Nezara viridula of the family Pentatomidae (Hirose et al., stages (nymphs) of K. resedae. The presence of symbionts at 2006; Prado et al., 2006; Prado & Almeida, 2009a), gamma- the anterior pole end of the oocytes (Fig. 1e) is indicative of proteobacterial symbiont from stinkbugs of the family the vertical transmission of these specific bacteria. As in Acanthosomatidae (Kikuchi et al., 2009) and also crypt- adults, the mycetome with endosymbionts occurs in all associated Candidatus Ishikawaella capsulatus from stink- larval stages over the midgut tract. The number of sym- bugs of the family Plataspidae (Fukatsu & Hosokawa, 2002; bionts increased from larval stage to stage, associated with Hosokawa et al., 2005). Interestingly, phylogenetic analysis the increasing size of the mycetome. Double hybridization indicated that the Kleidocerys symbiont was more separate performed with Kleido100 (Fig. 1f–h), together with the from the well-supported monophyletic groups of other bug universal probe EUB338, showed that the bulk of bacteria, symbionts such as Candidatus Ishikawaella capsulatus (pla- found in mycetome, belong to the Kleidocerys endosym- taspid bugs) and Candidatus Rosenkranzia clausaccus

Fig. 2. Phylogenetic position of the endosymbiont of Kleidocerys resedae. Consensus tree of the Bayesian interference with 48 sequences of the 16S rRNA gene [MRBAYES; 1328 bp (420 variable sites, 358 parsimony-informative), 60 000 000 generations, 600 000 trees, samplefreq = 1000, burnin = 15 000]. The tree has been rooted with Aeromonas jandaei and Pseudomonas aeruginosa as an outgroup. Support values 4 0.5 are indicated at the nodes.

Fig. 3. Phylogenetic position of the endosymbiont of Kleidocerys resedae. Consensus tree of the Bayesian interference with 16 sequences of the groEL gene [MRBAYES; 1574 bp (748 variable sites, 612 parsimony-informative), 1 000 000 generations, 10 000 trees, samplefreq = 100, burnin = 2500]. The tree has been rooted with Vibrio cholerae as an outgroup. Support values 4 0.5 are indicated at the nodes.

Fig. 4. Transmission electron microscopy of the mycetocytes, including the gammaproteobacterial endosymbiont of Kleidocerys resedae. (a) Rod- shaped symbiotic bacteria densely packed near the nucleus. Scale bar = 2.5 mm. (b) High magniﬁcation of cross-sections of Kleidocerys symbiont showing the typical morphology. Scale bar = 0.5 mm. M, mitochondrion; N, nucleus; S, symbiont.

(acanthosomatid stinkbugs), and exhibited a much closer dia, the P-symbiont of tse-tse ﬂy Glossina brevipalpis (Aksoy, phylogenetic relationship with other obligate endocellular 1995; Aksoy & Rio, 2005), and Candidatus Nardonella spp., symbiotic bacteria of diverse insects, ﬁrst of all W. glossini- the endosymbiont of weevils (Dryophthoridae) (Lefe`vre

c 2010 Federation of European Microbiological Societies FEMS Microbiol Ecol 73 (2010) 408–418 Published by Blackwell Publishing Ltd. All rights reserved Primary endosymbiont of birch catkin bug Kleidocerys resedae 415 et al., 2004; Conord et al., 2008). In both symbioses, the accelerated molecular evolution and reduced genome size mycetome with endosymbionts is positioned near the mid- (Mira & Moran, 2002; Wernegreen et al., 2002; Moran et al., gut, as in Kleidocerys (Nardon et al., 2002). To date, the 2008). Interestingly, the same peculiar genetic traits were functional role of these symbiotic bacteria has not been verified for extracellular gut symbionts of stinkbugs (Hoso- elucidated completely, but the obligate mutualist W. glossi- kawa et al., 2006; Kikuchi et al., 2009). The enhanced AT nidia seems to play a multifaceted role in the digestion, content of the 16S rRNA gene (51.96%) and the groEL gene reproduction and immunity processes of its host (Pais et al., (64.36%) of Kleidocerys symbiotic bacteria, in comparison 2008). with their free-living relatives, argues for a comparable In addition to harbouring a different kind of symbiont, molecular evolution. Current analysis of the symbiont the form of transmission differs. According to the endocel- genome size will shed more light on this. lular existence in mycetome, the Kleidocerys symbiont is Although the function of Kleidocerys symbiont is not yet transmitted transovarially to offspring (Fig. 1e), in contrast known, it seems possible that the bacteria aid in the to the posthatch symbiont transmission mechanism, which degradation of the diet or supply essential nutrients (amino requires the symbionts to survive outside the hosts for part acids, vitamins) to the host that the insect can neither of their life cycle (Buchner, 1965; Fukatsu & Hosokawa, synthesize itself nor obtain from its diet in sufficient 2002; Hosokawa et al., 2005; Prado et al., 2006; Kaltenpoth quantities. Sucking on plant seed by Kleidocerys is in et al., 2009). For the transmission of the Kleidocerys sym- contrast to Buchner’s theory, who pointed out that insects biont, the oocyte has to pass a belt-shaped infection zone in will predominantly have a nutrition-relevant symbiosis, each ovary (Schneider, 1940). A specific ‘lubricating organ’ living as they do on nutritionally unbalanced diets such as for egg smearing, as described for acanthosomatid stinkbugs plant sap or vertebrate blood (Buchner, 1965; Kikuchi et al., (Kikuchi et al., 2009), is therefore not developed. These 2008). However, the important functional roles of sym- results indicate that the way in which symbionts are bionts became especially apparent in bugs with extracellular harboured and transmitted is more meaningful than the symbiosis, whose symbionts could be removed experimen- phylogenetic relationship of the hosts, suggesting that an tally from the gut. When deprived of gut symbiotic bacteria, independent evolution of symbiotic associations has oc- insects showed retarded growth, sterility and/or mortality curred in different bug lineages. (Muller,¨ 1956; Huber-Schneider, 1957; Schorr, 1957; Chang, Nevertheless, the symbiont phylogeny generally reflects 1974; Abe et al., 1995). Comparable observations were made the host phylogeny within a group of host animals, as a in pentatomid and acanthosomatid stinkbugs, where sym- result of strict vertical transmission of the symbiont through biotic-free bugs exhibited a lower adult emergence rate, host generations (Moran et al., 1993). These stable host prolonged development time and an abnormal morphology –symbiont associations exist primarily in symbiotic rela- (Kikuchi et al., 2009; Prado & Almeida, 2009b). tions with obligate intracellular bacteria. However, strict The detection of alphaproteobacterial endosymbionts of host–symbiont cospeciations were also discovered for gut the genus Wolbachia and Rickettsia is not unusual for symbiotic bacteria in plataspid and acanthosomatid bugs, heteropteran bugs (Kikuchi & Fukatsu, 2003; Weinert et al., although they are not isolated in the body cavity and are 2009), but was first described for the genus Kleidocerys. vulnerable to invasion and replacement by foreign micro- Comparison with GenBank revealed that the Wolbachia organisms (Buchner, 1965). In contrast, in other extracellu- strain in K. resedae exhibited the highest similarity to the lar gut symbiotic associations, such as those in alydid Wolbachia endosymbiont (supergroup B) of parasitoid wasp stinkbugs and termites, the symbiont phylogeny does not N. vitripennis (Hymenoptera; Pteromalidae), where different mirror the host phylogeny (Hongoh et al., 2005; Kikuchi Wolbachia strains caused cytoplasmatic incompatibility et al., 2005). Whether there is a host–symbiont cospeciation (Bordenstein & Werren, 1998). Additional, K. resedae was with seed bugs of genus Kleidocerys or members of the also infected by a Rickettsia strain that showed high similarity subfamily Ischnorhynchinae remains to be examined in to the Rickettsia endosymbiont of stone beetle C. dactyliperda future studies. According to the description of the same (Coleoptera; Scolytinae). Similar to Wolbachia, rickettsiae symbiotic accommodation by Schneider (1940) for Kleido- influenced oogenesis in stone beetle (Zchori-Fein et al., cerys ericae, which so far has not been available for further 2006). Such reproductive aberrations have not been detected investigations, host–symbiont cospeciation also seems to be in K. resedae so far, where a well-balanced sex ratio was possible for bugs of the family Lygaeidae. demonstrated. In this context, further research is required to As a result of their insular intracellular lifestyle as well as investigate in more detail the influence of Wolbachia and their small population size and the tight bottleneck caused Rickettsia on Kleidocerys spp. by the predominantly vertical transmission mechanisms, There may be further, hidden, less abundant bacteria that obligate endocellular symbiotic bacteria are subjected to a were missed by cloning or selection of RFLP types, but genetic drift, including AT-biased nucleotide composition, comparison of hypothetical RFLP fragment sets of closely

FEMS Microbiol Ecol 73 (2010) 408–418 c 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 416 S.M. Kuchler¨ et al. related bacteria with the Kleidocerys primary symbiont Bordenstein SR & Werren JH (1998) Effects of A and B Wolbachia showed no similarities in the RFLP pattern (DISTINCTIENZ; and host genotype on interspecies cytoplasmic incompatibility http://www.bioinformatics.org). Furthermore, the fluores- in Nasonia. Genetics 148: 1833–1844. cent in situ double hybridization showed no evidence of Braendle C, Miura T, Bickel R, Shingleton AW, Kambhampati S bacteria other than Rickettsia and Wolbachia. & Stern DL (2003) Developmental origin and evolution According to the ‘‘Ad Hoc Committee for the re-evalua- of bacteriocytes in the aphid-Buchnera symbiosis. PLoS Biol tion of the species definition in bacteriology’’ (Stackebrandt 1: E21. et al., 2002), authors propose the designation ‘Candidatus Buchner P (1965) Endosymbiosis of Animals with Plant Kleidoceria schneideri’ for the intracellular symbiotic bac- Microorganisms. Interscience Publishers, New York. teria of K. resedae, on the basis of the distinct genetic, Chang KP (1974) Effects of elevated temperature on the phylogenetic and microbiological traits described above. mycetome and symbiotes of the bed bug Cimex lectularis The generic name establishes the relationship with the host (Heteroptera). J Invertebr Pathol 23: 333–340. Chang KP & Musgrave AJ (1970) Ultrastructure of Rickettsia-like organism, whereas the specific name is in honour of microorganisms in the midgut of a plant bug, Stenotus Gerhard Schneider, who first described the symbiosis in seed binotatus Jak (Heteroptera: Miridae). Can J Microbiol 16: bug K. resedae. 621–622. 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