Phylogenetic Characterization of Wolbachia Symbionts Infecting Cimex lectularius L. and Oeciacus vicarius Horvath (Hemiptera: Cimicidae) JASON L. RASGON1



The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD 21205

J. Med. Entomol. 41(6): 1175Ð1178 (2004)

ABSTRACT Wolbachia symbionts are obligate intracellular bacteria that cause host reproductive alterations in many arthropods and Þlarial nematodes. We identiÞed Wolbachia symbionts in the cliff swallow bug (Oeciacus vicarious Horvath) and the human bed bug (Cimex lectularius L.) (Hemiptera: Cimicidae) by polymerase chain reaction (PCR) ampliÞcation and sequencing using WolbachiaspeciÞc 16S rDNA and FtsZ primers. Phylogenetic analyses using Bayesian, maximum likelihood, and maximum parsimony algorithms indicated, with strong support, that (1) Wolbachia infections in these two cimicid hosts form a monophyletic group, and (2) the Wolbachia strains detected belong to the F clade, previously associated with termites, weevils, and Þlarial nematodes. KEY WORDS Wolbachia, Cimicidae, phylogenetics, classiÞcation, symbiosis

Wolbachia are maternally inherited bacterial endosymbionts in the ␣ proteobacteria. They have a wide host range (infecting insects, terrestrial crustaceans, arachnids, and Þlarial nematodes) and are associated with diverse alterations in host reproductive biology. There are six major Wolbachia clades or “supergroups” (AÐF) based on phylogenetic clustering of FtsZ gene sequences (Lo et al. 2002). A, B, and E infect diverse arthropods, and C and D infect nematodes (Stouthamer et al. 1999, Vandekerckhove et al. 1999). The F clade has a broader host range than the other groups and has been detected infecting both arthropods and nematodes (Lo et al. 2002). Insects in the family Cimicidae are obligatory hematophagous ectoparasites of birds, bats, and humans. Four decades ago, Wolbachia-like bacterial inclusions were observed by microscopy in several cimicid species (Usinger 1966). More recently, Wolbachia infection was detected by PCR in the human bed bug (Cimex lectularius L.). However, the phylogenetic placement of this Wolbachia symbiont was not investigated (Hypsa and Aksoy 1997). In this study, we used molecular methods to identify and phylogenetically characterize the Wolbachia infecting C. lectularius and Oeciacus vicarius Horvath (the cliff swallow bug). We sequenced several bacterial nuclear genes and used sequence data to examine phylogenetic relationships of Wolbachia strains infecting these cimicids com-


Corresponding author, e-mail: [email protected] Department of Entomology, University of California Davis, One Shields Avenue, Davis, CA 95616. 2

pared with representative members of all six recognized Wolbachia supergroups. Materials and Methods Insects Examined. Adult O. vicarius were obtained from abandoned cliff swallow nests collected underneath a bridge located ⬃2 mi north of Davis, CA (Yolo Co.; 38⬚36⬘0.17⬙ N; 121⬚51⬘31.47⬙ W) in September 2000. Nests were brought back to the University of California at Davis (UCD) and broken into small pieces, and O. vicarius were picked out of the debris. O. vicarius were stored at ⫺80⬚C until processed for DNA extraction. Adult C. lectularius were obtained from the National Institutes of Health human bed bug colony, which has been in colony for ⬎10 yr (J. Valenzuela, personal communication). Insects were transported live to UCD, killed at ⫺80⬚C, and stored at that temperature until processed for DNA extraction. We did not attempt to sex insects. DNA Extraction. Oeciacus vicarius and C. lectularius genomic DNA was extracted with DNEasy spin columns (Qiagen, Valencia, CA). Individual bugs were homogenized in 200 ␮l PBS, and DNA was extracted using the manufacturerÕs suggested protocol for cultured cells. Genomic DNA was eluted using the manufacturerÕs provided buffer and stored at ⫺20⬚C until used for PCR ampliÞcation. PCR Amplification and Sequencing. All PCR ampliÞcation was performed using a Perkin-Elmer GeneAmp 9700 thermocycler (PE Applied Biosystems, Foster City, CA). PCR ampliÞcation of Wolbachia 16S rDNA, and FtsZ sequences was car-

0022-2585/04/1175Ð1178$04.00/0 䉷 2004 Entomological Society of America



ried out using previously described primers and conditions (OÕNeill et al. 1992, Holden et al. 1993). Infected colony specimens of Culex pipiens (LIN strain) (Rasgon and Scott 2003) were included in all reactions as positive controls. A sample containing deionized water in place of template DNA was included in all reactions as a negative control. Readyto-go (RTG) PCR beads (Amersham-Pharmacia Biotech, Piscataway, NJ) were used for all reactions. For sequencing, ampliÞed PCR fragments were separated by agarose gel electrophoresis, puriÞed with Qiaquick PCR puriÞcation spin columns (Qiagen), and directly sequenced in both directions on an ABI Prism 377 DNA sequencer with BigDye chemistry (Perkin-Elmer Applied Biosystems, Foster City, CA). Phylogenetic Analysis. Homology searches of the GenBank database for obtained sequences were performed using the Basic Local Alignment Search Tool (BLAST). Retrieved sequences were aligned with manual correction using Clustal X (Thompson et al. 1997). As analyses of individual 16S and FtsZ data sets gave qualitatively similar results (data not shown), we combined both data for greater resolution. Phylogenetic analyses were conducted using Bayesian, maximum likelihood (ML), and maximum parsimony (MP) methods. For Bayesian and ML analyses, the GTR⫹I⫹G model was selected as the most appropriate evolutionary model of DNA substitution using Modeltest v. 3.06 (Posada and Crandall 1998). ML and MP analyses were carried out using PAUP* v. 4.01b 10 (Swofford 1998). Tree support was evaluated by bootstrapping (1,000 replications). Bayesian analyses were conducted using MrBayes v. 3.0 (Huelsenbeck and Ronquist 2001). We ran simulations for 500,000 generations, sampling every 10 generations, for a total of 50,000 trees. The Þrst 40,000 trees were considered the burn-in and were discarded. We constructed a 50% majority-rule consensus tree from the remaining 10,000 trees. Results and Discussion Using Wolbachia-speciÞc 16S rDNA primers (OÕNeill et al. 1992), we successfully ampliÞed an 850-bp fragment from DNA extracted from whole O. vicarius (n ⫽ 10/10) and an 806-bp fragment from C. lectularius (n ⫽ 3/3). Using general primers for a portion of the Wolbachia FtsZ gene (Holden et al. 1993), we were able to successfully amplify a 721-bp fragment from O. vicarius (n ⫽ 5/5) and a 735-bp fragment from C. lectularius (n ⫽ 3/3). FtsZ and 16S PCR fragments were puriÞed and directly sequenced. For both 16S and FtsZ fragments, all sequences within species were identical. There were no ambiguous peaks to suggest the presence of multiple Wolbachia strains in either species. GenBank homology searches of 16S and FtsZ sequences indicated that the symbionts of both O. vicarius and C. lectularius were most closely related to the Wolbachia symbiont of Kalotermes flavicollis, a termite (16S: both 99% homology, FtsZ: both 97% homology).

Vol. 41, no. 6

Table 1. GenBank accession numbers for 16S and FtsZ sequences used in this analysis Host


Accession no. (16S)

Accession no. (FtsZ)

Anaplasma marginale Ehrlichia chaffeensis Diabrotica lemniscata Drosophila simulans (Riv) Culex pipiens Gryllus interger Laodelphax striatellus Dirofilaria immitis Dirofilaria repens Onchocerca ochengi Brugia malayi Brugia pahangi Litmosoides sigmodontis Folsomia candida Kalotermes flavicollis Microcerotermes sp Rhinocyllus conicus Mansonella ozzardi Oeciacus vicarius Cimex lectularius

Outgroup Outgroup A A B B B C C C D D D E F F F F F F

AF414876 AF147752 AY007547 X64264 X61768 U83097 AB039036 Z49261 AJ276500 AF172401 AF051145 AF093511 AF069068 AF179630 Y11377 AJ292347 M85267 AJ279034 AY091456 AY316361

AJ010274 AF221944 AY007555 U28178 U28209 AF011269 AB039038 AJ010272 AJ010273 AJ010268 AJ010269 AJ010270 AJ010271 AJ344216 AJ292345 AJ292346 Ñ Ñ AY091457 AY316362

The two strains were closely related to one another (16S: 99% homology, FtsZ: 98% homology). GenBank accession numbers are listed in Table 1. Phylogenetic analyses using individual data sets gave similar results except that the placement of the Wolbachia symbiont of Folsomia candida (clade “E”) could not be resolved with certainty (data not shown). We therefore conducted phylogenetic analysis on a combined 16S and FtsZ dataset, which included 646 bp of the 16S sequence and 508 bp of the FtsZ sequence. The alignment contained taxa from all six supergroups and two outgroup taxa (Table 1). The total 1,154-bp alignment contained 358 variable sites, of which 233 were parsimony-informative. Wolbachia FtsZ sequences are not available for the F-group strains infecting the weevil Rhinocyllus conicus or the nematode Manzonella ozzardi. We treated these omissions as missing data in our analysis. Trees generated by Bayesian, ML, and MP algorithms indicated general concordance with one another. Wolbachia are expected to form a monophyletic group when they have diverged dependently with hosts that are related by descent. All three analyses strongly supported the monophyly of the O. vicarius and C. lectularius symbionts (bootstrap or posterior probability support ⱖ 80) within the F group (Fig. 1) and indicate that, in these cimicids, dependent Wolbachia divergence has likely occurred. Alternatively, the two insects may share related Wolbachia strains because of horizontal transmission from one cimicid to the other. For example, infection with similar Wolbachia strains by horizontal transmission has been hypothesized in leafhoppers that acquired the symbionts by feeding on the same host plant (Mitsuhashi et al. 2002). Our analysis indicates that, although arthropod Wolbachia infections in the A, B, and E clades form a monophyletic group (bootstrap or posterior probability support: 55Ð97), arthropod-infecting Wolbachia

November 2004



Fig. 1. Rooted phylogenetic tree of arthropod and nematode Wolbachia symbionts based on 16S and FtsZ sequences. Taxon names represent host species. Tree topology was estimated using Bayesian, ML, and MP algorithms (CI: 0.70, RI: 0.68). Each supergroup is identiÞed by a letter (AÐF). OG, outgroup. Numbers at tree nodes represent Bayesian posterior probability values (top), ML bootstrap values for 1,000 replicates (middle), and MP bootstrap values for 1,000 replicates (bottom).

strains in the F clade likely originated from an independent horizontal transfer event (clade support ⱖ90) than the A-B-E grouping. It is likely that there has also been extensive horizontal transmission within the F clade, similar to what has been observed in the A and B clades (Stouthamer et al. 1999), because the various invertebrate host groups within the F clade are not closely related. High levels of host diversity encompassing both arthropods and nematodes have not been observed for other Wolbachia supergroups and indicate that F-group Wolbachia may be more plastic in their possible host range or may have experienced more frequent horizontal transmission events during their evolutionary history than the other supergroups. Acknowledgments We thank L. Goddard for assistance with O. vicarius collections, J. Valenzuela for providing C. lectularius, and S. Brady, P. Ward, P. Cranston, and P. Gullan for assistance with phylogenetic analysis. Funding was provided by NIH (GM20092 to J.L.R.) and the California Department of Agriculture and Natural Resources (248M to T.W.S.).

References Cited Holden, P. R., J. F. Brookfield, and P. Jones. 1993. Cloning and characterization of an FtsZ homologue from a bacterial symbiont of Drosophila melanogaster. Mol. Gen. Genet. 240: 213Ð220. Huelsenbeck, J. P., and F. Ronquist. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754 Ð755. Hypsa, V., and S. Aksoy. 1997. Phylogenetic characterization of two transovarially transmitted endosymbionts of the bedbug Cimex lectularius (Heteroptera: Cimicidae). Insect Mol. Biol. 6: 301Ð304. Lo, N., M. Casiraghi, E. Salati, C. Bazzocchi, and C. Bandi. 2002. How many Wolbachia supergroups exist? Mol. Biol. Evol. 19: 341Ð346. Mitsuhashi, W., T. Saiki, W. Wei, H. Kawakita, and M. Sato. 2002. Two novel strains of Wolbachia coexisting in both species of mulberry leafhoppers. Insect Mol. Biol. 11: 577Ð584. O’Neill, S. L., R. Giordano, A.M.E. Colbert, T. L. Karr, and H. M. Robertson. 1992. 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc. Natl. Acad. Sci. U.S.A. 89: 2699 Ð2702.



Posada, D., and K. A. Crandall. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14: 917Ð 818. Rasgon, J. L., and T. W. Scott. 2003. Wolbachia and cytoplasmic incompatibility in the California Culex pipiens mosquito species complex: parameter estimates and infection dynamics in natural populations. Genetics 165: 2029 Ð2038. Stouthamer, R., J. A. Breeuwer, and G. D. Hurst. 1999. Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu. Rev. Microbiol. 53: 71Ð102. Swofford, D. L. 1998. PAUP* 4.0 Ñphylogenetic analysis using parsimony. Sinauer, Sunderland, MA. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL-X Windows

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interface: ßexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res. 15: 4876 Ð 4882. Usinger, R. L. 1966. Monograph of Cimicidae. Entomological Society of America, College Park, MD. Vandekerckhove, T. T., S. Watteyne, A. Willems, J. G. Swings, J. Mertens, and M. Gillis. 1999. Phylogenetic analysis of the 16S rDNA of the cytoplasmic bacterium Wolbachia from the novel host Folsomia candida (Hexapoda, Collembola) and its implications for wolbachial taxonomy. FEMS Microbiol. Lett. 180: 279 Ð286. Received 26 January 2004; accepted 12 July 2004.

Phylogenetic Characterization of Wolbachia Symbionts ...

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