diversity of wolbachia endosymbionts in heteropteran bugs

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2003, p. 6082–6090 Vol. 69, No. 100099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.10.6082–6090.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Diversity of Wolbachia Endosymbionts in Heteropteran BugsYoshitomo Kikuchi1,2* and Takema Fukatsu2

Natural History Laboratory, Faculty of Science, Ibaraki University, Mito 310-8512,1 and Institute forBiological Resources and Functions, National Institute of Advanced Industrial

Science and Technology (AIST), Tsukuba 305-8566,2 Japan

Received 8 April 2003/Accepted 1 August 2003

An extensive survey of Wolbachia endosymbionts in Japanese terrestrial heteropteran bugs was performed byPCR detection with universal primers for wsp and ftsZ genes of Wolbachia, cloning of the PCR products,restriction fragment length polymorphism analysis of infecting Wolbachia types, and molecular phylogeneticcharacterization of all the detected Wolbachia strains. Of 134 heteropteran species from 19 families examined,Wolbachia infection was detected in 47 species from 13 families. From the 47 species, 59 Wolbachia strains wereidentified. Of the 59 strains, 16 and 43 were assigned to A group and B group in the Wolbachia phylogeny,respectively. The 47 species of Wolbachia-infected bugs were classified into 8 species with A infection, 28 specieswith B infection, 2 species with AA infection, 3 species with AB infection, 5 species with BB infection, and 1species with ABB infection. Molecular phylogenetic analysis showed little congruence between Wolbachiaphylogeny and host systematics, suggesting frequent horizontal transfers of Wolbachia in the evolutionarycourse of the Heteroptera. The phylogenetic analysis also revealed several novel lineages of Wolbachia. Basedon statistical analyses of the multiple infections, we propose a hypothetical view that, in the heteropteran bugs,interactions between coinfecting Wolbachia strains are generally not intense and that Wolbachia coinfectionshave been established through a stochastic process probably depending on occasional horizontal transfers.

The genus Wolbachia is a bacterial group that belongs to theorder Rickettsiales in the �-subdivision of the class Proteobac-teria. The members of Wolbachia obligatorily live inside thecells of arthropods and filarial nematodes and are transmittedthrough egg cytoplasm of the hosts. Many Wolbachia endosym-bionts cause reproductive alterations of their arthropod hosts,such as cytoplasmic incompatibility, parthenogenesis, femini-zation, and male killing. On account of the maternal inheri-tance of Wolbachia, these reproductive symptoms are regardedas selfish strategies of the symbionts whereby the frequency ofinfected females increases in host populations, often at theexpense of host fitness (24, 33, 37). Extensive PCR surveys ofdiverse insect taxa revealed high infection frequencies world-wide: 16.9% (26 of 154 species) in neotropic Panama (40);21.7% (18 of 83) in Britain (42); and 19.3% (28 of 145) inNorth America (39). PCR surveys of specific insect taxa alsodetected considerable levels of Wolbachia infection: 23.5% (4of 17 species) in stalk-eyed flies (8); 50.0% (25 of 50) in ants(36); 57.9% (11 of 19) in rose gallwasps (25); 29.2% (7 of 24)in Acraea butterflies (16); 28.1% (25 of 89) in mosquitoes (20);and 17.2% (11 of 64) in oak gallwasps and their inquilines (28).The universal prevalence of Wolbachia infection is likely at-tributed to the ability of manipulating host reproduction.

Since Wolbachia endosymbionts are difficult to culture, de-tection and characterization of the bacteria have been princi-pally performed by PCR-based techniques. The most populargene for bacterial characterization, 16S ribosomal DNA, isalmost useless for differentiation of Wolbachia strains due to apaucity of nucleotide substitutions. Therefore, the diversity of

Wolbachia endosymbionts has been analyzed by using fast-evolving genes, such as ftsZ and wsp. Based on ftsZ sequences,most of Wolbachia from arthropods are classified into twomajor clades, A group and B group (41). The A and B groupsare further divided into a number of subgroups based on wspsequences (44).

A single insect individual may be infected with more thanone strain of Wolbachia. Previous extensive surveys of naturalinsect communities detected low levels (1.2 to 5.8%) of mul-tiple Wolbachia infections (28, 36, 39, 40, 42). However, thesevalues cannot be regarded as true multiple-infection frequen-cies. In these studies, multiple infections were detected by adiagnostic PCR technique using specific primers that can dif-ferentiate between A group and B group of Wolbachia. Thetechnique detects only AB double infections, whereas AA andBB double infections are erroneously recognized as single in-fections. Using the technique, it is impossible to detect tripleinfections. Therefore, these reported values of multiple Wol-bachia infections are no doubt underestimates. In fact, when 89mosquito species were analyzed by using 12 sets of specificprimers for known wsp subgroups of Wolbachia, the diagnosticPCR of much better resolution unveiled a remarkably higherfrequency, 15.7% (14 of 89 species), of double infections, con-sisting of 9.0% (8 of 89) AB, 5.6% (5 of 89) AA, and 1.1% (1of 89) BB infections (20). It should be noted, however, that thisapproach tends to fail to detect novel Wolbachia strains be-cause the specific primers were designed for already-knownwsp sequences. Therefore, a thorough survey independent ofsuch specific PCR primers would lead to a deeper grasp ofWolbachia diversity in natural insect communities.

The Heteroptera, known as true bugs, is one of the mostdiverse insect groups with incomplete metamorphosis. Manyheteropteran bugs are known to harbor extracellular symbioticbacteria in their midgut caeca. The symbiotic bacteria are, in

* Corresponding author. Mailing address: National Institute of Ad-vanced Industrial Science and Technology, AIST Tsukuba Central 6,1-1-1 Higashi, Tsukuba 305-8566, Japan. Phone: 81-29-861-6087. Fax:81-29-861-6080. E-mail: [email protected].

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general, maternally inherited to the offspring by superficialcontamination of the eggshell with excretion containing thebacteria (2, 3, 7, 31). In several heteropteran bugs, deprivationof the symbiont was reported to result in retarded growthand/or mortality of the nymphs, suggesting some importantrole of the symbiont for the host (1, 5, 11, 22, 32). On the otherhand, intracellular symbionts of heteropteran bugs have beenvery poorly investigated, except for those of blood-suckingreduviid bugs and bedbugs (13, 14, 26).

In this study we conducted an extensive survey of Wolbachiainfection in Japanese terrestrial heteropteran bugs. Two hun-dred twenty-six field-collected heteropteran bugs, representing134 species from 19 families, were subjected to PCR detectionwith universal primers for Wolbachia, cloning of the PCR prod-ucts, restriction fragment length polymorphism (RFLP) anal-ysis of infecting Wolbachia types, and molecular phylogeneticcharacterization of all the detected Wolbachia strains. In total,36 single infections, 10 double infections, and 1 triple infectionwere detected, and 59 Wolbachia strains, some of which be-longed to novel lineages, were identified.

MATERIALS AND METHODS

Materials. The terrestrial heteropteran bugs used in this study are listed inTable 1. They were collected in 2001 and 2002 in Japan, except for the samplesof the Acanthosomatidae that were collected in 1994 and 1995. These bugs werepreserved in acetone immediately after collection until molecular analysis (4).

DNA extraction. DNA was individually extracted from 1 to 10 insects for eachspecies. Females were preferentially used if available. Small bugs were subjectedto whole-body extraction, whereas large bugs were dissected and their abdomensor ovaries were extracted. The tissues were homogenized in 200 �l of lysis buffer(10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.1 M NaCl, 0.5% SDS, 0.2 mg ofProteinase K/ml) and were incubated at 56°C for 2 h. The lysate was extractedwith 200 �l of PCI (phenol:chloroform:isoamyl alcohol [25:24:1]). DNA wasrecovered from the purified lysate by ethanol precipitation and was dissolved in200 �l of TE buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA).

Specific PCR detection. Diagnostic PCR was performed using specific primersfor two Wolbachia genes. The primers ftsF (5�-GTATGCCGATTGCAGAGCTTG-3�) and ftsR (5�-GCCATGAGTATTCACTTGGCT-3�) yielded a 0.8-kb seg-ment of ftsZ (10). The primers wspF (5�-GGGTCCAATAAGTGATGAAGAAAC-3�) and wspR (5�-TTAAAACGCTACTCCAGCTTCTGC-3�) yielded a0.6-kb segment of wsp (21). To check the quality of DNA samples, a 1.5-kbsegment of insect mitochondrial ribosomal DNA was amplified using the primersMtrA1 and MtrB1 (6). PCRs were conducted using Takara TaqDNA polymerase(Takara) and its supplemented buffer system under a temperature profile of 95°Cfor 4 min followed by 35 cycles of 95°C for 30 s, 50°C (ftsZ) or 55°C (wsp) for 30 s,and 72°C for 30 s. The PCR products were electrophoresed on Tris-acetate-EDTA (TAE)-agarose gels for 15 min, stained with ethidium bromide, andobserved on a UV transilluminator. DNA prepared from ovaries of the Wolba-chia-infected bruchid beetle Callosobruchus chinensis (21) was used as a positivecontrol sample.

Cloning, RFLP typing, and sequencing of wsp gene segment. PCR products ofthe wsp gene segment were directly cloned with TA cloning vector pT7Blue(Takara) and Escherichia coli DH5�-competent cells (Takara) by using an am-picillin and 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside blue-white selec-tion system. To check the length of the inserted DNA fragment, white coloniesexpected to contain inserted plasmid were directly subjected to PCR using theprimers wspF and wspR. When a PCR product of expected size (0.6 kb) wasobtained, the product was double digested with the restriction endonucleasesClaI and DraI and was electrophoresed in TAE-agarose gels for typing of the wspclone. More than three clones from each of all RFLP types were culturedovernight in 3 ml of Luria-Bertani medium containing ampicillin and weresubjected to plasmid extraction by using a QIAprep-Spin Miniprep kit (QIA-GEN). The purified plasmids were eluted with 50 �l of distilled water and wereused for sequencing. A dye terminator-labeled cycle sequencing reaction wasconducted with DNA Sequencing kit FS (Perkin Elmer) and two sequencingprimers, Univ19 (5�-GTTTTCCCAGTCACGACGT-3�) and Rev20 (5�-AGCTATGACCATGATTACGC-3�), under a temperature profile of 95°C for 4 minfollowed by 30 cycles of 95°C for 30 s, 50°C for 1 min, and 60°C for 4 min. The

products were analyzed with an ABI PRISM 377 DNA sequencer (PerkinElmer).

Molecular phylogenetic analysis. The wsp gene sequences determined weresubjected to molecular phylogenetic analysis together with wsp gene sequences ofWolbachia representatives retrieved from the DDBJ nucleotide sequence data-base. A multiple alignment of the wsp sequences was generated by the programpackage Clustal W (34) and then was manually realigned. Phylogenetic treeswere constructed by the neighbor-joining method using Clustal W. Bootstraptests were performed with 1,000 replications.

Randomization test. A randomization test was performed using pairwise ge-netic distances between 20 wsp gene sequences from 10 doubly infected bugs. A20 by 20 distance matrix was constructed using Kimura’s two parameter model(19) packaged in Clustal W (34). From 190 pairwise distances in the matrix, 10distances were randomly sampled and averaged to obtain a mean distance. Thesampling was repeated 10,000 times, by which a null distribution of the meandistance was generated. The observed mean distance calculated for the 10 pairsof coinfecting Wolbachia strains was statistically evaluated in comparison withthe null distribution.

Nucleotide sequence accession numbers. The wsp gene sequences determinedin this study were deposited in the DDBJ nucleotide sequence database, and theaccession numbers are shown in boldface in Fig. 1.

RESULTS

Detection of Wolbachia infection. By using specific PCR de-tection of ftsZ and wsp genes, Wolbachia infection was detectedin 47 of 134 heteropteran species examined. There was nodiscrepancy between the ftsZ results and wsp results. Thesespecies belonged to 13 of 19 heteropteran families surveyed(Tables 1 and 2).

Identification of Wolbachia infection types. The PCR prod-ucts of wsp gene segments were cloned and subjected to RFLPtyping. Of the 47 samples, some showed only one type of RFLPpattern, suggesting either single infection or cryptic doubleinfection. Others exhibited two types of RFLP pattern, indi-cating double infection. One sample, Charagochilus angusticol-lis, showed three distinct RFLP patterns, suggesting triple in-fection. More than three clones for each of the RFLP typeswere subjected to DNA sequencing. At this stage, some of thesingle RFLP types turned out to contain two distinct se-quences, implying double infection undetected by the RFLPanalysis. In total, 59 wsp sequences were identified from the 47samples. These sequences were assigned to either A group orB group of Wolbachia based on the sequence similarity. Of the59 sequences, 16 and 43 were placed in A group and B group,respectively. As a result, the 47 species of Wolbachia-infectedbugs were classified into 8 species with A infection, 28 specieswith B infection, 2 species with AA infection, 3 species with ABinfection, 5 species with BB infection, and 1 species with ABBinfection (Tables 1 and 2).

Molecular phylogenetic analysis. A phylogenetic tree of allthe Wolbachia strains identified from the heteropteran bugswas constructed based on the wsp sequences (Fig. 1). Wolba-chia strains obtained from the same insect families were notclustered into distinct groups but were scattered throughoutthe phylogenetic tree. Wolbachia strains obtained from thesame insect genera were also scattered on the tree, althoughseveral congenic clusters (for example, Megacopta cribraria 1and Megacopta punctatissima 1, Cletus punctiger and Cletustrigonus, and Elasmucha putoni and Elasmucha amurensis)were identified. Wolbachia strains obtained from the sameinsect individuals, comprising multiple infections, were notclustered at all (Fig. 2). The phylogenetic analysis revealedseveral novel lineages of Wolbachia strains. For example, next

VOL. 69, 2003 WOLBACHIA IN HETEROPTERAN BUGS 6083

TABLE 1. Distribution of Wolbachia by Japanese terrestrial heteropteran species

Family and speciesc OriginaType of

Wolbachiainfectionb


Wolbachiainfectionb

MiridaeHarpocera orientalis MTPlagiognathus collaris TKAdelphocoris suturalis TKApolygus lucorum TK BApolygus pulchellus TKArbolygus sp. TKCharagochilus angusticollis TK ABBLygus rugulipennis SP (0/6)Stenodema calcarata TKTrigonotylus caelestialium TK (0/5)

NabidaeNabis stenoferus TK BBNabis kinbergii HJ B

ReduviidaeEctrychotes andreae HJPeirates turpis TKCydnocoris russatus KIAgriosphodrus dohrni HJVelinus nodipes TKSphedanolestes impressicollis KZ

TingidaeCantacader japanicus TKGaleatus spinifrons KM BStephanitis pyrioides MT B (3/5)

AradidaeNeuroctenus castaneus TK (0/5)

BerytidaeYemma exilis TK A (1/2)

MalcidaeChauliops fallax MT

LygaeidaeTropidothorax belogolowi HJAethalotus nigriventris IGCaenocoris dimidiatus IGSpilostethus hospes IG BB (1/4)Graptostethus servus IGOncopeltus nigriceps IGThunbergia sanguinaria IGNysius plebeius KM BBPylorgus colon TK ABGeocoris proteus TK BPiocoris varius KM BBPachygrontha antennata TK A (2/2)Lethaeus assamensis MT BPachybrachius luridus TKParomius exiguus TK B (8/10)Togo hemipterus TK ABParaeucosmetus pallicornis NH BBHorridipamera inconspicua TK BHorridipamera nietneri NH BPanaorus japonicus TK (0/2)Panaorus albomaculatus TKMetochus abbreviatus KZ

ScutelleridaeCantao ocellatus IGPhilia miyakonus IGCalliphara nobilis IGCalliphara exellens IGEucorysses grandis SM (0/2)

Continued on facing page

Poecilocoris lewisi TKSolenosthedium chinense IG

PentatomidaeGonopsis affinis KMDybowskyia reticulata TKScotinophara horvathi TKScotinophara lurida TK AAGraphosoma rubrolineatum TKErthesina fullo HJ (0/2)Aelia fieberi TK (0/4)Halyomorpha halys MT (0/4)Palomena angulosa KIDolycoris baccarum JE (0/9)Carpocoris purpureipennis TKEysarcoris lewisi SP (0/5)Eysarcoris aeneus TKEysarcoris ventralis TKEysarcoris annamita KM B (3/3)Carbula humerigera KIEurydema rugosa TKEurydema dominulus HJ BAgonoscelis femoralis IG BPlautia crossota MT (0/6)Nezara antennata TKHomalogonia obtusa KMMenida violacea KIPiezodorus hybneri MTBathycoelia indica IGPentatoma japonica KIRhynchocoris humeralis IGEocanthecona furcellata IGAndrallus spinidens NHZicrona caerulea TB

LargidaePhysopelta cincticollis IGPhysopelta gutta TK

PyrrhocoridaeAntilochus coqueberti IGPyrrhocoris sibiricus KZPyrrhocoris sinuaticollis TK BDysdercus cingulatus IG (0/2)Dysdercus poecilus IG (0/3)Dysdercus decussatus IG (0/2)

CoreidaeMolipteryx fuliginosa HNAcanthocoris sordidus TK BLeptoglossus australis IGDasynus coccocinctus IG ANotobitus meleagris KS B (2/2)Hygia opaca TK B (6/6)Hygia lativentris TK BHomoeocerus dilatatus MTHomoeocerus unipunctatus KM B (1/4)Homoeocerus marginiventris NHAnacanthocoris striicornis TKParadasynus spinosus IGPlinachtus bicoloripes TKPlinachtus basalis IGCletus trigonus TK BCletus punctiger TK B (3/6)Cletus rusticus HN B

AlydidaeLeptocorisa chinensis TK

6084 KIKUCHI AND FUKATSU APPL. ENVIRON. MICROBIOL.

to the subgroup Con, wsp sequence from Paromius exiguusconstituted an isolated lineage, and wsp sequences from E.putoni and E. amurensis formed a distinct clade. The mostnotable was the identification of “Bugs” subgroup consisting of11 wsp sequences. Except for two sequences from a dragonfly,Perithemis tenera, and a wasp, Ceroptres cerri, the well-definedbut diverse clade was composed of nine wsp sequences fromheteropteran bugs (Agonoscelis femoralis, Eurydema dominu-lus, Nysius plebeius, Orius minutus, Orius strigicollis, Paraeucos-metus pallicornis 2, Piocoris varius 1, Pyrrhocoris sinuaticollis,Spirostethus hospes 2) that represented four families (Antho-coridae, Lygaeidae, Pentatomidae, and Pyrrhocoridae).

Expected and observed frequencies of double infections.From the 134 heteropteran species examined, 16 strains ofA-type and 43 strains of B-type Wolbachia were identified.Provided that 11.0% (16 of 146) and 29.5% (43 of 146) areregarded as frequencies of A type and B type in the heterop-teran bugs, respectively, frequencies of double infections wereexpected to be 1.2% for AA, 3.2% for AB, and 8.7% for BB onan assumption of random combination. Observed double in-fection frequencies were 1.5% (2 out of 134) for AA, 2.2% (3out of 134) for AB, and 3.7% (5 out of 134) for BB (Table 2).The differences between the expected and observed valueswere statistically not significant (�2 � 5.29, df � 2, P � 0.071).

Relatedness between Wolbachia strains and double infec-tions. To know whether coinfecting Wolbachia strains tend tobe closely or distantly genetically related, a randomization testwas applied to 20 wsp sequences from 10 doubly infected in-sects (Fig. 3). The genetic distances between coinfecting Wol-bachia strains showed no statistically significant bias (P �0.61).

DISCUSSION

Wolbachia infection in the heteropteran bugs. Our extensivesurvey of Wolbachia infection in 134 species of Japanese ter-restrial heteropteran bugs revealed a high infection frequencyat 35.1% (47 out of 134). This result clearly indicates thatWolbachia infection is prevailing in the heteropteran speciesdiversity, as has been reported for other insect groups (8, 16,20, 25, 28, 36, 39, 40, 42). The value of 35.1% is, however, nodoubt an underestimate, because quite limited numbers ofindividuals and populations were examined in this study. Todate, no studies have been conducted on the effects of Wolba-chia infection in heteropteran insects. To understand themechanism for maintaining the high infection frequency, phe-notypic effects of Wolbachia infection on the heteropteranbugs, such as fitness consequences, vertical transmission rates,and intensity of cytoplasmic incompatibility and other repro-ductive phenotypes, should be investigated.

Multiple Wolbachia infection in the heteropteran bugs. Ourapproach, independent of group-specific PCR primers, identi-fied a considerable frequency of multiple infection at 8.2% (11of 134) in the heteropteran bugs. The frequency was remark-ably higher than the multiple infection frequencies at 1.2 to5.8% reported in previous studies where only AB double in-fections were detected by diagnostic PCR (28, 36, 39, 40, 42).The frequency of AB infection in this study was 2.2%, which isalmost comparable to the frequencies of 1.2 to 5.8%. There-fore, the higher frequency of 8.2% was probably not due tohigher multiple-infection rates in the heteropteran bugs butwas due to improved detection of non-AB-type multiple infec-tions. In spite of the improved detectability, however, the es-

TABLE 1—Continued


Wolbachiainfectionb


Wolbachiainfectionb

Riptortus clavatus AN B (4/9)Riptortus linearis NH

RhopalidaeLeptocoris rufomarginata IGRhopalus maculatus TB BStictopleurus punctatonervosus TK B (3/3)

UrostylidaeUrostylis sp. TK

PlataspidaeCoptosoma parvipictum KS ACoptosoma sphaerula IG (0/3)Megacopta punctatissima TK ABMegacopta cribraria NH AABrachyplatys subaeneus NH B

a The samples were collected from AN, Ano Mie; HJ, Honjo Saga; IG, Ishigaki Okinawa; HN, Hitachinaka Ibaraki; JE, Joetsu Niigata; KI, Kitaibaraki Ibaraki; KM,Kasama Ibaraki; KS, Kagoshima Kagoshima; KZ, Kanzaki Saga; MK, Mashike Hokkaido; MT, Mito Ibaraki; NH, Naha Okinawa; OB, Obihiro Hokkaido; OT, OtaruHokkaido; SM, Shimizu Kouchi; SP, Sapporo Hokkaido; SW, Suwa Nagano; TB, Tomobe Ibaraki; TK, Tsukuba Ibaraki; TM, Tomakomai Hokkaido.

b Type of Wolbachia infection: A or B single infections, AA, AB or BB double infections or ABB triple infection. If multiple individuals were tested for a species,the number infected out of the number tested is indicated in parentheses.

c The following numbers of species for each family group were infected: Miridae, 2 out of 10 (20%); Nabidae, 2 out of 2 (100%); Reduviidae, 0 out of 6 (0%);Tingidae, 2 out of 3 (67%); Aradidae, 0 out of 1 (0%); Berytidae, 1 out of 1 (100%); Malcidae, 0 out of 1 (0%); Lygaeidae, 12 out of 22 (55%); Scutelleridae, 0 outof 7 (0%); Pentatomidae, 4 out of 30 (13%); Largidae, 0 out of 2 (0%); Pyrrhocoridae, 1 out of 6 (17%); Coreidae, 9 out of 17 (53%); Alydidae, 1 out of 3 (33%);Rhopalidae, 2 out of 3 (67%); Urostylidae, 0 out of 1 (0%); Plataspidae, 4 out of 5 (80%); Cydnidae, 2 out of 3 (67%); Acanthosomatidae, 5 out of 11 (45%).

CydnidaeAdomerus triguttulus TK AChilocoris piceus TK AChilocoris piceus TK AMacroscytus japonensis TK

AcanthosomatidaeElasmostethus humeralis SPElasmostethus brevis SP AElasmucha dorsalis TMElasmucha putoni SP B (2/2)Elasmucha amurensis MK B (2/2)Elasmucha signoreti OT ASastragala esakii SW BSastragala scutellata TSAcathosoma denticaudum OBAcathosoma labiduroides KI (0/2)Acathosoma haemorrhoidale angulatum SP


timated multiple-infection frequencies are still likely to beunderestimates. Although we selected the restriction endo-nucleases ClaI and DraI for the best detectability of wsp se-quence polymorphisms, some multiple infections may fail to berecognized by the RFLP analysis.

Putative horizontal transfers of Wolbachia. Wolbachia endo-symbionts are maternally transmitted through host generationsby vertical transmission. However, phylogenetic relationshipsof Wolbachia strains are generally not concordant with those oftheir host insects (30, 41, 44), which strongly suggests thathorizontal transfers of Wolbachia between unrelated host or-ganisms may have taken place at a considerable frequency overevolutionary time. Although the mechanism of horizontaltransfer in natural conditions is unknown, a plausible hypoth-esis is that parasitoids may be involved in the process (9, 12,23). In the heteropteran bugs, little congruence was foundbetween the Wolbachia phylogeny and the host systematics(Fig. 1 and 2), supporting frequent horizontal transfers ofWolbachia in the evolutionary course of the Heteroptera. Inseveral cases, closely related congenic bugs possessed closelyrelated wsp sequences, which may favor the idea of host-sym-biont cospeciation of relatively recent origin. However, thepattern can also be explained by horizontal transfer of Wolba-chia between closely related host species that often utilizesimilar ecological niches and share common parasitoids. In thiscontext, survey of Wolbachia in hymenopteran and strep-sipteran parasitoids that exploit heteropteran bugs will be ofinterest.

Identification of novel lineages of Wolbachia. From the het-eropteran bugs we identified 59 wsp sequences, some of whichwere conceived to represent novel lineages of Wolbachia. No-tably, a novel clade of Wolbachia tentatively named Bugs sub-group, whose members are predominantly associated with het-eropteran bugs, was identified (Fig. 1). Since the Wolbachiaphylogeny in the group did not reflect the host systematics, theassociation cannot be ascribed to host-symbiont cospeciation.Wolbachia of the group might be adapted to the internal en-vironment of the bugs and/or might be readily vectored be-tween bug lineages by parasitoids. However, the integrity ofthe group requires further verification. As more wsp sequencesare reported from diverse insect taxa, it appears possible thatthe group would be diluted and disintegrated by the sequencesfrom nonheteropteran insects.

Interaction between coinfecting Wolbachia endosymbionts.When different endosymbionts share the common habitat ofthe host body, it is expected that various types of interactionsmay arise between the symbionts. The frequent occurrence ofmultiple Wolbachia infections in the heteropteran bugs pro-vides an opportunity to test the ideas concerning microbiolog-ical and ecological interactions between coexisting endosymbi-onts.

Competitive exclusion between Wolbachia strains? Sinceavailable resources and space are strictly limited in the hostbody, intersymbiont competition may result in exclusion ofeither of the symbionts. If Wolbachia coinfections are generallyunstable due to such competitive exclusion, it is expected thatobserved frequencies of multiple infections in host populationsmay be lower than expected. In the heteropteran bugs, ex-pected double infection frequencies were calculated to be1.4% for AA infection, 3.8% for AB infection, and 10.3% forBB infection. The values were not significantly different fromthe observed frequencies of 1.5% for AA infection, 2.2% forAB infection, and 3.7% for BB infection. Therefore, compet-itive exclusion between coinfecting Wolbachia strains was notdetected in this study, although the possibility that weak an-tagonistic interactions do exist cannot be ruled out. It may benotable that the observed frequency of BB infection (3.7%)was remarkably lower than the expected one (10.3%).

Coinfecting Wolbachia strains genetically closely or distantlyrelated? If intersymbiont competition leads to intrahost nichedivision, differential tissue tropism of the symbionts may beobserved, and different types of symbionts may be favored tocoexist. If Wolbachia coinfections tend to require such func-tional divergence, it is expected that genetic divergence be-tween the coexisting symbionts may be greater than expected.On the contrary, similar types of symbionts may tend to coexist

TABLE 2. Distribution of Wolbachia in Japanese terrestrialheteropteran families

Family

Infection type and no. positive

TotalUninfected A B AA AB BB Triple

(ABB)

Miridae 8 0 1 0 0 0 1 10Nabidae 0 0 1 0 0 1 0 2Reduviidae 6 0 0 0 0 0 0 6Tingidae 1 0 2 0 0 0 0 3Aradidae 1 0 0 0 0 0 0 1Berytidae 0 1 0 0 0 0 0 1Malcidae 1 0 0 0 0 0 0 1Lygaeidae 10 1 5 0 2 4 0 22Largidae 2 0 0 0 0 0 0 2Pyrrhocoridae 5 0 1 0 0 0 0 6Coreidae 8 1 8 0 0 0 0 17Alydidae 2 0 1 0 0 0 0 3Rhopalidae 1 0 2 0 0 0 0 3Urostylidae 1 0 0 0 0 0 0 1Plataspidae 1 1 1 1 1 0 0 5Cydnidae 1 2 0 0 0 0 0 3Scutelleridae 7 0 0 0 0 0 0 7Pentatomidae 26 0 3 1 0 0 0 30Acanthosomatidae 6 2 3 0 0 0 0 11

Total 87 8 28 2 3 5 1 134

FIG. 1. Molecular phylogenetic analysis of 59 wsp sequences identified from 43 Japanese terrestrial heteropteran species. A total of 656 alignednucleotide sites were subjected to the analysis. On the right side are shown the names of groups and subgroups of Wolbachia according to Werrenet al. (41) and Zhou et al. (44). The bootstrap values higher than 70% are shown at the nodes. Scientific names of the host insects are indicated.The accession numbers are in brackets, and the host insect taxa are described in parentheses. Numbers following the scientific names indicatemultiple Wolbachia infection in the host insects. Boldface means the data was obtained in this study.


on the grounds that the symbionts have to adapt to the sameenvironment in the host body. If so, it is expected that geneticdivergence between the coinfecting Wolbachia strains may besmaller than expected. Contrary to either of the expectations,the randomization test indicated that genetic divergence ofcoinfecting Wolbachia strains exhibited no significant differ-ence from the expected value (Fig. 3). In the heteropteranbugs, therefore, relatedness between the coinfecting Wolbachiastrains was neither close nor distant but was almost at thebackground level. It should be noted, however, that the fre-quencies of coinfection are inherently underestimated andtherefore may be affected by the bias.

Tentative conclusion. These results suggest that, in the het-eropteran bugs, interactions between coinfecting Wolbachiastrains are generally not intense and that Wolbachia coinfec-tions have been established through a stochastic process prob-ably depending on occasional horizontal transfers. Recentstudies have suggested the occurrences of putative genetic

recombination between Wolbachia strains (17, 27, 38), whichmight also contribute to such coinfections and diversity ofWolbachia endosymbionts. It should be noted, however, thatthose arguments above are based merely on the global patternsof combination and phylogenetic relationship of coinfectingWolbachia strains. Future detailed investigation of specific het-eropteran bugs infected with multiple Wolbachia strains wouldreveal various types of ecological, physiological, and microbi-ological interactions, such as differential tissue tropism re-ported in a bruchid beetle (15).

Perspective. Owing to this study, the Heteroptera is nowrated among the insect taxa whose Wolbachia infection anddiversity are best documented. A number of notorious agricul-tural pests are known from the Heteroptera, and some of themcan be conveniently maintained in the laboratory by usinggrains, seeds, potted plants, and/or artificial diets (18, 29, 35,43), which will provide excellent model systems to investigatehost-symbiont and symbiont-symbiont interactions. Although

FIG. 2. Molecular phylogenetic analysis of 23 wsp sequences identified from 10 doubly infected heteropteran bug and 1 triple-infectedheteropteran bug. A and B on the left side indicate the groups of Wolbachia (41). On the right side Wolbachia strains detected from the same hostinsects are connected with lines.


many heteropteran bugs are known to harbor symbiotic bac-teria in the gut, microbiological nature and biological functionof the symbionts are, in general, poorly understood (2, 3, 5).Not only interactions between coinfecting Wolbachia strainsbut also interactions between Wolbachia and gut symbiont willbe of interest.

ACKNOWLEDGMENTS

We thank H. Higuchi, N. Ijichi, M. Ishizaki, K. Itoh, T. Kashima, A.Kikuchi, K. Kohno, S. Kudo, S. Moriya, M. Takai, T. Takemoto, andT. Tsuchida for providing bug samples, A. Sugimura, H. Ouchi, S.Tatsuno, S. Suo, and K. Sato for technical and secretarial assistance,and M. Yokoyama for programming and J. Kojima for encouragement.

This research was supported by the Program for Promotion of BasicResearch Activities for Innovation Biosciences (ProBRAIN) of theBio-Oriented Technology Research Advancement Institution.

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diversity of wolbachia endosymbionts in heteropteran bugs

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