INVERTEBRATE MICROBIOLOGY

Distribution Patterns of Wolbachia Endosymbiontsin the Closely Related Flower Bugs of the Genus Orius:Implications for Coevolution and Horizontal Transfer

Masaya Watanabe & Yohsuke Tagami & Kazuki Miura &

Daisuke Kageyama & Richard Stouthamer

Received: 31 August 2011 /Accepted: 7 March 2012 /Published online: 27 March 2012# Springer Science+Business Media, LLC 2012

Abstract Vertical transmission is the primary route of theendosymbiont Wolbachia for its own spread among inver-tebrate hosts, but horizontal transmission between differenthosts is believed to have occurred multiple times. However,it is not well known how Wolbachia commonly spreadamong closely related hosts. We focused on the closelyrelated species of the minute pirate bugs belonging to thegenus Orius, which are important biological control agentsin agricultural crops because they are the most useful naturalenemy of various tiny pests, such as thrips. Here, we exam-ined five Orius species (Orius sauteri, Orius nagaii, Oriusminutus, Orius strigicollis, and Orius tantillus) from eightgeographic localities in Japan for Wolbachia infection. Two

distinct strains, wOus1 and wOus2, were detected based onWolbachia surface protein (wsp) gene sequencing. Further-more, multilocus sequence typing revealed that each of thestrains comprised two variants that differed in a singlenucleotide. The overall distribution patterns of the two Wol-bachia strains were found to differ among host species:prevalent double infection with wOus1 and wOus2 in O.strigicollis; fixation of single infection with wOus2 in O.nagaii; occurrence of single infection with wOus1 in O.sauteri; prevalence of single infection with wOus1 in O.minutus with an exception in a single population; and lackof Wolbachia infection in O. tantillus. Such differences inthe distribution patterns of Wolbachia may reflect the evo-lutionary history of Wolbachia infection among Orius spe-cies and/or ecological and physiological differences amongthe Orius species that determine the invasiveness and main-tenance of the two Wolbachia strains.

Introduction

The symbiotic bacterium Wolbachia pipientis, the only spe-cies of the genus Wolbachia (alpha-Proteobacteria), residesin invertebrate cells and is transmitted from mothers tooffspring [16, 36, 42, 43, 45]. Wolbachia can manipulatethe reproduction of its hosts in various tactical ways, such asfeminization of males, induction of thelytokous partheno-genesis, male-killing and cytoplasmic incompatibility (CI)[29, 35, 40]. These advantageous properties allow Wolba-chia to spread and be maintained at high frequencies in hostpopulations. A recent meta-analysis of Wolbachia surveysestimated that 66 % of insect species harbor this bacterium[12]. Wolbachia has also been found in other invertebrates,such as spiders, mites, crustaceans and filarial nematodes [1,6, 8]. Such ubiquitous Wolbachia infection in arthropods is

M. Watanabe :K. Miura (*)Graduate School of Biosphere Sciences, Hiroshima University,6-12-1 Nishifukatsu,Fukuyama 721-8514, Japane-mail: miurak@affrc.go.jp

Y. TagamiFaculty of Agriculture, Shizuoka University,836 Ohya, Suruga-ku,Shizuoka 422-8529, Japan

K. MiuraLaboratory of Insect Pest Control, National Agricultural ResearchCenter for Western Region (WeNARC),6-12-1 Nishifukatsu,Fukuyama 721-8514, Japan

D. KageyamaInsect-Microbe Research Unit, National Institute ofAgrobiological Sciences (NIAS), 1–2 Oowashi,Tsukuba, Ibaraki 305-8634, Japan

R. StouthamerDepartment of Entomology, University of California,Riverside, CA 92521, USA

Microb Ecol (2012) 64:537–545DOI 10.1007/s00248-012-0042-x

considered to depend not only on its stability during verticaltransmission within host lineages but also on multiple his-torical events of horizontal transmission between differenthost lineages [44].

However, recent studies have demonstrated that recom-bination between different Wolbachia strains is particular-ly common in the Wolbachia surface protein (wsp) genesequence and that the wsp gene is under strong diversify-ing selection [3, 21, 26, 31, 41]. Hence, this gene isconsidered to be unsuitable for reliable strain character-ization when evaluated alone. To resolve this issue, amultilocus sequence typing (MLST) system using fivehousekeeping genes (ftsZ, coxA, fbpA, gatB and hcpA)has been established [4].

Incongruence between the phylogenies of Wolbachiagenes (such as wsp and ftsZ) and those of host genes hasrepeatedly been observed and led researchers to assume thathorizontal transmission of Wolbachia between different hostspecies has occurred multiple times [6, 10, 23, 33, 43, 51].Recent analyses on certain spiders and ants based on theWolbachia MLST genes have clearly shown that the Wol-bachia and host phylogenies are incongruent [5, 11]. Morespecifically, the same or closely related Wolbachia strainshave been found in different host species that share the sameecological niche or have host–parasite or predator–preyinteractions [23, 34]. On the other hand, the Wolbachiaphylogeny based on the MLST genes was concordant withthe host species phylogeny in some cases, suggesting thatvertical inheritance of Wolbachia from a common ancestorhas also occurred [15, 20]. However, it remains unclear howcommonly such coevolutionary processes operate betweenWolbachia and its hosts.

The genus Orius (Hemiptera: Anthocoridae) comprisespolyphagous predators of various agricultural pests, such asthrips. In Japan, the subgenus Heterorius (i.e., Orius sauteri,Orius nagaii, Orius minutus, and Orius strigicollis) andthree other species, Orius tantillus, Orius miyamotoi, andOrius atratus occur [47–49]. The Heterorius species arecommon and distributed in a wide area across Japan. Onthe other hand, O. tantillus and O. atratus are only found inOkinawa Island, and O. miyamotoi is only found in restrict-ed areas of northern and western Kyushu. The four speciesof the subgenus Heterorius often occur sympatrically onvarious flowering plants, such as Trifolium repens and Sol-idago altissima [14, 19, 28, 50] and have similar food prey,including thrips, aphids, and hatchlings and larvae of vari-ous insects. Recently, O. strigicollis, a species commerciallyused as a biological control agent [46], was found to beinfected with at least two Wolbachia strains, wOus1 andwOus2, based on PCR and sequencing analyses using wspgene primers [39]. Crossing experiments showed thatwOus1 caused very weak or no CI while wOus2 causedstrong CI [39]. In this context, an investigation ofWolbachia

infection in closely related species in the genus Orius is ofgreat interest from an evolutionary viewpoint.

In the present study, Wolbachia infection was examinedin five Orius species found in eight geographic localities inJapan. Based on MLST analyses, we characterized the Wol-bachia strains found in the Orius species and estimated theirphylogenetic relationships. Moreover, we evaluated the dis-tribution patterns of the Wolbachia strains within and be-tween populations and species of the genus Orius thatinhabit Japan. Based on our findings, possible routes ofWolbachia invasion in the genus Orius are discussed.

Materials and Methods

Insect Sampling

The five Orius species, O. sauteri, O. nagaii, O. minutus, O.strigicollis, and O. tantillus, were collected from 19 popu-lations across Japan (Table 1; Fig. 2). Dr. Toru Shimizu(Ryukyu Sankei Co. Ltd.) sampled adults of all five speciesin Tsukuba (TK), Kochi (KC1), Kumamoto (KM), andOkinawa (OK). Dr. Masahiro Iguchi (Wakayama Agricul-tural Exp. Stn.) sampled adults of three species (O. sauteri,O. minutus, and O. strigicollis) in Wakayama (WK).Masaya Watanabe sampled adults of two species (O. sauteriand O. strigicollis) in Kasaoka (KS) and Kochi (KC2).Yohsuke Tagami sampled adults of two species (O. sauteriand O. strigicollis) in Shizuoka (SZ). All insect samplingwas carried out during the period from 2002 to 2010.

DNA Extraction and Species Identification

DNA was extracted from each individual (abdomen andlegs) by crushing with a clean plastic rod in 32 μl of STEbuffer (5 N NaCl, 500 mM EDTA (pH 8.0), 1 M Tris–HCl(pH 8.0)) and incubated with 2 μl of proteinase K (0.5mg/ml) at 56 °C for 2 h. The homogenate was heated to99.9 °C for 5 min to inactivate the proteinase K and thenused as a template for PCR. To identify the species of thebugs, diagnostic multiplex PCR, which amplifies internaltranscribed spacer (ITS)-1 of the nuclear ribosomal DNAgene, was carried out using the Orius-specific primers F2(5′-GTC GCT ACT ACC GAT TGA ATG G-3′), R2 (5′-GTG TCC TGC AGT TCA CAT GG-3′), st-F (5′-GTGGTG CCT TCG TGA GAA A-3′), sa-R2 (5′-ATT CTTGGT CCG ATT TTT GG-3′), and na-F (5′-CGG ACCCGA ACT CTC GTAT-3′) [13]. Each species was identifiedby the size and number of the PCR bands [13]. Each genefragment was amplified in a total volume of 33 μl contain-ing 16.5 μl of ×2 AmpliTaq Gold PCR Master Mix (PEApplied Biosystems, Tokyo, Japan), 1.3 μl of forward andreverse primers (10 pmol μl−1), and 10.0 μl of sterile water.

538 M. Watanabe et al.

The PCR amplification was carried out in an ABI thermo-cycler (PCR System 9700; PE Applied Biosystems) with thefollowing thermal profile: 95 °C for 10 min; 28 cycles of94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min; 72 °Cfor 10 min. Samples in which the ITS-1 gene failed tobe amplified were excluded from the analysis. The PCRproducts were resolved in a 1.5 % agarose gel, stainedwith ethidium bromide and visualized under an UVtransilluminator.

PCR, Cloning, and Sequencing of wsp

For each of four Orius species (O. sauteri, O. nagaii, O.minutus, and O. strigicollis), two or three individuals fromdifferent populations were taken and subjected to cloningand sequencing of the Wolbachia wsp gene. The Wolbachiawsp sequences were amplified using the Wolbachia univer-sal primers wsp81F (5′-TGG TCC AAT AAG TGA TGAAGA AAC-3′) and wsp691R (5′-AAA AAT TAA ACGCTA CTC CA-3′) [7] in the same reaction volume describedabove. The PCR amplifications were conducted using thefollowing temperature profile: 95 °C for 10 min; 35 cyclesof 94 °C for 1 min, 55 °C for 1 min 30 s, and 72 °C for 1 min30 s; 72 °C for 1 min 30 s; hold at 4 °C. The amplifiedproducts were ligated into a T-easy vector (Promega, Mad-ison, WI). The four colonies obtained from each individualwere incubated with ampicillin-containing LB medium

overnight and purified 50-μl aliquots of the plasmids wereeluted using a QIAprep-Spin Miniprep Kit (Qiagen, Hilden,Germany). The plasmids were directly sequenced using thesequencing primers SP6 (5′-TAT TTA GGT GAC ACTATAG-3′) and T7 (5′-TAA TAC GAC TCA CTA TAG GG-3′)and a BigDye DNA Sequencing Kit (PE Applied Biosys-tems). The reaction products were analyzed using an ABIPRISM 310 Genetic Analyzer or 3130x (PE Applied Bio-systems). Each subclone of the partial sequences was editedand connected using Sample Manager (PE Applied Biosys-tems) or CLC DNAWorkbench (CLC Japan, Tokyo, Japan).Multiple alignments were performed manually using theClustal W program [38]. The nucleotide sequences havebeen deposited in the DDBJ/EMBL/GenBank databases(accession numbers, AB094354–AB094368).

Diagnostic PCR for Wolbachia Strains

Specific PCR detection of the two Wolbachia strains(wOus1 and wOus2) was conducted using specific Wolba-chia primers designed from the wsp sequences found inOrius species. The wsp sequence of wOus1 was amplifiedusing the primer set kosF (5′-TAA ATA CTT CTG AAACAA ATG TTG-3′) and wsp691R (5′-AAA AAT TAA ACGCTA CTC CA-3′) [7], while the wsp sequence of wOus2was amplified using the primer set naeF (5′-GAT GTA GTATCT TCT GAT GAC AAG-3′) and taiR (5′-GGA CGA

Table 1 Collection sites of five Orius species in Japan

Species Collection site Abbreviation Year Collector

Orius sauteri Kumamoto, Kumamoto Prefecture KM 2002 Dr. Toru Shimizu 32.8 N, 130.7 E

Kochi,, Kochi Prefecture KC1 2002 Dr. Toru Shimizu 33.6 N, 133.5 E

Kasaoka, Okayama Prefecture KS 2010 Dr. Masaya Watanabe 34.5 N, 133.5 E

Wakayama, Wakayama Prefecture WK 2008 Dr. Masahiro Iguchi 34.2 N, 135.2 E

Shizuoka, Shizuoka Prefecture SZ 2008 Dr. Yohsuke Tagami 35.0 N, 138.4 E

Tsukuba, Ibaraki Prefecture TK 2002 Dr. Toru Shimizu 36.1 N, 140.1 E

Orius minutus Kumamoto, Kumamoto Prefecture KM 2002 Dr. Toru Shimizu 32.8 N, 130.7 E

Kochi,, Kochi Prefecture KC1 2002 Dr. Toru Shimizu 33.6 N, 133.5 E

Wakayama, Wakayama Prefecture WK 2008 Dr. Masahiro Iguchi 34.2 N, 135.2 E

Tsukuba, Ibaraki Prefecture TK 2002 Dr. Toru Shimizu 36.1 N, 140.1 E

Orius nagaii Kochi,, Kochi Prefecture KC1 2002 Dr. Toru Shimizu 33.6 N, 133.5 E

Tsukuba, Ibaraki Prefecture TK 2002 Dr. Toru Shimizu 36.1 N, 140.1 E

Orius strigicollis Okinawa, Okanawa Prefecture OK 2002 Dr. Toru Shimizu 26.2 N, 127.7 E

Kochi,, Kochi Prefecture KC1 2002 Dr. Toru Shimizu 33.6 N, 133.5 E

Kochi,, Kochi Prefecture KC2 2008 Dr. Masaya Watanabe 33.3 N, 133.4 E

Kasaoka, Okayama Prefecture KS 2008 Dr. Masaya Watanabe 34.5 N, 133.5 E

Wakayama, Wakayama Prefecture WK 2008 Dr. Masahiro Iguchi 34.2 N, 135.2 E

Shizuoka, Shizuoka Prefecture SZ 2008 Dr. Yohsuke Tagami 35.0 N, 138.4 E

Orius tantillus Okinawa, Okanawa Prefecture OK 2002 Dr. Toru Shimizu 26.2 N, 127.7 E

OK Okinawa, Okinawa Prefecture, KM Kumamoto, Kumamoto Prefecture, KC1 Kochi, Kochi Prefecture, KC2 Nankoku, Kochi Prefecture, KSKasaoka, Okayama Prefecture, WK Wakayama, Wakayama Prefecture, SZ Shizuoka, Shizuoka Prefecture, TK Tsukuba, Ibaraki Prefecture

Wolbachia Infection in Orius Species 539

TGA TCT CTT TAG TAG-3′). Diagnostic PCR was per-formed using the thermal profile of the Wolbachia universalPCR as described above.

MLST Analyses of Wolbachia Strains

The MLST gene sequences of three individuals singlyinfected with wOus1 or wOus2 in each Orius species wereamplified using standard primers and PCR protocols (avail-able at http://www.pubmlst.org/wolbachia/). The sequenceswere aligned automatically by the Vector NTI Advance ver.10.1 program (Invitrogen InforMax, Frederick, MD). Am-biguous bases and single-point polymorphisms wereassessed by performing a second amplification and rese-quencing. The Wolbachia strains were assigned a sequencetype (ST), and defined as the combination of five integerscorresponding to the allele numbers at the five MLST loci(allelic profile). By convention, a strain is defined as aWolbachia isolate from a single host population. Strainsidentical at the five alleles were assigned the same ST. Thehost and strain information has been submitted to the MLSTdatabase (http://pubmlst.org/wolbachia/). The nucleotidesequences have been deposited in the DDBJ/EMBL/Gen-Bank databases (accession numbers, AB499736–AB499745and AB597162–AB597163).

Recombination Analyses

Recombination analyses were conducted on wsp and concat-enated MLST gene alignments using the MaxChi method,implemented in the RDP3 program [25]. The parameters wereset as follows: triplets were scanned using different values ofthe fraction of variable sites per window, a Bonferroni correc-tion was applied, and 1,000 permutations were generated. Thehighest acceptable P value cut-off was set to 0.05.

Phylogenetic Analyses

The phylogenetic relationships of the Wolbachia strains inthe Orius species were based on the nucleotide sequences ofthe concatenated MLST gene and MLST allelic profiles.MLST sequence alignments were performed using ClustalW [38] in MEGA4 [37]. Neighbor-joining trees were con-structed using the MEGA4 or START2 program [18] basedupon unambiguously aligned sites using Kimura’s two-parameter correction [22] with support for the tree topologyassessed by bootstrap resampling (1,000 replicates). Wolba-chia data for species/genera other than Orius were takenfrom a database of previous MLST studies [4, 27, 32]. Thesequences of the concatenated MLST genes of Brugiamalayi and Cimex lectularius were used as outgroups insome analyses. The COI sequences in the Orius specieswere obtained from GenBank.

Vertical Transmission Rates

In each of the wOus1 single-infected, wOus2 single-infectedand wOus1 and wOus2 double-infected strains, 3- and 4-day-old male and female pairs were allowed to oviposit for4 days onto a cutting of the plant Sedum rubrotinctum with adiet of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae)eggs in a glass vial (1-cm diameter×5-cm length). Afteroviposition, the females were screened for Wolbachia usingwOus1- and wOus2-specific primers as described above.Larvae were reared on E. kuehniella eggs with cottonsoaked with distilled water. The emerged adults were sexedbased on the morphology of the abdominal tip under adissecting microscope, and then subjected to the diagnosticPCR for Wolbachia. Insects were maintained in the labora-tory under conditions of a 16-h light/8-h dark photoperiod,60 % humidity and 25 °C.

Results

Two Wolbachia Strains Detected in Four Orius Species

Four of the five Orius species (O. sauteri, O. nagaii, O.minutus, and O. strigicollis) were found to be positive forWolbachia by diagnostic PCR usingWolbachia-specific wspprimers. To characterize theWolbachia, the PCR products ofthe wsp sequences amplified from the four infected Oriusspecies were subjected to cloning and sequencing. Thesequencing data from 40 colonies suggested that the fourspecies of Orius harbored two different Wolbachia strains:wOus1 and wOus2. The wsp sequences of wOus1 or wOus2in the four species of Orius were identical to each other(AB094354–AB094368).

According to the MLST scheme, two closely-related STs(ST-144 and ST-211, designatedwOus1a andwOus1b, respec-tively) were found forwOus1 and two closely related STs (ST-143 and ST-212, designated wOus2a and wOus2b, respective-ly) were found for wOus2. wOus1a and wOus1b only differedin a single base pair of coxA (wOus1a, coxA-331G andwOus1b, coxA-331A). Similarly, wOus2a and wOus2b onlydiffered in a single base pair of hcpA (wOus2a, hcpA-257C andwOus2b, hcpA-257T). All four STs belonged to supergroup Bon theWolbachia phylogeny (Fig. 1a). None of the fiveMLSTgenes ofwOus1 shared any alleles with other STs (Fig. 1b andWolbachia MLST database). On the other hand, two of thefive MLST genes (coxA and hcpA) of wOus2 shared alleleswith other STs (Fig. 1b and WolbachiaMLST database). Thesequence of hcpA of wOus2a (allele 40 according to theMLST database) is particularly common, and found in avariety of insects [32]. MaxChi analyses indicated no recom-bination events among two STs based on either single gene orconcatenated MLST data set alignments (P>0.05).

540 M. Watanabe et al.

Distribution Patterns of the Two Wolbachia Strainsamong Orius Populations

Nineteen local populations of the five Orius specieswere examined for their Wolbachia infection status.Some individuals were singly infected with wOus1 in

three species: O. sauteri, O. minutus,and O. strigicollis.The infection frequencies of wOus1 were relativelymoderate and variable: O. sauteri, 29–83 % (Fig. 2a);O. minutus, 80–100 % (Fig. 2b); O. strigicollis, 0–25 %(single infection state), 23–100 % (double infectionstate), and 23–100 % (total) (Fig. 2d). Single infections

Figure 1 a Neighbor-joining tree of 45 Wolbachia STs and repre-sentatives of the two supergroups A and B, based on the concat-enated alignment of MLST loci (2,079 bp) and b the MLSTallelic profiles (right columns). The numbers above the branchesindicate a bootstrap value of >70 % for 1,000 replicates. Each

name next to a branch indicates the name of the host species(single asterisk, [4]; double asterisks, [32]; triple asterisks, [27]).The bars indicate 0.01 (a) and 0.1 (b) substitutions per site,respectively

Wolbachia Infection in Orius Species 541

with wOus2 were found in three species: O. minutus,O. strigicollis, and O. nagaii. The frequencies of indi-viduals infected with wOus2 were very high and lessvariable: O. minutus, 92 % (Fig. 2b); O. nagaii, 100 %(Fig. 2c); O. strigicollis, 0–77 % (single infection state),23–100 % (double infection state), 75–100 % (total)(Fig. 2d). All O. strigicollis populations were infected,and all possible types of populations existed: single infectionwith either wOus1 or wOus2, and double infection with bothwOus1 andwOus2. The infection frequencies inO. strigicollisand O. nagaii totaled 100 % in all populations. InO. minutus,

onlywOus1was detected in three of four populations. In sharpcontrast, only wOus2 was detected in the TK population ofO. minutus.

Vertical Transmission Rates

A total of 41 offspring in wOus1-infected strains, 44 offspringin wOus2-infected strains, and 45 offspring in wOus1 andwOus2-infected strains were screened. The vertical transmis-sion fidelities of both the single-infected and double-infectedstrains were perfect (Table 2).

KM(11)

KM(5)

KC1(12)

c) O. nagaii

b) O. minutus

a) O. sauteri

73%

27%

KC1(7)

71%

29%

KS(22)

WK(11)

SZ(18)

TK(12)

55%

45%

50%

50% 83%

17%

KC1(10)

WK(5)

TK(12)

TK(12)

100%

100%

100%

90%

80%

92%20%

10%

8%

36%

64%

e) O. tantillus

d) O. strigicollis

OK(4)

KC1(5)

(11)

KC2(38)

KS(31)

WK(20)

SZ(13)

OK

100%

81%

87%

85%

77%

25%

25%

9%9%

13%

15%

23%

100%300km

50%

Figure 2 Infection frequencies of the two Wolbachia stains in O.sauteri (a), O. minutus (b), O. nagaii (c), O. strigicollis (d), andO. tantillus (e) across Japan. OK Okinawa, Okinawa Prefecture,KM Kumamoto, Kumamoto Prefecture, KC1 Kochi, Kochi Prefec-ture, KC2 Nankoku, Kochi Prefecture, KS Kasaoka, OkayamaPrefecture, WK Wakayama, Wakayama Prefecture, SZ Shizuoka,

Shizuoka Prefecture, TK Tsukuba, Ibaraki Prefecture. The numb-ers of examined individuals are given in parentheses. Black,doubly infected with wOus1 and wOus2; dark gray, singlyinfected with wOus2; light gray, singly infected with wOus1;white, uninfected

542 M. Watanabe et al.

Discussion

Current Distribution Patterns of wOus1 and wOus2 in OriusSpecies

The infection frequencies of Wolbachia strain wOus1 werefound to vary in wild populations of O. strigicollis, O.sauteri, and O. minutus. On the other hand, the infectionfrequencies of a second Wolbachia strain, wOus2, werefound to be very high in wild populations of O. strigicollis,O. nagaii, and O. minutus.

In this study,Wolbachia detection was performed using theabdomen of Orius species. Therefore, possible detection ofendosymbionts of its prey, such as thrips, lepidopterans, andmites that remained undigested in the gut of Orius cannot beruled out. Indeed, endosymbionts of the whitefly Bemisiatabaci were detected in the gut of its parasitoid [9]. If thiswas the case, various strains of Wolbachia would be foundbecauseOrius species feed on a wide variety of insects. Basedon the limited number of Wolbachia strains (wOus1 andwOus2) found in this study irrespective of the host speciesand geographic localities, we consider that wOus1 and wOus2are the endosymbionts of Orius and thatWolbachia detectionoriginating from undigested prey is very unlikely.

Our previous study revealed thatwOus1 induces very weakor no CI while wOus2 induces strong CI [39], and suchdifferences in the intensity of CI can explain the markeddifferences in the infection frequencies of wOus1 and wOus2.

In O. strigicollis, a large number of individuals weredoubly infected with wOus1 and wOus2 (86/111; 77 %).Our previous study revealed that the infection density ofwOus1 in the ovaries of O. strigicollis is higher than that ofwOus2 [39] and that the transmission rate of wOus1 frommothers to offspring in laboratory populations is 100 %(Table 2). There are no significant differences in fecunditybetween all infection statuses (i.e., uninfected line, singlyinfected line, and doubly infected line) [39]. Therefore,despite the low level of CI in wOus1, the high level of CI

in wOus2 may have allowed the spread of wOus1 via ahitchhiking effect in some populations of O. strigicollis.

Curiously, the distribution patterns of wOus1 and wOus2seemed to be quite different between populations in somespecies. In O. strigicollis, double infection with wOus1 andwOus2 was commonly detected in all populations, exceptSZ. In SZ, the proportion of doubly infected individuals wasless than 25 %. In O. minutus, western populations (KM,KC1, and WK) were singly infected with wOus1, while aneastern population (TK) was singly infected with wOus2.Various factors such as host genetic background, climate,

Table 2 Vertical transmissionrates of w Ous1 and w Ous2 inOrius strigicollis

Parent type Pair number Male Female

w Ous1 (n) w Ous2 (n) w Ous1 (n) w Ous2 (n)

w Ous1+2 1 100 % (6) 100 % (6) 100 % (8) 100 % (8)

2 100 % (8) 100 % (8) 100 % (4) 100 % (4)

3 100 % (5) 100 % (5) 100 % (10) 100 % (10)

w Ous1 1 100 % (6) – 100 % (11) –

2 100 % (6) – 100 % (5) –

3 100 % (5) – 100 % (11) –

w Ous2 1 – 100 % (9) – 100 % (7)

2 – 100 % (6) – 100 % (10)

3 – 100 % (8) – 100 % (5)

b) wOus2

92

99

84

0.01

Orius minutus

Orius strigicollis

Orius sauteri

Orius nagaii

Orius tantillus

Nezara viridula(Outgroup)

a) wOus1

92

99

84

0.01

Orius minutus

Orius strigicollis

Orius sauteri

Orius nagaii

Orius tantillus

Nezara viridula(Outgroup)

***

*

wOus1a (ST144)

wOus1a (ST144)

wOus1b (ST143)

*

***

*

wOus2b(ST212)

wOus2a (ST211)

wOus2a (ST211)

*

Figure 3 Hypothesized evolutionary histories of wOus1 (a) andwOus2 (b) among five Japanese Orius species. As in the host phylog-eny, a neighbor-joining tree based on the partial sequences of a mito-chondrial COI region (368 bp) is given. Bootstrap values of >70 % for1,000 replicates are given above the branches. The host species andWolbachia strains are given at the terminal nodes. Solid arrows, ac-quisition of Wolbachia; dotted arrow, horizontal transmission of Wol-bachia; arrowhead, one nucleotide substitution detected in thesequenced Wolbachia genes

Wolbachia Infection in Orius Species 543

prey, and predators may differ slightly between populationsand may be responsible for the differences in the distributionpatterns of wOus1 and wOus2. In the adzuki bean beetle,Callosobruchus chinensis, the infection densities of Wolba-chia are known to be strongly affected by the nucleargenotypes [24]. As performed in the case of C. chinensis[24], similar experiments testing artificial transfer of Wolba-chia between Orius populations (through hybrid introgres-sion or microinjection) may reveal the possible involvementof the host genetic background in the different sustainabilityof the two Wolbachia strains.

History of Infection and Spread of wOus1 and wOus2Among Orius Species

By what routes did wOus1 and wOus2 invade the Orius spe-cies? The presence of similar strains ofWolbachia in the closelyrelated species can be explained by three different historicalprocesses: (1) vertical transmission via the common ancestor inthe speciation processes [15], (2) horizontal transmission [2, 5],and (3) introgressive hybridization between hosts [17, 30].

First, the transmission of Wolbachia between Orius spe-cies by introgressive hybridization can be excluded. Accord-ing to our preliminary sequencing data for a mitochondrialcytochrome b region, each of the species (O. strigicollis, n0102; O. sauteri, n012; O. nagaii, n08; and O. minutus, n08) clearly formed a monophyletic clade irrespective of theWolbachia infection (unpublished data). If Wolbachia hadtransferred by introgressive hybridization, mitochondrialhaplotypes would be shared by different species with thesame infection status. Therefore, the remaining possibilitiesare vertical transmission from the common ancestor andhorizontal transmission induced by, for example, host–par-asitoid interactions and predator–prey interactions.

Regarding wOus1, the most likely possibility is that theancestral Orius species of O. minutus, O. strigicollis, and O.sauteri was infected once with the common ancestor ofwOus1a and wOus1b, and that this Wolbachia strain per-sisted through vertical transmission and host speciation(Fig. 3a). Another possibility is that the common ancestorof all Orius species was infected with wOus1, but Wolba-chia was subsequently lost in O. nagaii and O. tantillus. InO. sauteri or the common ancestor of O. minutus and O.strigicollis, one mutational change in coxA of wOus1 shouldhave occurred after speciation (from wOus1a to wOus1b orfrom wOus1b to wOus1a, respectively). However, the pos-sibility remains that wOus1 was horizontally transferredbetween different species through host–parasitoid interac-tions, predator–prey interactions or cannibalism.

For wOus2, the possibilities of vertical transmission andhorizontal transmission seem equally likely. One possibility isthat the common ancestor of all Orius species was infectedwithwOus2, butWolbachiawas subsequently lost inO. sauteri

and O. tantillus. Another possibility is that the infection oc-curred first inO. nagaii (as the infection frequency ofwOus2 is100% in this species) and that thisWolbachiawas horizontallytransferred to the common ancestor of O. minutus and O.strigicollis (Fig. 3b). There are also other possibilities, suchas the opposite direction of transfer from the common ancestorof O. minutus and O. strigicollis to O. nagaii, and multipleoccurrences of horizontal transfer. It is clear at least that onemutational change in hcpA of wOus2 should have occurred inO. minutus after speciation (from wOus2a to wOus2b).

We inferred the association between Wolbachia andOrius species based on a phylogenetic tree that consistedof only five Orius species originating from Japan. However,there are more than 60 species in the genus Orius thatinhabit Asia, Europe, and North America [47–49] and thephylogenetic relationships among many of these species aretotally unknown. In future studies, extensive examinationsof other Orius species around the world may allow us tounderstand the evolutionary associations between Oriusspecies and Wolbachia with higher resolution.

Acknowledgments We would like to thank Drs. Laura Baldo,Satoko Narita, and Keiko Niijima for their valuable comments on themanuscript, and Dr. Yuuki Kawasaki for helpful advice on MLSTanalyses. We also thank Dr. Toru Shimizu and Mr. Masahiro Iguchiand Masao Ito for providing insects. This research was partially sup-ported by a Grant-in-Aid for JSPS Fellows to Masaya Watanabe.

References

1. Bandi C, Anderson TJC, Genchi C, Blaxter ML (1998) Phylogenyof Wolbachia in filarial nematodes. Proc R Soc Lond B 265:1–7

2. Baldo L, Werren JH (2007) Revisiting Wolbachia supergrouptyping based on WSP: spurious lineages and discordance withMLST. Curr Microbiol 55:81–87

3. Baldo L, Bordenstein S, Wernegreen JJ, Werren JH (2006) Wide-spread recombination throughout Wolbachia genomes. Mol BiolEvol 23:437–449

4. Baldo L, Dunning Hotopp JC, Jolley KA, Bordenstein SR, BiberSA, Choudhury RR, Hayashi C, Maiden MC, Tettelin H, WerrenJH (2006) Multilocus sequence typing system for the endosymbi-ont Wolbachia pipientis. Appl Environ Microbiol 72:7098–7110

5. Baldo L, Nadia A, Ayoub NA, Hayashi CY, Russell JA, StahlhutJK, Werren JH (2008) Insight into the routes of Wolbachia inva-sion: high levels of horizontal transfer in the spider genus Agele-nopsis revealed by Wolbachia strain and mitochondrial DNAdiversity. Mol Ecol 17:557–569

6. Bouchon D, Rigaud T, Juchault P (1998) Evidence for widespreadWolbachia infection in isopod crustaceans: molecular identifica-tion and host feminization. Proc R Soc Lond B 265:1081–1090

7. Braig HR, Zhou W, Dobson S, O’Neill SL (1998) Cloning andcharacterization of a gene encoding the major surface protein of thebacterial endosymbiont Wolbachia pipientis. J Bacteriol180:2373–2378

8. Breeuwer JAJ, Jacobs G (1996) Wolbachia: intra-cellular manipu-lators of mite reproduction. Exp Appl Acarol 20:421–434

9. Chiel E, Zchori-Fein E, Inbar M, Gottlieb Y, Adachi-Hagimori T,Kelly SE, Asplen MK, Hunter MS (2009) Almost there:

544 M. Watanabe et al.

transmission routes of bacterial symbionts between trophic levels.PLoS One 4:e4767

10. Cordaux R, Michel-Salzat A, Bouchon D (2001) Wolbachia infec-tion in crustaceans: novel hosts and potential routes for horizontaltransmission. J Evol Biol 14:237–243

11. Frost CL, Ferna H, Ndez-Mari N, Smith NJE, Hughes OH (2010)Multiple gains and losses of Wolbachia symbionts across a tribe offungus-growing ants. Mol Ecol 19:4077–4085

12. Hilgenboecker K, Hammerstein P, Schlattmann P, TelschowA,WerrenJH (2008) How many species are infected with Wolbachia?—a statis-tical analysis of current data. FEMS Microbiol Lett 281:215–220

13. Hinomoto N, Muraji M, Noda T, Shimizu T, Kawasaki K (2004)Identification of five Orius species in Japan by multiplex polymer-ase chain reaction. Biol Control 31:276–279

14. Hinomoto N, Nagamori S, Kakimoto K, Shimizu T, Higaki T,Muraji M, Noda T, Kawasaki K (2009) Molecular identificationand evaluation of Orius species (Heteroptera: Anthocoridae) asbiological control agents. JSRQ 43:281–288

15. Jaenike J, Stahlhut JK, Boelio LM, Unckless RL (2010) AssociationbetweenWolbachia and SpiroplasmawithinDrosophila neotestacea:an emerging symbiotic mutualism? Mol Ecol 19:414–425

16. Jeyaprakash A, Hoy MA (2000) Long PCR improves WolbachiaDNA amplification: wsp sequences found in 76 % of sixty-threearthropod species. Insect Mol Biol 9:393–405

17. Jiggins FM (2003) Male-killing Wolbachia and mitochondrialDNA: selective sweeps, hybrid introgression and parasite popula-tion dynamics. Genetics 164:5–12

18. Jolley KA, Feil EJ, Chan MS, Maiden MC (2001) Sequence typeanalysis and recombinational tests (START). Bioinformatics17:1230–1231

19. Kakimoto K, Inoue H, Yoshida T (2002) Seasonal changes inpopulation density and species composition of Orius spp. in mul-berry fields. Jpn J Appl Entomol Zool 46:209–215 (in Japanesewith English summary)

20. Kawasaki Y, Ito M, Miura K, Kajimura H (2010) Superinfection offive Wolbachia in the alnus ambrosia beetle, Xylosandrus germa-nus (Blandford) (Coleoptera: Curuculionidae). Bull Entomol Res100:231–239

21. Keller GP, Windsor DM, Saucedo JM, Werren JH (2004) Repro-ductive effects and geographical distributions of two Wolbachiastrains infecting the Neotropical beetle, Chelymorpha alternansBoh. (Chrysomelidae, Cassidinae). Mol Ecol 13:2405–2420

22. Kimura M (1980) A simple method for estimating evolutionaryrates of base substitutions through comparative studies of nucleo-tide sequences. J Mol Evol 16:111–120

23. Kittayapong P, JamnonglukW, Thipaksorn A, Milne JR, SindhusakeC (2003) Wolbachia infection complexity among insects in thetropical rice-field community. Mol Ecol 12:1049–1060

24. Kondo N, Shimada M, Fukatsu T (2005) Infection density ofWolbachia endosymbiont affected by co-infection and host geno-type. Biol Lett 1:488–491

25. Martin DP, Williamson C, Posada D (2005) RDP2: recombinationdetection and analysis from sequence alignments. Bioinformatics21:260–262

26. Malloch G, Fenton B (2005) Super-infections of Wolbachia inbyturid beetles and evidence for genetic transfer between A andB super-groups of Wolbachia. Mol Ecol 14:627–637

27. Narita S, Shimajiri Y, Nomura M (2009) Strong cytoplasmicincompatibility and high vertical transmission rate can explainthe high frequencies of Wolbachia infection in Japanese popula-tions of Colias erate poliographus (Lepidoptera: Pieridae). BEntomol Res 99:385–391

28. Ohno K, Takemoto H (1997) Species composition and seasonaloccurrence of Orius spp. (Heteroptera: Anthocoridae), predaciousnatural enemies of Thrips palmi (Thysanoptera: Thripidae) in egg-plant fields and surrounding habitats. Appl Entomol Zool 32:27–35

29. O’Neill SL, Hoffmann AA, Werren JH (1997) Influential passen-gers. Inherited microorganisms and arthropod reproduction. Ox-ford University Press, Oxford

30. Raychoudhury R, Baldo L, Oliveira DCSG, Werren JH (2009)Modes of acquisition of Wolbachia: horizontal transfer, hybridintrogression, and codivergence in the Nasonia species complex.Evolution 63:165–183

31. Reuter M, Keller L (2003) High levels of multiple Wolbachiainfection and recombination in the ant Formica exsecta. Mol BiolEvol 20:748–753

32. Russell JA, Goldman-Huertas B, Moreau CS, Baldo L, StahlhutJK, Werren JH, Pierce NE (2009) Specialization and geographicisolation among Wolbachia symbionts from ants and lycaenidbutterflies. Evolution 63:624–640

33. Schilthuizen M, Stouthamer R (1997) Horizontal transmission ofparthenogenesis-inducing microbes in Trichogramma wasps. ProcR Soc Lond B 264:361–366

34. Stahlhut JK, Desjardins CA, Clark ME, Baldo L, Russell JA, WerrenJH, Jaenike J (2010) The mushroom habitat as an ecological arena forglobal exchange of Wolbachia. Mol Ecol 19:1940–1952

35. Stouthamer R, Breeuwer JA, Hurst GD (1999) Wolbachia pipien-tis: microbial manipulator of arthropod reproduction. Annu RevMicrobiol 53:71–102

36. Tagami Y, Miura K (2004) Distribution and prevalence of Wolba-chia in Japanese populations of Lepidoptera. Insect Mol Biol13:359–364

37. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: MolecularEvolutionary Genetics Analysis (MEGA) software version 4.0.Mol Biol Evol 24:1596–1599

38. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improv-ing the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position specific gap penalties andweight matrix choice. Nucleic Acids Res 22:4673–4680

39. Watanabe M, Miura K, Hunter MS, Wajnberg E (2011) Superin-fection of cytoplasmic incompatibility-inducing Wolbachia is notadditive in Orius strigicollis (Hemiptera: Anthocoridae). Heredity106:642–648

40. Werren JH (1997) Biology of Wolbachia. Annu Rev Entomol42:587–609

41. Werren JH, Bartos JD (2001) Recombination in Wolbachia. CurrBiol 11:431–435

42. Werren JH, Windsor DM (2000) Wolbachia infection frequenciesin insects: evidence of a global equilibrium? Proc R Soc Lond B267:1277–1285

43. Werren JH, Windsor D, Guo LR (1995) Distribution of Wolbachiaamong neotropical arthropods. Proc R Soc Lond B 262:197–204

44. Werren JH, Baldo L, Clark ME (2008) Wolbachia: master manip-ulators of invertebrate biology. Nat Rev Microbiol 6:741–751

45. West SA, Cook JM, Werren JH, Godfray HCJ (1998)Wolbachia intwo insect host–parasitoid communities. Mol Ecol 7:1457–1465

46. Yano E (2004) Recent development of biological control and IPMin greenhouses in Japan. J Asia-Pacific Entomol 7:5–11

47. Yasunaga T (1997) The flower bug genus Orius Wolff (Hetero-ptera: Anthocoridae) from Japan and Taiwan, Part I. Appl EntomolZool 32:355–364

48. Yasunaga T (1997) The flower bug genus Orius Wolff (Hetero-ptera: Anthocoridae) from Japan and Taiwan, Part II. Appl Ento-mol Zool 32:379–386

49. Yasunaga T (1997) The flower bug genus Orius Wolff (Hetero-ptera: Anthocoridae) from Japan and Taiwan, Part III. Appl Ento-mol Zool 32:387–394

50. Yasunaga T, Kashio T (1993) Taxonomy and identification ofJapanese Orius species. Plant Prot 47:180–183 (in Japanese)

51. Zhou W, Rousset F, O’Neill SL (1998) Phylogeny and PCR basedclassification of Wolbachia strains using wsp gene sequences. ProcR Soc Lond B 265:509–515

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