MOLECULAR BIOLOGY/GENOMICS Molecular (Sub)Grouping of Endosymbiont Wolbachia Infection Among Mosquitoes of

1 2 1 1 3 KUN-HSIEN TSAI, JIH-CHING LIEN, CHIN-GI HUANG, WEN-JER WU, AND WEI-JUNE CHEN

J. Med. Entomol. 41(4): 677Ð683 (2004) ABSTRACT Wolbachia are maternally inherited bacteria that infect a wide range of as well as Þlarial worms. The infection usually results in reproductive distortions of the host, primarily cytoplasmic incompatibility, parthenogenesis, and feminization. This study showed that Wolbachia infection (15/29; 51.72%) was prevalent among Þeld-caught mosquitoes in Taiwan. Three species were identiÞed as having Wolbachia A infection, eight species as having Wolbachia B, and four other species were dually infected by both groups. Each Wolbachia isolate from different mosquitoes was further divided into a speciÞc subgroup. However, there were still some isolates that did not belong to any known subgroup, suggesting that more subgroups remain to be identiÞed. Investigation of tissue tropism in either Aedes albopictus (Skuse) or Armigeres subalbatus (Coquillett) revealed that Wol- bachia were extensively distributed within the host, although the ovary was most susceptible to infection. This report provides preliminary features of molecular relationships among Wolbachia groups of mosquitoes from Taiwan.

KEY WORDS endosymbiont, mosquito, tissue tropism, Wolbachia

THE ENDOSYMBIONT Wolbachia are intracellular rickett- 1995b, Zhou et al. 1998). The type species, Wolbachia sia-like bacteria that are maternally inherited and nor- pipientis, was Þrst identiÞed in the mosquito Culex mally harbored in a number of arthropods (Rigaud pipiens (L.) complex (Hertig and Wolbach 1924). It and Juchault 1993, 1995; Breeuwer and Jacobs 1996; has been found that Wolbachia infects almost 100% of OÕNeill et al. 1997; Werren 1997; Bourtzis and OÕNeill wild-caught mosquitoes of the Cx. pipiens complex 1998). According to previous reports, 16.9% of Neo- (Sinkins et al. 1995). Wolbachia is normally main- tropical (Werren et al. 1995a), 35.7% of dipter- tained in nature through vertical transmission (Wer- ans (Werren et al. 1995a, Bourtzis et al. 1996), 35%Ð ren 1997). However, horizontal transmission of Wol- 40% of mites (Breeuwer and Jacobs 1996), and up to bachia has been mentioned; this apparently sheds light 46.3% of terrestrial isopods (Bouchon et al. 1998) are on mosquito control because Wolbachia is expected to infected with Wolbachia. Moreover, it was recently drive a deleterious gene into and to reduce the density found that Wolbachia could infect various nematode of natural populations of mosquitoes (Beard et al. Þlarial worms (Sironi et al. 1995). Undoubtedly, Wol- 1993, Sinkins et al. 1997, Beard et al. 1998, Curtis and bachia is one of the most ubiquitous endosymbionts Sinkins 1998). described to date (Werren et al. 1995a). Wolbachia IdentiÞcation of Wolbachia has been convention- causes reproductive distortions, primarily cytoplasmic ally dependent on electron microscopy because of incompatibility (CI), feminization of genetic males, difÞculties with in vitro culture (Wright and Barr 1980, and induction of parthenogenesis in infected arthro- Hayes and Burgdorfer 1981, Louis and Nigro 1989). pods (Werren 1997). Of these, CI is the most common Polymerase chain reaction (PCR) ampliÞcation and feature occurring in the mosquito. The reproductive sequencing are routine techniques used in the iden- distortions caused by Wolbachia infection are known tiÞcation of Wolbachia. At present, various genes, in- to result in embryonic death and subsequent failure of cluding wsp (Wolbachia outer surface protein) (Zhou egg hatch due to disruptions during early events of et al. 1998), 16S rDNA (Breeuwer et al. 1992, OÕNeill fertilization (Hoffmann and Turelli 1997). et al. 1992, Rousset et al. 1992, Bensaadi-Merchermek It is believed that Wolbachia is a monophyletic as- et al. 1995, Clark et al. 2001), ftsZ (Holden et al. 1993, semblage belonging to the alpha-subdivision of the Clark et al. 2001), and dnaA protein sequence Proteobacteria (OÕNeill et al. 1992, Werren et al. (Bourtzis et al. 1994) are extensively used for the purpose of Wolbachia detection and have shown tre- 1 Department of Entomology, National Taiwan University, Taipei mendous advantages in its phylogenic analysis. 106, Taiwan. Wolbachia occurs commonly in natural mosquito 2 Institute of Preventive Medicine, National Defense Medical Cen- ter, San-Hsia, Taipei 237, Taiwan. populations (Sinkins et al. 1995, Kittayapong et al. 3 Department of Public Health and Parasitology, College of Med- 2000). As mentioned, Wolbachia may play a role in the icine, Chang Gung University, Kwei-San, Tao-Yuan 333, Taiwan. control of disease vectors, especially the mosquito,

0022-2585/04/0677Ð0683$04.00/0 ᭧ 2004 Entomological Society of America 678 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 41, no. 4 due to its potential role in reducing the Þtness of the 3Ј; reverse, 5Ј-CCACTTAACTCTTTCGTTTG-3Ј) for host population (Beard et al. 1993, 1998). Thus, it is group B. Another primer pair was wsp81 F/691R worthwhile to extensively investigate the occurrence (wsp 81F, 5Ј-TGGTCCAATAAGTGATGAAGAAAC- and distribution of Wolbachia in mosquitoes in nature. 3Ј; wsp691R, 5Ј-AAAAATTAAACGCTACTCCA-3Ј) Herein, we describe the status of Wolbachia infection from which 590Ð632 bp of cDNA was synthesized among mosquitoes in Taiwan based on molecular data (Zhou et al. 1998). derived from PCR-based techniques, including DNA Subgrouping of Wolbachia. The wsp gene fragment sequencing. was used as the target for further division of each group of Wolbachia. In this experiment, all PCR prod- Materials and Methods ucts obtained from the wsp 81F/691R primer pair were used as the template to amplify a shorter gene frag- Mosquitoes. Larvae of 29 mosquito species belong- ment that served as the gene marker speciÞc to sub- ing to nine genera were collected in the Þeld from groups, including AlbA, Aus, Haw, Mel, Mors, Pap, Riv, various parts of Taiwan and then reared in the labo- and Uni for group A and CauB, Con, Dei, and Pip for ratory as described previously (Chen et al. 2000). group B. The PCR products ampliÞed from secondary Mosquito identiÞcation was carried out on the basis of PCR ranged from 379 to 556 bp in length (Zhou et al. morphological characteristics illustrated by Lien 1998). (1978). To cure mosquitoes of the Wolbachia infec- Visualization of PCR Products. PCR products of the tion, newly eclosed larvae were reared in water con- wsp gene fragment derived from 81F/691R primers taining diluted tetracycline as described by Yen and were separated on a 2% (wt:vol) agarose gel and then Barr (1973). stained with ethidium bromide. Banding patterns on DNA Extraction and PCR. DNA was extracted from the gel were photographed with a Polaroid camera. A tissues dissected from at least Þve mosquitoes of each recheck was carried out with higher concentrations of test, except for Mansonia uniformis (Theobald) and the DNA template of negative cases. All results, except Uranotaenia novobscura Barraud, by using a DNA ex- those for which the 12S -speciÞc gene was not traction kit (Viogene, Sunnyvale, CA). Twenty mi- successfully detected, were subjected to further anal- croliters of reaction mix contained 1 ␮l of extracted ysis. The band of the target cDNA fragment was sub- DNA, 12.5 ␮l of double distilled H O, 2 ␮lof10ϫ 2 sequently excised from the gel and puriÞed using a gel buffer (Klen Tag), 2 ␮l of 25 mM MgCl , 0.5 ␮lof 2 extraction kit (Viogene). The puriÞed cDNA derived dNTPs (10 mM each), 1 ␮l each of 2 pM forward and three individuals of each species were directly se- reverse primers, and1UofTag DNA polymerase quenced using a Prism automated DNA sequencing kit (Klen Tag). All reactions in this study were run in an ERICOMP thermocycler (PowerBlock System, San from the Applied Biosystems (Foster City, CA). Diego, CA) under the following settings: one cycle Wolbachia Detection from Mosquito Tissues. Tis- (1 min at 94ЊC, 1 min at 55ЊC, and 3 min at 72ЊC), 35 sues, including muscles, guts, testes, salivary glands, cycles (15 s at 94ЊC, 1 min at 55ЊC, and 3 min at 72ЊC), and ovaries were dissected from Þve mosquito larvae and one cycle (15 s at 94ЊC, 1 min at 55ЊC, and 10 min or 2-d-old adults (unfed males and females) of either at 72ЊC). The universal primers (12SAI-forward, 5Ј- Ae. albopictus or Ar. subalbatus. Individual tissues AAACTAGGATTAGATACCCTATTAT-3Ј; 12SBI-re- were Þrst rinsed with phosphate-buffered saline (pH verse, 5Ј-AAGAGCGACGGGCGATGTGT-3Ј) were 7.4) to avoid contamination between tissues and were used to amplify a cDNA fragment of insect mtDNA (of then processed for DNA extraction as described Ϸ400 bp) (Simon et al. 1991). This primer pair served above. The presence of Wolbachia in tissues was dem- as a control to assess the quality of the template DNA onstrated by ampliÞcation of a speciÞc gene fragment extracted from mosquito tissues. In each test, tissues by using the primer pair of wsp 81F/691R (Zhou et al. from Wolbachia-infected and tetracycline-treated 1998). Three replicates have been applied in the de- Armigeres subalbatus (Coquillett) were used as the tection of this study. positive and negative controls, respectively. Primer Pairs Used to Amplify Wolbachia-Specific Genes. Three primer pairs were used to amplify Wol- Results bachia-speciÞc genes in this study. The 99 F/994R primers (99 F, 5Ј-TTG TAG CCT GCT ATG GTA TAA Identification of Wolbachia Infection in Ae. albop- CT-3Ј; 994R, 5Ј-GAA TAG GTA TGA TTT TCA TGT- ictus. Wolbachia infection was deÞnitively demon- 3Ј) were designed from the hypervariable V1 and V6 strated in Ae. albopictus by checking ovaries through regions of the 16S rRNA gene of Wolbachia pipiens; a series of gene ampliÞcations with various primer from which we anticipated to obtain a 900-bp cDNA pairs, including the universal Wolbachia-speciÞc 16S fragment (OÕNeill et al. 1992). The fragment (Ϸ955Ð rDNA (900 bp), the group A-speciÞc 16S rDNA (259 957 bp) ampliÞed from the ftsZ gene was obtained by bp), the group B-speciÞc 16S rDNA (259 bp), the using the primer pair ftsZ F/R (Holden et al. 1993, group A-speciÞc ftsZ gene (955Ð957 bp), the group Sinkins et al. 1995), which consisted of ftsZA (forward, B-speciÞc ftsZ gene (955Ð957 bp), the universal Wol- 5Ј-CTCAAGCACTAGAAAAGTCG-3Ј; reverse: 5Ј- bachia-speciÞc wsp gene (Ϸ590Ð632 bp), the group TTAGCTCCTTCGCTTACCTG-3Ј) for group A and A-speciÞc wsp gene (556 bp), and the group B-speciÞc ftsZB (forward, 5Ј-CCGATGCTCAAGCGTTAGAG- wsp gene (442 bp) (Fig. 1). July 2004 TSAI ET AL.: MOLECULAR (SUB)GROUPING OF Wolbachia INFECTION 679

Fig. 1. PCR-ampliÞed products from the ovary of Ae. albopictus. Lanes 1 and 12, 100-bp DNA ladder; lane 2, insect 12S rRNA gene (400 bp); lane 3, 16S rDNA, A-speciÞc Wolbachia (259 bp); lane 4, 16S rDNA, B-speciÞc Wolbachia (259 bp); lane 5, universal Wolbachia 16S rDNA (900 bp); lane 6, ftsZ gene, A-speciÞc Wolbachia (Ϸ955Ð957 bp); lane 7, ftsZ gene, B-speciÞc Wolbachia (Ϸ955Ð957 bp); lane 8, the universal Wolbachia wsp gene (Ϸ590Ð632 bp); lane 9, the wsp gene, A-speciÞc Wolbachia (556 bp); lane 10, the wsp gene, B-speciÞc Wolbachia (442 bp); and lane 11, negative control (tetracycline-treated).

Wolbachia Infection among Field-Caught Mosqui- infection) could not be further classiÞed to any known toes. Field-caught mosquitoes were identiÞed to nine subgroup (Table 2). genera. Except for Anopheles and Heizmannia, Wol- Tissue Tropism of Wolbachia within the Mosquito. bachia infection was seen in seven other mosquito Tissues, including the pharynx, salivary gland, esoph- genera, including Aedes, Armigeres, Culex, Malaya, agus, cecum, trachea, stomach, Malpighian tubules, Mansonia, Tripteroides, and Uranotaenia with infection anterior hindgut, rectum, testis, accessory gland, and rates of 20% (1/5), 100% (2/2), 50% (6/12), 100% ovary, were dissected out from either Ae. albopictus or (1/1), 100% (1/1), 100% (2/2), and 66.67% (2/3), Ar. subalbatus mosquitoes (adults and larvae). A respectively. Of the 29 mosquito species tested, 15 cDNA fragment of Ϸ590Ð632 bp ampliÞed from the (51.72%) were infected with Wolbachia by showing wsp gene occurred in most tissues from both mosqui- speciÞc wsp fragments (Table 1); all wsp fragments toes (Table 3). The strongest band representing the (except those in Culex mimeticus Noe´ and Mn. uni- wsp gene fragment usually occurred in the preparation formis) were sequenced and shown to be Wolbachia- of ovaries. speciÞc (accession nos. AY462852Ð5, and AY462857Ð 67). Of these species, Ar. subalbatus, Tripteroides aranoides (Theobald), and Triperoides bambusa Discussion (Yamada) were infected with group A (3/15; 20%), This study showed that 51.72% (15/29) of mosquito whereas Cx. mimeticus, Culex murrelli Lien, Culex neo- species collected in Taiwan were positive for Wol- mimulus Lien, Culex pipiens molestus Forskal, Culex bachia infection. In addition to 26.67% of mosquitoes quinquefasciatus Say, Culex bicornutus Theobald, simultaneously infected by groups A and B, more Uranotaenia novobscura Barraud, and Uranotaenia an- mosquito species were shown to be infected by group nandalei Barraud were infected with group B (8/15; B (53.33%) than group A (20%). Recently, eight spe- 53.33%). Four species, Ae. albopictus, Armigeres cies of mosquitoes were recorded as being Wolbachia- omissus (Edwards), Malaya genurostris Liecester, and infected in Taiwan but not in other Southeast Asian Mn. uniformis were dually infected with both group A countries (Kittayapong et al. 2000), implying that and B (4/15; 26.67%) (Table 1). Wolbachia infection, especially group B, has become Subgroups of Wolbachia. Wolbachia A harbored in extensively established among mosquitoes of Taiwan. Ae. albopictus belonged to the subgroup AlbA, In addition, Þve species of Culex were all identiÞed to whereas that in Ml. genurostris and Mn. uniformis be- be infected by Wolbachia B, whereas two species of longed to subgroups Pap and Riv, respectively. Wol- Tripteroides were infected by Wolbachia A. bachia B harbored in Cx. neomimulus, Cx. pipiens mo- This study also identiÞed dual infection with lestus, Cx. quinquefasciatus, Cx. bicornutus, Tp. Wolbachia groups A and B in Ae. albopictus, Ar. omis- aranoides, Ae. albopictus, and Ar. omissus were iden- sus, Ml. genurostris, and Mn. uniformis. This is consis- tiÞed as the subgroup Pip, whereas that in Ur. novob- tent with the fact that an insect may naturally harbor scura, Ur. annandalei, and Mn. uniformis were identi- more than one strain of Wolbachia (superinfection) Þed to the subgroup Con (Table 2). However, (Breeuwer et al. 1992, Rousset et al. 1992, Rousset and Wolbachia harbored in Cx. mimeticus, Cx. murrelli Solignac 1995). In fact, more than one-fourth of spe- (group B), Ar. subalbatus, Tp. bambusa (group A), Ar. cies identiÞed with Wolbachia may have actually been omissus, and one subgroup of Ml. genurostris (dual superinfected (Werren et al. 1995a,b). A stable triple 680 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 41, no. 4

Table 1. Infection of Wolbachia in mosquitoes of Taiwan

16S rRNA ftsZ Gene wsp Gene Genus (subgenus) species Universal A-type B-type A-type B-type Universal A-type B-type Anopheles (Anopheles) An. sinensis (6)a ϪϪ Anopheles (Cellia) An. maculatu (3) ϪϪ Aedes (Finlaya) Ae. elsiae vicarius (6) ϪϪ Ae. japonicus shintienensis Tsai and Lien (6) ϪϪ Ae. togoi (Theobald) (3) ϪϪ Aedes (Stegomyia) Ae. Aegypti (100) ϪϪ Ae. Albopictus (50) ϩ ϩϩϩϩ ϩ ϩϩ Armigeres (Armigeres) Ar. subalbatus (100) ϩϩ ϩ ϩϩ Armigeres (Leicesteria) Ar. omissus (20) ϩ ϩϩϩϩ ϩ ϩϩ Culex (Culex) Cx. mimeticus (3) ϩϩϩϩϩ Cx. murrelli (3) ϩϩϩϩϩ Cx. neomimulus (3) ϩϩϩϩϩ Cx. pipiens molestus (6) ϩϩϩϩϩ Cx. quinquefasciatus (6) ϩϩϩϩϩ Cx. tritaeniorhynchus summorosus (9) ϪϪ Culex (Culiciomyia) Cx. pallidothorax Theobald (5) ϪϪ Culex (Eumelanomyia) Cx. foliatus Brug (6) ϪϪ Cx. okinawae Bohart (9) ϪϪ Culex (Lophoceraomyia) Cx. bicornutus Theobald (5) ϩϩϩϩϩ Culex (Lutzia) Cx. fuscanus Wiedemann (2) ϪϪ Cx. halifaxii Theobald (2) ϪϪ Heizmannia (Heizmannia) Hz. taiwanensis Lien (10) ϪϪ Malaya Ml. genurostris Leicester (10) ϩ ϩϩϩϩ ϩ ϩϩ Mansonia (Mansonioides) Mn. uniformis (2) ϩ ϩϩϩϩ ϩ ϩϩ Tripteroides (Rachionotomyia) Tp. aranoides (4) ϩϩ ϩ ϩϩ Tripteroides (Tripteroides) Tp. bambusa (Yamada) (3) ϩϩ ϩ ϩϩ Uranotaenia (Pseudoficalbia) Ur. maxima (3) ϪϪ Ur. novobscura (1) ϩϩϩϩϩ Uranotaenia (Uranotaenia) Ur. annandalei (20) ϩϩϩϩϩ

a Number of specimens tested. infection of Wolbachia in Drosophila has even been vectors, including Culex tritaeniorhynchus Dyar and generated by an experimental injection (Rousset et al. Aedes aegypti (L.) have never been identiÞed as har- 1999). However, it is still not clear whether compe- boring Wolbachia (Sinkins et al. 1997, Kittayapong tition between different bacteria may actually limit et al. 2000). One explanation for this phenomenon the persistence of dual or multiple infections (Rousset is that these mosquitoes are presumed to be unsuit- et al. 1999). Until recently, any individual host in- able for supporting Wolbachia reproduction. Never- fected by more than two Wolbachia strains had not theless, eubacteria-speciÞc genes, including 16S and been described in nature (Nirgianaki et al. 2003). New 23S rDNAs, were recently identiÞed from Anopheles molecular markers may be more efÞcient at diagnos- gambiae Giles (Brown et al. 2001). This implies that a ing multiple infections. eubacterial symbionts other than Wolbachia may re- Wolbachia were detected in seven of nine genera of side in Anopheles mosquitoes (Brown et al. 2001) and mosquitoes examined in this study. The absence of possibly in other currently “Wolbachia-free” species as Wolbachia from Anopheles and Heizmannia is believed well. to be the result of differential intracellular environ- The wsp gene is known to possess Ϸ10 times the ments, such as competition from other bacterial en- variability of the ftsZ gene (Werren et al. 1995b, Zhou dosymbionts of these mosquitoes (West et al. 1998, et al. 1998, van Meer and Stouthamer 1999) and is thus Kittayapong et al. 2000). In addition, some disease more suitable for elucidation of evolutionary relation- July 2004 TSAI ET AL.: MOLECULAR (SUB)GROUPING OF Wolbachia INFECTION 681

Table 2. Subgroups of Wolbachia based on the wsp gene among other reproductive distortions in a host. In insects mosquitoes of Taiwan infected by Wolbachia, host Þtness can actually be enhanced by increased host fertility (Wade and Wolbachia subgroupa Species Chang 1995, Poinsot and Merc¸ot 1997, Hariri et al. AB1998). However, such infection in some cases may Single infection decrease host Þtness through reduced female fecun- Armigeres (Armigeres) dity (Hoffmann et al. 1990) or deleterious effects Ar. subalbatus * Culex (Culex) under speciÞc conditions (Hoffmann et al. 1990). Cx. mimeticus * More observations are needed to elucidate why CI is Cx. murrelli * only seen in Culex, Aedes, and Armigeres mosquitoes Cx. neomimulus Pip (Yen and Barr 1973, Wright and Barr 1980, Jamnon- Cx. pipiens molestus Pip Cx. quinquefasciatus Pip gluk et al. 2000, Dobson and Rattanadechakul 2001). Culex (Lophoceraomyia) Wolbachia is highly infective to reproductive tissues Cx. bicornutus Pip and may be deleterious to the reproduction of infected Tripteroides (Rachionotomyia) hosts in certain cases (OÕNeill et al. 1997, Werren 1997, Tp. aranoides * Tripteroides (Tripteroides) Bourtzis and OÕNeill 1998). However, heavy infection Tp. bambusa * of Wolbachia in nervous and muscle tissues has been Uranotaenia (Pseudoficalbia) shown in Drosophila (Min and Benzer 1997) and other Ur. novobscura Con insects (Dobson et al. 1999), indicating that nonre- Uranotaenia (Uranotaenia) Ur. annandalei Con productive tissues also are susceptible to Wolbachia Dual infection (Min and Benzer 1997). The current study showed Aedes (Stegomyia) that a variety of somatic tissues of Ae. albopictus and Ae. albopictus AlbA Pip Ar. subalbatus were actually infected by Wolbachia, Armigeres (Leicesteria) Ar. omissus * Pip although the intensities of infection were varied. Malaya Among these tissues sampled, the ovary seemed to be Ml. genurostris Pap * the most suitable site for Wolbachia infection. In con- Mansonia trast, Wolbachia-infected salivary glands were only Mn. uniformis Riv Con infected at a moderate intensity. The relevance of this a Gene fragments for identiÞcation of Wolbachia A included Mel, observation will be better understood once it is de- AlbA, Mors, Riv, Uni, Haw, Pap, and Aus, whereas those for Wolbachia termined whether Wolbachia is involved in interfer- B included Con, Dei, Pip, and CauB. ence with arboviral replication or even transmission * The species was not subgrouped. by a mosquito vector. Furthermore, the broad spec- trum of tissue tropism renders it possible to use Wol- ships among Wolbachia isolates. Even so, some wsp- bachia to manipulate host populations as long as it can positive Wolbachia could not be appropriately placed express antipathogen proteins in mosquito vectors into any known subgroup, suggesting that some (Beard et al. 1993, Sinkins et al. 1995, Beard et al. 1998). Wolbachia isolates actually belonged to a subgroup not described previously (Zhou et al. 1998). It would be interesting to determine whether coinfection of Acknowledgments two subgroups of Wolbachia also could cause CI or This work was in part supported by a grant (NSC92-2815- C-182-029-B) from the National Science Council, Taiwan, Table 3. Tissue tropism of Wolbachia within Ae. albopictus and Republic of . Ar. subalbatus, based on amplification of a wsp gene fragment

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