Molecular (Sub)Grouping of Endosymbiont Wolbachia Infection Among Mosquitoes of Taiwan

MOLECULAR BIOLOGY/GENOMICS Molecular (Sub)Grouping of Endosymbiont Wolbachia Infection Among Mosquitoes of Taiwan 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 arthropods 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 mosquito 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 insects (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 insect-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.

Load more