Madhu, M.

Puttaraju, H.P.

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NotePresence of Wolbachia endosymbionts in diﬀerent silkwormspecies and races and in their uzi ﬂy parasitesThe genus Wolbachia is composed of endosymbioticbacteria that occur in the cytoplasm of reproductivetissues and other tissues in many arthropod species.These bacteria are capable of causing reproductive al-terations in their hosts such as cytoplasmic incompati-bility (Barr, 1980; Breeuwer et al., 1992; O’Neill et al.,1992), parthenogenesis in wasps (Stouthamer et al.,1993), and feminization of males in isopods (Roussetet al., 1992). In addition, these bacteria may cause rapidspeciation in some arthropods (Werren, 1997). Extensivehorizontal transmission of Wolbachia has occurredamong insect taxa, including between diﬀerent orders ofinsects (Werren et al., 1995b).Sericulture is an industry based on the rearing ofsilkworms (Lepidoptera) on host plants for the raw silkproduction. Among the parasites that attack silkworms,the most important is the uzi ﬂy (Diptera: Tachinidae),an endoparasite that causes considerable mortality(Siddappaji and Channabasavanna, 1990). The initimateassociation of the uzi ﬂy and silkworm provided anopportunity to determine whether there was horizontaltransmission of Wolbachia endosymbionts betweenthese two insects.In previous studies, systematic surveys for Wolbachiahave been carried out using the polymerase chain reac-tion (PCR) to detect 16S rDNA, ftsZ, and wsp genes(Werren et al., 1995a). Using this technology, horizontaltransmission has been shown to occur in some insecthost-parasitoid systems (Werren et al., 1995b). In thepresent study, we used PCR to detect the presence ofwsp gene in diﬀerent silkworm races and in its uzi ﬂyparasites. We further compared the PCR products todetermine whether there was horizontal transmissionbetween the uzi ﬂy and silkworms.The diﬀerent silkworm species and races and theirparasitoid uzi ﬂies screened for Wolbachia are listed inTables 1 and 2. Total genomic DNA from the silkwormsand ﬂies was prepared for PCR ampliﬁcation followingthe standard protocol (Sambrook et al., 1989). PCR wasdone in 25 ll reaction volume containing 1 ll of sampleDNA, 2.5 ll 10 buﬀer (Genei), 0.5 ll dNTPs (10mMeach), 1 ll 26 lM forward primer and 1 ll 35 lM reverseprimer, and 0.5 ll Taq DNA polymerase (Genei). Dis-tilled, deionized water was added to a ﬁnal volume of25 ll. The reaction mixture was prepared in one batchand then added to each sample. PCR ampliﬁcation wasdone under the following thermal proﬁle: 92 C 30 s,52 C 30 s, and 75 C 30 s per cycle for 30 cycles (MJResearch PTC-200). PCR products were separated byelectrophoresis in a 1.5% agarose gel to determine thepresence and size of ampliﬁed DNA, afterwhich the gelswere photographed on a UV transilluminator.The general wsp primers used for PCR ampliﬁcationwere as follows:wsp 81F (50 TGG TCC AAT AAG TGA TGA AGAAAC) and wsp 691R (50 AAA AAT TAA ACG CTACTC CA).By use of these wsp primers, a wsp gene fragment wasdetected in diﬀerent uzi ﬂy populations of E. sorbillans(Diptera: Tachinidae) and Blepharipa zebina (Diptera:Tachinidae). The mobilities of DNA fragments thatwere ampliﬁed using DNA from diﬀerent ﬂy popula-tions are shown in Figs. 1 and 2. The prominent reactionproducts were represented by single bands of the ex-pected size of approximately 600 bp. Using the sameprimers, however, we obtained no PCR products fromthe silkworm samples (Figs. 1 and 2).Braig et al. (1998) found that the primer combinationof wsp 81F and wsp 691R was able to amplify wsp genefragments from diﬀerent strains of Wolbachia. By usingthe same general wsp primers, Zhou et al. (1998) am-pliﬁed fragment of the wsp gene from 28 Wolbachiastrains. They reported that these primers amplify a DNAfragment ranging from 590 to 632, the size depending onthe individual Wolbachia strain. These primers onlyampliﬁed fragments from infected insects. Thus, the re-sults we obtained in the present study using wsp primersdemonstrate that Wolbachia was present in E. sorbillans(see also, Madhu and Puttaraju, 2001) and B. zebina. Itis also evident from this study that Wolbachia was notpresent in any of the silkworm species or races.Several routes of horizontal transfer have been dis-cussed (Hurst et al., 1992) and there is evidence ofcross-infection in predator–prey (Jolianowicz and Hoy,1996) and host–parasitoid systems (Werren et al.,1995b). O’Neill et al. (1992) and Rousset et al. (1992)Journal of Invertebrate Pathology 79 (2002) 120–122www.academicpress.com0022-2011/02/$ - see front matter  2002 Elsevier Science (USA). All rights reserved.PII: S0022 -2011 (02 )00031-9suggested that Wolbachia strains could be transmittedhorizontally between insect taxa, because closely re-lated bacteria have been found in distantly relatedhosts. Werren et al. (1995b) proposed that transmissionbetween parasitic insects and their hosts is one vehiclefor intertaxon transmission of Wolbachia. An obvioustest of this hypothesis is to show that parasitoids andtheir host insects share Wolbachia strains that are moreclosely related. For example, Nasonia giraulti and N.longicornis are parasitoid wasps harbour B groupWolbachia that are closely related to those of theirpreferred Protocallipliorid ﬂy hosts. A second possibleexchange between parasitoid and host involves Dro-sophila melanogaster and the drosophilid larval par-asitoid Asobara tabida. These two species share nearlyidentical Wolbachia. However, using a PCR assay,West et al. (1998) surveyed for Wolbachia infection in82 insect species from two host–parasitoid communi-ties. One host–parasitoid community was based aroundleaf-mining Lepidoptera, and the other around aphids.They found that phylogenetic analyses of the sequencedata suggested host–parasitoid transfer of Wolbachiawas not the major route through which the species theyexamined became infected. Since in the present studywe found Wolbachia in the uzi ﬂies, but not in any ofthe silkworm species or races, we conclude that theWolbachia organisms in the ﬂies are not transmitted tothe silkworms.Table 2Populations of the uzi ﬂies E. sorbillans and B. zebina and theirWolbachia infection status as determined by PCR using wsp primersSpecies Locality PCR results(+/))E. sorbillans Ramanagar (Karnataka) +E. sorbillans Sira (Karnataka) +E. sorbillons Madlaugiri (Karnataka) +E. sorbillans Mandya (Karnataka) +E. sorbillans Chitradurga (Karnataka) +E. sorbillans Ananthpur (Andhrapradesh) +B. zebina Bastar (Madhyapradesh) +Table 1Silkworm species and races and their Wolbachia infection status determined by the PCR using wsp primersSilkworm species/race Voltinism Place of origin PCR results (+/))Mulberry silkworm (Bombyx mori) (Lepidoptera: Bombycidae)RacePure Mysore Multivoltine Karnataka )Nistari Multivoltine West Bengal )Sarupat Multivoltine Assam )Moria Multivoltine Assam )Guangnong Multivoltine China )NB-1 Bivoltine Karnataka )NB-7 Bivoltine Karnataka )NB4D2 Bivoltine Karnataka )KA Bivoitine West Bengal )HU-204 Bivoltine China )Non-mulberry silkworms (species) (Lepidoptera: Bombycidae)Antheraea mylitta (Temperate Tasar) Bivoltine & Trivoltine India )Antheraea proylei (Temperate Tasar) Univoltine India )Antheraea roylei (Temperate Tasar) Univoltine India )Antheraea pernyi (Temperate Tasar) Univoltine China )Samia cynthia ricini (Eri) Mu1tivoltine India )Antheraea assamensis (Muga) Multivoltine India )Fig. 1. PCR screening for the presence of Wolbachia in races of themulberry silkworm, B. mori, and its parasite the uzi ﬂy, E. sorbillansusing wsp primers. Lane 1, HindIII–EcoR1 digest as markers. Lanes 2–9, B. mori samples from the following races, respectively: Lane 2, PureMysore; 3, Nistari; 4, Sarupat; 5, Moria; 6, Guangnong; 7, NB-1; 8,NB-7; 9, NB4D2. Lanes 10–13, E. sorbillans samples from the fol-lowing sites, respectively, Lane 10, Ramanagar; 11, Sira; 12, Mad-hugiri; and 13, Mandya. Lanes 14 and 15, B. mori samples from racesKA and HU-204, respectively.H.P. Puttaraju, M. Madhu / Journal of Invertebrate Pathology 79 (2002) 120–122 121Keywords: PCR; Silkworm species; Bombyx mori; Uziﬂies; Exorista sorbillans; Blepharipa zebina; Wolbachia;wsp gene; Cytoplasmic incompatibility; HorizontaltransmissionReferencesBarr, A.R., 1980. Cytoplasmic incompatibility in natural populationsof a mosquito, Culex pipiens. Nature (London) 283, 71–72.Breeuwer, J.A.J., Stouthamer, R., Burns, D.A., Pelletier, D.A.,Weisburg, W.G., Werren, J.H., 1992. Phylogeny of cytoplasmicincompatibility microorganisms in the parasitoid wasp genusNasonia (Hymenoptera: Pteromalidae) based on 16s ribosomalDNA sequences. Insect Mol. Biol. 1, 25–36.Braig, H.R., Zhou, W., Dobson, S., O’Neill, S.L., 1998. Cloning andcharacterization of a gene encoding the major surface protein of thebacterial endosymbiont Wolbachia pipientis. J. Bacteriol. 180,2373–2378.Hurst, G.D.D., Hurst, L.D., Majerus, M.E.N., 1992. Selﬁsh genesmove sideways. Nature. 356, 659–660.Jolianowicz, D.L., Hoy, M.A., 1996. Wolbachia in a predator–preysystem: 16s ribosomal DNA analysis of two phytoseiids (Acari:Phytoseiidae) and their prey (Acari: Tetranychidae). Ann. Ento-mol. Soc. Am. 89, 435–441.Madhu, M., Puttaraju, H.P., 2001. Cytological and molecular evidencefor Walbachia infection in uziﬂies of exorista species. Cytologia 66,197–203.O’Neill, S.L., Giordano, R., Colbert, A.M.E., Karr, T.L., Robertson,H.M., 1992. 16S rRNA phylogenetic analysis of the bacterialendosymbionts associated with cytoplasmic incompatibility ininsects. Proc. Natl. Acad. Sci. USA 89, 2699–2702.Rousset, F., Bouclion, D., Pintureau, B., Juchault, P., Solignac, M.,1992. Wolbachia endosymbionts responsible for various altera-tions of sexuality in arthropods. Proc. R. Soc. (London) B 250,91–98.Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning Alaboratory manual. Cold Spring Harbor Laboratory Press, NewYork.Siddappaji, C., Channabasavanna, G.P., 1990. The Indian uzi ﬂy,Exorista bombycis. A parasitoid of the mulberry silkworm. IndianJ. Seric. 29, 119–137.Stouthamer, R., Breeuwer, J.A.J., Luck, R.F., Werren, J.H., 1993.Molecular identiﬁcation of microorganisms associated with par-thenogenesis. Nature (London) 361, 66–68.Werren, J.H., 1997. Biology of Wolbachia. Annu. Rev. Entomol. 42,587–609.Werren, J.H., Windsor, D., Guo, L.R., 1995a. Distribution ofWolbachia among neotropical arthropods. Proc. R. Soc. (London)B 262, 197–204.Werren, J.H., Zhang, W., Guo, L.R., 1995b. Evolution and phylogenyof Wolbachia: reproductive parasites of arthropods. Proc. R. Soc.(London) B 251, 55–63.West, S.A., Cook, J.M., Werren, J.H., Godfray, H.C.J., 1998.Wolbachia in two insect host–parasitoid communities. Mol. Ecol.7, 1457–1465.Zhou, W., Rousset, F., O’Neill, S.L., 1998. Phylogeny and PCR-basedclassiﬁcation of Wolbachia strains using wsp gene sequences. Proc.R. Soc. (London) B 265, 509–515.H.P. Puttaraju*M. MadhuDepartment of SericultureBangalore UniversityBangalore 560 056IndiaE-mail address: puttarajuhp@hotmail.com(H.P. Puttaraju)Received 16 January 2001; accepted 9 May 2002Fig. 2. PCR screening for the presence of Wolbachia in species of non-mulberry silkworms and their uzi ﬂy parasites, E. sorbillans and B.zebina using wsp primers. Lane 1, HindIII–EcoR1 digest as markers.Lanes 2 and 3, respectively, samples from E. sorbillans from Chitrad-urga and Anathpur. Lane 4, sample from the silkworm, A. mylitta.Lane 5, sample from the uzi ﬂy, B. zebrina. Lanes 6–10, samples fromnon-mulberry silkworms: 6, A. proylei; 7, A. roylei; 8, A. pernyi; 9,S. cynthia ricini; and 10, A. assamensis.* Corresponding author. Fax: +91-80-3219295.122 H.P. Puttaraju, M. Madhu / Journal of Invertebrate Pathology 79 (2002) 120–122

Presence of Wolbachia endosymbionts in different silkworm species and races and in their uzi fly parasites

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Presence of Wolbachia endosymbionts in different silkworm species and races and in their uzi fly parasites

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