Parasitol Res (2007) 100:479–485 DOI 10.1007/s00436-006-0309-6 ORIGINAL PAPER Supergroup F Wolbachia bacteria parasitise lice (Insecta: Phthiraptera) Catherine Covacin & Stephen C. Barker Received: 10 May 2006 /Accepted: 8 August 2006 / Published online: 18 October 2006 # Springer-Verlag 2006 Abstract We studied six species of lice from three of the RNA gene (SSU rRNA) (Werren et al. 1995; Bandi et al. four suborders of lice. These lice were infected with 1998, 2001; Lo et al. 2002; Bordenstein and Rosengaus Wolbachia bacteria from supergroups A and F. This is the 2005). Phylogeny inferred from the genes that encode first report of an infection of supergroup F Wolbachia in citrate synthase (gltA), and a heat-shock protein 60 lice. To date, Wolbachia from supergroup F have been (groEL), is consistent with this taxonomy (eg. Casiraghi et found in filarial nematodes, Mansonella spp., and, rarely, in al. 2005). Supergroups A and B have been found only in insects. We inferred the phylogeny of the Wolbachia from arthropods (Werren et al. 1995), supergroups C and D only lice and representatives of all Wolbachia supergroups, with in filarial nematodes (Bandi et al. 1998), supergroup E only nucleotide sequences from the small subunit ribosomal in springtails, Folsomia candida, Mesaphorura italica, RNA gene (SSU rRNA). There was no evidence of Mesaphorura macrochaeta and Paratullbergia callipygos congruence between the taxon of louse and the Wolbachia (Vandekerckhove et al. 1999; Czarnetzki and Tebbe 2004), bacteria that infect lice. There is no evidence that supergroup F in filarial nematodes and on rare occasions in Wolbachia and their louse hosts co-evolved at least at the insects (Lo et al. 2002), supergroup G only in spiders level of Wolbachia supergroups. We propose a novel (Rowley et al. 2004) and supergroup H only in the Pacific mechanism for the horizontal transfer of Wolbachia dampwood termites, Zootermopsis angusticollis and Zoo- between different species of lice from birds: transfer of termopsis nevadensis. Wolbachia during phoresis by hippoboscid flies. Wolbachia bacteria often manipulate the reproductive success of their female hosts to increase transmission of the Wolbachia in populations of their hosts (Werren 1997; Introduction Stouthamer et al. 1999). The most common manipulation, cytoplasmic incapability, occurs when infected and unin- Wolbachia bacteria belong to the alpha-subdivision of the fected hosts mate (Yen and Barr 1971) and when hosts Proteobacteria and are closely related to the Rickettsia infected with different strains of Wolbachia mate (Yen and species. Wolbachia are obligate, intracellular bacteria that Barr 1971; Montchamp-Moreau et al. 1991; Rousset et al. are transmitted from female hosts to their offspring in the 1992a,b; Rousset and Solignac 1995; Perrot-Minnot et al. cytoplasm of the host’s eggs. Wolbachia have been placed 1996). Parthenogenesis induction (Stouthamer et al. 1990; in eight taxonomic groups, supergroups A to H, on the O’Neill et al. 1992), feminisation (Rousset et al. 1992a,b), basis of phylogenetic relationships inferred from the cell- male killing (Jiggins et al. 1998; Hurst et al. 1999), cycle protein FTSZ (ftsZ) and the small subunit ribosomal enhanced fecundity and fertility of infected hosts (Girin and Bouletreau 1995) and pathenogenicity in infected hosts : (Min and Benzer 1997) have also been attributed to C. Covacin (*) S. C. Barker Wolbachia infections. Parasitology Section, School of Molecular and Microbial Wolbachia infections may lead to low levels of genetic Sciences, The University of Queensland, Brisbane 4072, Australia diversity in populations of hosts, high levels of genetic e-mail: [email protected] divergence among populations of hosts and even speciation 480 Parasitol Res (2007) 100:479–485 in their hosts (Frati et al. 2004; Marshall 2004; Hiroki et al. studied before, Columbicola columbae, Bovicola ovis and 2005; Opijnen et al. 2005;Telschowetal.2005). Campanulotes bidentatus compar (Ischnocera) and Menac- Wolbachia strains that cause cytoplasmic incompatibility anthus stramineus and Hohorstiella lata (Amblycera); (2) in their hosts may sweep through populations of insects, e. inferred the phylogeny of the Wolbachia strains we found in g. populations of Drosophila simulans (Turelli and these species of lice and representatives of all Wolbachia Hoffman 1991). Mitochondrial DNA (mtDNA) and Wol- supergroups; and (3) assessed congruence between the bachia bacteria are maternally inherited. Thus, mtDNA and strains of Wolbachia that infected the species of lice and the Wolbachia bacteria phylogenies are often congruent (Turelli phylogeny of these lice. We studied the SSU rRNA gene of et al. 1992; Shoemaker et al. 2000; James et al. 2002). Wolbachia rather than the wsp gene, as studied by Perotti et Indeed, Wolbachia-driven selective sweeps of mtDNA al. (2004) and Kyei-Poku et al. (2005). The SSU rRNA haplotypes have been reported in populations of insects gene seems to evolve in Wolbachia, about 10 times slower (Turelli and Hoffman 1991;Roussetetal.1992a,b; than the wsp gene (Zhou et al. 1998) and thus may provide Solignac et al. 1994; James and Ballard 2000; Ballard and more resolution than the wsp gene, of the relationships of Dean 2001; Baudry et al. 2003). Baudry et al. (2003) found Wolbachia bacteria and their louse hosts. that a bird-nest blowfly, Protocalliphora siala, had two mtDNA haplotypes and that each mtDNA haplotype was associated with only one strain of Wolbachia. James and Materials and methods Ballard (2000) found that each mtDNA haplotype of D. simulans was associated with a distinct group of Wolbachia DNA was extracted from individual lice and groups of lice strains, and Behura et al. (2001) found a correlation with the DNeasy Tissue Kit (QIAGEN, Valencia, CA, between mtDNA haplotypes and Wolbachia strains in the USA). PCR reactions had 100–200 ng of DNA template, Asian rice gall midge, Orseolia oryzae. Behura et al. (2001) 2.5 μl of Reaction Buffer IV at 10× concentration (ABgene, found that all female hosts and only certain males were Epsom, UK), 2.25 μl of MgCl2 (25 mM), 1.1 μl of dNTPs infected with Wolbachia; the females and infected males (5 mM), 0.3 μl of each primer (10 μM), 0.1 μl Red had only type 1 mtDNA haplotype, whereas uninfected Hot®Taq polymerase (ABgene) and MilliQ water to a final males had mtDNA haplotype 2 or 3. volume of 25 μl. The first-round PCR conditions were: 92°C There have been two studies of Wolbachia in lice for 2 min for 1 cycle, 92°C for 37 s, 50°C for 37 s and (Insecta: Phthiraptera): Perotti et al. (2004) and Kyei-Poku 72°C for 2 min for 35 cycles followed by 72°C for 5 min et al. (2005). Perotti et al. (2004)usedSSUrRNA with the SSU rRNA primers: forward primer sequences and the nucleotide sequence of the outer surface 5′-GCTTAACACATGCAAG-3′ and reverse primer protein-encoding gene (wsp) to test for Wolbachia in the 5′-CCATTGTAGCACGTGT-3′. Two microliters from the head louse, Pediculus capitis (Anoplura), and the pig louse first-round PCR reaction was used in the second-round Haematopinus suis (Anoplura). Perotti et al. (2004) found PCR. Second-round PCR conditions were: 92°C for 2 min six strains of Wolbachia in these two species of lice from for 1 cycle, 92°C for 37 s, 45°C for 37 s and 68°C for sequences of the SSU rRNA gene and the wsp gene; 2 min for 35 cycles followed by 68°C for 5 min with however, the sequences from the SSU rRNA were not PCR primers designed to be specific for Wolbachia published. Kyei-Poku et al. (2005) used the wsp gene to pipientis (forward: 5′-TTGTAGCCTGCTATGGTATAACT- study Wolbachia in 19 species of lice from three of the four 3′ and reverse: 5′-GAATAGGTATGATTTTCATGT-3′). All suborders of lice: the Anoplura, Ischnocera and Amblycera. primers were from O’Neill et al. (1992). PCR products were Kyei-Poku et al. (2005) found 39 strains of Wolbachia in 19 prepared for nucleotide sequencing with the Wizard® SV Gel species of lice. Therefore, Perotti et al. (2004) and Kyei- and PCR Clean-up System (Promega). Nucleotide sequenc- Poku et al. (2005) found a total of 45 different strains of ing was with the ABI BigDye dideoxy chain termination Wolbachia in 20 species of lice. Both studies found more sequencing system (Applied Biosystems, Foster City, CA, than one strain (one to four strains) of Wolbachia in an USA). The cycling conditions for nucleotide sequencing individual louse. Moreover, Kyei-Poku et al. (2005) found were: 1 cycle of 94°C for 5 min and 29 cycles at 94°C for similar strains of Wolbachia in species of lice from different 20 s, 50°C for 15 s and 60°C for 4 min. The second-round suborders of lice. For example, strains of Wolbachia from PCR primers were the sequencing primers. All sequences the group Phhu were found in Pediculus humanus were generated by the Australian Genome Research Facility (Anoplura), the human body louse, and Saemundssonia with ABI 377, 3700 and MD MegaBACE (capillary) lari (Ischnocera), a bird louse from the herring gull. automated sequencers. To investigate further the relationships of lice and Contigs of nucleotide sequences were made with Wolbachia we: (1) studied the head louse P. capitis Sequencher 3.1.1 (Gene Codes, Ann Arbor, MI, USA). (Anoplura) and five species of lice that have not been Contigs were then compared with sequences in GenBank by Parasitol Res (2007) 100:479–485 481 Table 1 Wolbachia bacteria from lice (order Phthiraptera: Insecta) found in the present study and by Kyei-Poku et al. (2005) and Perotti et al. (2004) Suborder;