Supergroup F Wolbachia Bacteria Parasitise Lice (Insecta: Phthiraptera)

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; family Species of louse Host of louse Wolbachia Country of GenBank accession of louse supergroup origin (region) number (Wolbachia)

Present study Ischnocera; Campanulotes bidentatus Columba livia F Australia DQ498894 Philopteridae compar (domestic pigeon) (Queensland) Ischnocera; Columbicola columbae Columba livia F Australia DQ498891 Philopteridae (domestic pigeon) (Queensland) Amblycera; Hohorstiella lata Columba livia F Australia DQ498893 Menoponidae (domestic pigeon) (Queensland) Ischnocera; Bovicola ovis Ovis aries A Australia DQ498895 Trichodectidae (domestic sheep) (Queensland) Amblycera; Menacanthus Gallus gallus A Australia DQ498892 Menoponidae stramineus (domestic chicken) (Queensland) Anoplura; Pediculus capitis Homo sapiens (human) A Australia DQ498897–8 Pediculidae (head lice) (Queensland) Anoplura; Pediculus capitis Homo sapiens (human) A Australia DQ498896 Pediculidae (head lice) (Tasmania) Kyei-Poku et al. (2005) Anoplura; Echinophthirius horridus Phoca groenlandica A Russia AY331986 Echinophthiridae (harp seal) (Withe Sea) Anoplura; Echinophthirius horridus Phoca groenlandica A, B Norway (Jan AY331122, AY331123 Echinophthiridae (harp seal) Mayen Island) Anoplura; Linognathus africanus Capra hircus (goat) A Canada AY330317 Linognathidae Anoplura; Linognathus setosus Canis latrans (coyote) A, B Canada AY33032–3, AY330321 Linognathidae Anoplura; Linognathus setosus Canis familiaris B Canada AY330324 Linognathidae (domestic dog) Anoplura; Linognathus vituli Bos taurus (domestic B Canada AY330319 Linognathidae cattle) Anoplura; Pediculus humanus Homo sapiens (human) A Canada AY331112 Pediculidae (body/clothes lice) Anoplura; Pediculus humanus Homo sapiens (human) B USA AY331117 Pediculidae (body/clothes lice) Anoplura; Pediculus capitis Homo sapiens (human) A, B Canada AY331113, AY331114 Pediculidae (head lice) Anoplura; Pediculus capitis Homo sapiens (human) B Canada AY331115 Pediculidae (head lice) Anoplura; Pediculus capitis Homo sapiens (human) B USA AY331116 Pediculidae (head lice) Anoplura; Pthirus pubis Homo sapiens (human) A, B Italy AY331124–5, AY331126 Pthiridae (pubic lice) Anoplura; Polyplax serrata Mus musculus (mouse) B Canada AY331121 Polyplacidae Amblycera; Colpocephalum Pelecanus A, B Canada AY330308, AY330307 Menoponidae unciferum erythrorhynchos (American white pelican) Amblycera; Eidmanniella Phalacrocorax auritus A Canada AY330306 Menoponidae -pellucida (double-breasted cormorant) Amblycera; Trinoton Aix sponsa (wood duck) A Canada AY330315–6 Menoponidae querquedulae Ischnocera; Pectinopygus Pelecanus A, B Canada AY331120, AY331118 Philopteridae falloroni erythrorhynchos (American white pelican) 482 Parasitol Res (2007) 100:479–485

Table 1 (continued)

Suborder; family Species of louse Host of louse Wolbachia Country of GenBank accession of louse supergroup origin (region) number (Wolbachia)

Ischnocera; Pectinopygus tortoffi Pelecanus A Canada AY33039–11 Philopteridae erythrorhynchos (American white pelican) Ischnocera; Quadraceps sp. Larus argentatus A Canada AY330312–4 Philopteridae (herring gull) Ischnocera; Saemundssonia lari Larus argentatus A, B Canada AY331129–30, Philopteridae (herring gull) AY331131 Ischnocera; Bovicola bovis Bos taurus (domestic A Canada AY331128 Trichodectidae cattle) Ischnocera; Bovicola bovis Bos taurus B Italy AY331127 Trichodectidae (domestic cattle) Ischnocera; Eutrichophilus sp. Erethizon dorsatum A Canada AY330320 Trichodectidae (North American porcupine) Ischnocera; Trichodectes sp. Taxidea taxus A Canada AY330318 Trichodectidae (American badger) Perotti et al. (2004) Anoplura; Pediculus capitis Homo sapiens A Argentina AY596781 Pediculidae (head lice) (human) Anoplura; Pediculus capitis Homo sapiens A Argentina AY596782 Pediculidae (head lice) (human) Anoplura; Pediculus capitis Homo sapiens (human) A Wales AY596783–4 Pediculidae (head lice) Anoplura; Haematopinus suis Sus scrofa domestica AB Poland AY596785–AY596786 Haematopinidae (pig)

BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST/, Discussion Altschul et al. 1997). We then aligned our contigs with sequences from GenBank with Clustal X (Jeanmougin et al. Wolbachia supergroup F in Australian lice 1998). Unrooted genetic-distance phylogenetic trees were inferred by neighbor joining (NJ) in PAUP version 4.0b10 This is the first report of lice infected with Wolbachia from (Swofford 2002) with Kimura two-parameter model. The supergroup F. To date, supergroup F Wolbachia have been robustness of branches in these trees was tested by foundinfilarialnematodesMansonella sp., M. ozzardi,and 1,000 cycles of bootstrap resampling. seven species of insects, termites Kalotermes flavicollis, Microcerotermes sp. and Coptotermes sp., the bed bug Cimex lectularius, the cliff swallow bug Oeciacus vicarius,the Results weevil Rhinocyllus conicus and a hippoboscid fly Pseudo- lynchia canariensis (Bandi et al. 1997;Loetal.2002; We found Wolbachia from supergroup F in the small pigeon Rasgon and Scott 2004). This is also the first report of louse, C. bidentatus, the slender pigeon louse, Co. colum- Wolbachia from the chicken body louse, M. stramineus,the bae, and the pigeon body louse, H. lata, whereas Wolbachia sheepbodylouse,B. ovis, the small pigeon louse, from supergroup A was found in the sheep body louse, B. C. bidentatus, the pigeon body louse, H. lata,andtheslender ovis, the chicken body louse, M. stramineus, and the head pigeon louse, Co. columbae, and the first report of Wolbachia louse, P. capitis (Table 1, Fig. 1). There was variation in the from the head louse, P. capitis, from Australia. Wolbachia nucleotide sequences at 15 sites between Wolbachia from from supergroups A and B were found in the lice studied by supergroups A and F (Fig. 2). There was also variation Perotti et al. (2004) and Kyei-Poku et al. (2005), whereas we among sequences within supergroup F at two sites and at found Wolbachia from the supergroups A and F (Table 1, seven sites among the Wolbachia sequences from super- Fig. 1). Thus, lice are known to be infected with three of the groups A (Fig 2). eight supergroups of Wolbachia: supergroups A, B and F. Parasitol Res (2007) 100:479–485 483

Fig. 1 Phylogenetic tree inferred from SSU rRNA nucleotide sequences of Wolbachia bacteria that infect arthropods and filarial nematodes. Names are host species (arthropods and nematodes). Wolbachia supergroups are labelled A–H. Names in bold with an asterisk indicate new hosts (lice) from the present study. Bootstrap support is shown. Broken lines in supergroup A indicate hosts at similar positions in the tree

There is no evidence of congruence between the taxon of infected with Wolbachia from supergroups A, B and F, louse and the Wolbachia bacteria that infect these lice. For whereas the family Pediculidae (suborder Anoplura) is example, the family Menoponidae (suborder Amblycera) is infected with Wolbachia from supergroups A and B (Table 1).

Fig. 2 Alignment of sections of the SSU rRNA of the species of lice supergroup A. Shaded nucleotides indicate variation between super- from the present study. Circled nucleotides indicate an insertion within group A and supergroup F. Species names (lice) are in Table 1. Dash supergroup F. Boxed nucleotides indicate the variation within (––) indicates alignment gap, and N indicates no data 484 Parasitol Res (2007) 100:479–485

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