Quantitative analysis of the lytic cycle of WO phages infecting Wolbachia

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1 Appl Entomol Zool (0) 47: DOI 0.007/s ORIGINAL RESEARCH PAPER Quantitative analysis of the lytic cycle of WO phages infecting Wolbachia Seiichi Furukawa Kohjiro Tanaka Takashi Ikeda Takema Fukatsu Tetsuhiko Sasaki Received: 8 August 0 / Accepted: 9 August 0 / Published online: 8 September 0 Ó The Japanese Society of Applied Entomology and Zoology 0 Abstract WO is a temperate bacteriophage that infects Wolbachia, a maternally inherited endosymbiont of arthropods. WO has lysogenic and lytic cycles, the latter of which is an important process for the spread of WO infection. In this study, we measured the lytic activities of two WO phages, WOCauB and WOCauB, infecting a Wolbachia strain, wcaub. In the lytic cycle of WO, both ends of the prophage are ligated to create a junction sequence called attp in the phage genome. We performed real-time quantitative polymerase chain reaction to measure the amounts of attp sequences produced by WOCauB and WOCauB in wcaub-infected Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) and wcaub-infected insect cell lines. WOCauB produced the phage genome more actively than WOCauB in E. kuehniella, whereas WOcauB was more active than WOCauB in the cell lines, suggesting that the environment of host cells in which Wolbachia is harbored affects the lytic activity of WO phages. The lytic activity was constantly very low: the S. Furukawa Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan K. Tanaka T. Fukatsu Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan T. Ikeda Department of Reprogramming Science, Center for ips Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan T. Sasaki (&) Honeybee Science Research Center, Tamagawa University, Machida, Tokyo, Japan tsasaki@lab.tamagawa.ac.jp amounts of attp relative to the prophages were lower than in all measurements, which was discussed in conjunction with the intracellular life of Wolbachia. Keywords WO phage Wolbachia Lytic cycle attp site Real-time qpcr Introduction Wolbachia are maternally transmitted intracellular bacteria that infect a wide variety of insects and other arthropods, and manipulate the reproduction of their hosts. Wolbachiamediated reproductive alterations include cytoplasmic incompatibility (CI), male-killing, thelytokous parthenogenesis, and feminization (Stouthamer et al. 999). These reproductive phenotypes increase the number of Wolbachia-infected females or the relative fitness of the infected females against that of uninfected ones, thereby facilitating the spread of Wolbachia infection in the host population. One estimate suggested that approximately 40 % of insect species are infected with Wolbachia (Zug and Hammerstein 0), which would make Wolbachia among the most common endosymbionts in eukaryotes. Many Wolbachia strains carry temperate double-stranded DNA bacteriophages called WO. Diagnostic polymerase chain reaction (PCR) has suggested that more than 80 % of Wolbachia strains contain WO-related gene fragments (Gavotte et al. 007). This is notable because compared with free-living bacteria, intracellular symbiotic bacteria have fewer opportunities to encounter mobile genetic elements such as bacteriophages and transposons. In addition, the genome size of intracellular bacteria tends to decrease, and there may be selective pressures to delete repetitive or phage-related elements from the genome

2 40 Appl Entomol Zool (0) 47: (Frank et al. 00; Tamas et al. 00). Bacteriophages play multiple roles in the ecology and genome evolution of bacteria, for example, through their ability to mediate lateral gene transfer (Wommack and Colwell 000). Some bacteriophages are known to provide their host with beneficial genes (Brüssow et al. 004; Abedon and LeJeune 00). The wide spread distribution of WO phages despite the presence of reductive pressures implies that these phages may also carry genes beneficial to Wolbachia. Indeed, analysis of WO sequences has identified several ankyrin repeat and effector proteins that could interact with eukaryotic cells (Fujii et al. 004; Iturbe-Ormaetxe et al. 00; Sinkins et al. 00; Tanaka et al. 009). Temperate phages, including WO, have two replication pathways: lysogenic and lytic cycles. In the lysogenic cycle, phage DNA is integrated into the host genome in the form of a prophage. The prophage is passively replicated during the replication of the host genome. In the lytic cycle, the prophage is excised from the host genome, after which it replicates and produces phage particles that are released from the host cell to infect new host cells. Lytic and lysogenic phages potentially play separate roles in bacterial fitness: WO prophages may contain genes beneficial for Wolbachia, whereas their lytic replication may have deleterious effects on Wolbachia (Kent and Bordenstein 00). Bordenstein et al. (006) demonstrated a negative correlation between WO density and Wolbachia density in the parasitoid wasp, Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae), and a positive correlation between Wolbachia density and CI strength. Based on these observations, a phage density model of CI has been proposed in which lytic phage represses CI through a decrease in Wolbachia density as a result of cell lysis. To date, the lytic activity of WO has been estimated from real-time quantitative PCR (qpcr) of WO and Wolbachia genes (Bordenstein et al. 006; Biliske et al. 0; Bordenstein and Bordenstein 0). The WO gene is amplified from both the prophage integrated in the Wolbachia genome and the extrachromosomal phage genome in lytic replication. If the WO gene is in excess to the Wolbachia gene, this is considered to represent the phage genome in its lytic cycle. A more direct quantification of WO lytic activity would facilitate the analysis of infection dynamics of WO phages and provide further insights into the physiology and evolution of the tripartite association of WO, Wolbachia, and host insect. Two Wolbachia strains, wcaua and wcaub, naturally co-infect the almond moth Cadra cautella (Walker) (Lepidoptera: Pyralidae) and induce strong CI (Sasaki and Ishikawa 999). These Wolbachia stains were artificially transferred into the Mediterranean flour moth Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) in which they were segregated (Sasaki et al. 00). Fujii et al. (004) isolated WO particles from the transfected E. kuehniella lines and indicated that these Wolbachia strains contain complete WO prophages capable of producing particles. In addition, Tanaka et al. (009) determined the complete sequences of two prophages, WOCauB and WOCauB, from wcaub-infected E. kuehniella and also demonstrated that both ends of the prophage are ligated in the lytic cycle of WO, probably forming circular DNA. The ends of the prophage are called attr and attl sites, whereas the sequence created by the ligation of attl and attr is called attp site. The abundance of attp, which is present in the phage genome but not in the prophage, is expected to reflect the lytic activity of WO. In this study, real-time qpcr targeting the attp sequences was performed to estimate the lytic activities of the two WO phages in wcaub. Materials and methods Wolbachia-infected insects and cell lines E. kuehniella transfected with the Wolbachia strain wcaub (Sasaki et al. 00) and Wolbachia-uninfected E. kuehniella (Sasaki and Ishikawa 999) were grown on a diet consisting of wheat bran, dried yeast and glycerol (0:: w/w) at C under a 6-h light:8-h dark photoperiod. wcaub was transfected into two cell lines, Sf9 cells originating from Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) and NIAS-AeAl (AeAl) cells originating from Aedes albopictus (Skuse) (Diptera: Culicidae), using the shell vial technique (Dobson et al. 00; Furukawa et al. 008). The two cell lines were maintained at 8 C in 0 ml cell culture flasks containing IPL-4 medium (Gibco) supplemented with 0 % heat-inactivated fetal bovine serum (Gibco). Southern blot analysis DNA was extracted from the ovaries of wcaub-infected E. kuehniella by phenol extraction followed by ethanol precipitation. Approximately 8 lg of DNA was digested with restriction endonucleases, HpaI, HindIII, and ScaI, separated on a 0.8 % agarose gel, and transferred onto a GeneScreen Plus membrane (PerkinElmer). To prepare probes, flanking regions of WOCauB (AB478) and WOCauB (AB4786) were amplified by PCR. The primers used to amplify the flanking region of WOcauB (8 bp) were 0 -CAG CGC TAT AGT TAA AAG TTA CCG G- 0 and 0 -TCA ATC AAT CTT CTG TTA CCT CCA AAA A- 0, and those used to amplify the flanking region of WOcauB (8 bp) were 0 -CTG CAG ATA

3 Appl Entomol Zool (0) 47: AGT GTT GCT TAA TAG ACT G- 0 and 0 -GCT TGT AAT GCG CTA GGT AAA GAA TC- 0. The PCR products were labeled with Alkphos Direct Labeling Modules (GE Healthcare), and hybridization was performed according to the manufacturer s recommendations. The chemiluminescent image was analyzed in the Versa Doc imaging system (Bio-Rad). Preparation of DNA for real-time qpcr DNA was extracted from wcaub-infected and uninfected E. kuehniella, and wcaub-infected insect cell lines using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer s instructions. Each E. kuehniella DNA sample was prepared from a single individual. Cell line DNA was extracted from approximately 9 0 cells after the cells were maintained at the experimental temperatures for day. DNA was also extracted from phage particles partially purified from approximately g of wcaub-infected E. kuehniella adult moths according to the method of Fujii et al. (004). Real-time qpcr Real-time qpcr was performed targeting the attl and attp sites of WOCauB and WOCauB and targeting the 6S rrna gene of wcaub (AB608) in the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The 6S rrna gene was amplified using the primers, 0 - AAC TGA GAT ACG GTC CAG A- 0 and 0 -GAT CAG GCT TTC GCC CAT- 0, and the PCR product was detected using a fluorescent TaqMan probe, 0 -FAM-GGC AGC AGT GG*G GAA TA-TAMRA- 0 (G*, a modified nucleotide). The attl and attp sites were amplified using the primers shown in Fig. and Table. attl of WO- CauB was amplified using primers and, whereas that of WOCauB was amplified using primers 4 and. Primers and were used to amplify attp of WOCauB. Three attp sequences produced by WOCauB were amplified using primer and either primer 6, 7, or 8. All PCR products of the attl and attp sites were detected using a common probe, 0 -FAM-TTG ATC AAA ATA GCC ATA ATA GAA TTA TGC AAT GTT GTA C-TAMRA- 0, which hybridized to a conserved sequence at the attl ends of the two WO phages. The cycle conditions were an initial 0-min hold at 9 C followed by 0 cycles of 9 C for s, C for 0 s, and 7 C for min. The specific amplification of each target region was confirmed by agarose gel electrophoreses, in which a single band of expected size was detected. The amounts of target sequences were determined using dilutions of the cloned plasmids containing the PCR products as standard samples. The amounts of attp relative to those of attl were log transformed and analyzed by two-way analysis of variance (ANOVA) following Bartlett s test for equal variance. Results Analysis of prophages WOCauB has a 4,06-bp genome encoding 47 predicted open reading frames (ORFs), and WOCauB has a a att P b a b c att P att L att R att L att R 8 Hp Hi Hi Hi Hp Sc Hp Hp Sc. 9.4 Fig. Schematic structure of WOCauB (a) and WOCauB (b). In the lytic cycle, both ends of the prophage are ligated to form the attp sequence in the phage genome. Three types of phage genomes are produced by WOCauB. Gray boxes indicate the probes used in Southern hybridization. Restriction endonuclease sites around the probe region are also shown (Hp, HpaI; Hi, HindIII; Sc, ScaI). Arrows with numbers represent the primers used in real-time quantitative polymerase chain reaction, the sequences of which are listed in Table. Bidirectional arrows indicate the regions from which the TaqMan probe was designed

4 bcopies relative to 4 Appl Entomol Zool (0) 47: Table Polymerase chain reaction primers used to amplify the attl and attp sequences of WOcauB and WOcauB Primer 0 -CTCTCTGGATTCTGCAACAGTT- 0 Primer 0 -TCACTTGTATATAGTATTTCGCATAAAAAGTC- 0 Primer 0 -TCATCATTTCTCTTTGTTGTTTTCAAA- 0 Primer 4 0 -CGAAATGGCTACTGTTGTATACTTACA- 0 Primer 0 -TCACTTGTATATAGTATTTCGCATAAAAAGTC- 0 Primer 6 0 -GAAAAAGAATTTCGTGGATTTGGT- 0 Primer 7 0 -AAAGGGTTTTTCAGCAAAGTAGAATTT- 0 Primer 8 0 -AGTCAGTCTTAAGTCCAGCGGC- 0 a 6s rrna gene c att L att P att P (purified) Copies relative to 6s rrna gene att L a b c a b c att P att P (purified) Fig. Real-time quantitative polymerase chain reaction targeting attl and attp. a DNA extracted from purified WO phage particles. attl and attp of WOCauB (b) and WOCauB (c) were quantified in the whole tissues of wcaub-infected Ephestia kuehniella larvae. The amount of the target sequence relative to that of the rrna gene is expressed as the mean ± SD (n = 4). attp was also quantified in the purified WO particles 4,078-bp genome encoding 46 predicted ORFs (Tanaka et al. 009). An integrase gene is encoded at one end of each prophage, and this end is tentatively called attl (Fig. ). Southern blot analysis using probes that hybridized to specific sequences on the two prophages suggested that each prophage was present at its unique site on the wcaub genome (Tanaka et al. 009). However, whether the entire wcaub population in E. kuehniella or part of the population contains the prophages remained unknown. Prior to quantifying the attp sequences of the phages, real-time qpcr targeting the attl sites was performed using DNA extracted from the whole tissues of wcaubinfected E. kuehniella larvae to examine whether the two prophages are uniformly present in wcaub populations. The amounts of attl sites were normalized by those of the rrna gene, i.e., a single-copy gene in the wcaub genome (Furukawa et al. 008). The relative amounts of attl sites were approximately.0, suggesting that almost all wcaub cells carry the prophages (Fig. ). The uniform presence of the two prophages was also confirmed by Southern blot analyses using probes that hybridized to flanking regions of the prophages. The sizes of detected bands were consistent with those expected from the restriction enzyme sites around the attl sites (Figs., ). Signals that might be derived from the wcaub genome without prophages were not detected. Amplification of the attp sequence of WOCauB In the lytic cycle, attl and attr are ligated to form an attp site in the phage genome. To amplify the attp sequence of WOCauB, primers that hybridize to both ends of the prophage in an outward direction were designed (Fig. ). Diagnostic PCR using the primers amplified a DNA fragment of the expected size, and its identity was confirmed by sequencing. To perform real-time qpcr targeting the attp, DNA samples were prepared from wcaub-infected E. kuehniella larvae, WO particles purified from infected insects (Fig. a), and Wolbachia-uninfected larvae. The attp sequence was detected in the infected larvae by real-time qpcr, although the amount relative to that of the rrna gene was lower than (Fig. b). When DNA from the partially purified phage particles was used, the amount of attp relative to that of the rrna gene was 49.0 (Fig. b). attp was not detected in the uninfected larvae (data not shown). These results indicate that attp of the phage genome is successfully quantified by the qpcr technique. Amplification of the attp sequences of WOCauB At least three different phage genomes are produced from the WOCauB prophage, and these are tentatively called WOCauB-a, -b, and -c. These three types are derived from

5 Appl Entomol Zool (0) 47: a 0 WOCauB 0 recombination between an attl site and three different attr sites (Tanaka et al. 009). To quantify each of the three WOCauB genomes, three primer sets were designed: a common primer specific to the attl region and three primers that hybridize to each attr region (Fig. ). These primer sets amplified DNA fragments of expected sizes, and their identities were confirmed by sequencing. All the three types were detected in the infected larvae by real-time qpcr. Their abundance relative to that of the rrna gene was \ (Fig. c). The relative abundance was elevated by more than fold when DNA extracted from the partially purified phage particles was used. None of the three types was amplified from the uninfected larvae (data not shown). Quantification of the attp sequences in wcaub-infected E. kuehniella The abundances of the four attp sequences were measured in larvae, pupae, and adults of E. kuehniella, and also in the gonads of adults (Fig. 4). WOCauB was consistently the most abundant type in all the experimental groups, whereas WOCauB-b was the least abundant. Some differences in the attp titers were observed between different experimental groups. However, the amount of attp relative to that of attl was constantly lower than Two-way ANOVA (F: attp type, F: insect tissue) revealed significant differences in both the attp type (F =.7, p \ 0.00) and insect tissue (F =., p \ 0.00) without a significant interaction of the two factors (F =.4, p = 0.). b WOCauB Fig. Southern blot analyses of the flanking regions of the WOCauB (a) and WOCauB (b) prophages in the wcaub genome. Restriction endonucleases used are given at the top, and probes are shown in Fig. Relative amount of att P sequence (x 0-4 ) 0 abc Female Larva abc abc abc abc abc Male abc Female Male Female Male Ovary Testis Pupa Adult Gonad Quantification of the attp sequences in wcaub-infected insect cell lines abc Fig. 4 Measurement of attp sequences in the tissues of wcaubinfected Ephestia kuehniella by real-time quantitative polymerase chain reaction. The amount of attp relative to that of attl is shown as the mean ± SD (n = 4). The four types of attp sequences are indicated at the bottom of each column: WOCauB, a WOCauB-a, b WOCauB-b, and c WOCauB-c Relative amount of attp sequence (x 0 - ) abc abc abc abc abc abc 8 C 8 C 7 C 8 C 8 C 7 C SF9 AeAl Fig. Measurement of attp sequences in two wcaub-infect insect cell lines, Sf9 and AeAl cells, maintained at different temperatures. The amount of attp relative to that of attl is shown as the mean ± SD (n = 4). The four types of attp sequences are indicated at the bottom of each column: WOCauB, a WOCauB-a, b WOCauB-b, and c WOCauB-c To further examine the effects of the environment of host cells on the lytic activity of WO, the four attp sequences were quantified in two wcaub-infected insect cell lines, Sf9 cells derived from S. frugiperda and AeAl cells from A. albopictus (Fig. ). The lytic activities in the cell lines were much lower than in E. kuehniella: the relative amount of attp did not exceed The pattern of attp production in the cell lines was remarkably different from that in E. kuehniella in that the WOCauB-a type was consistently the most abundant type in the cell lines.

6 44 Appl Entomol Zool (0) 47: The effect of temperature on the induction of lytic cycles was also examined by maintaining the cells at three different temperatures: 8, 8, and 7 C. Two-way ANOVA (F: attp type, F: incubation temperature) revealed significant differences (F =., p \ 0.00 and F = 7.0, p \ 0.00) without a significant interaction in Sf9 cells. In AeAl cells, the differences were significant (F =.4, p \ 0.00 and F = 9.7, p \ 0.00) and there was a significant interaction (F =.8, p \ 0.0). Discussion The lytic activity of WO phages was measured by real-time qpcr targeting the attp sequences exclusively present in the phage genome excised from the prophage. Lytic activities of temperate phages are usually measured by plaque assay using host cells. However, the plaque assay is not applicable to WO phages because Wolbachia is unculturable outside the insect cells. In wcaub, at least four different WO genomes are produced by two prophages, and they were separately quantified using type-specific PCR primers. This is the first report of type-specific quantification of the WO genome without the effect of confounding the prophage. This method enables quantification of even very small amounts of WO genome. Previous analysis of the WO genome extracted from purified phage particles suggested that the genome consists of double-stranded linear DNA (Fujii et al. 004). To date, the process by which the mature WO genome is produced has not been elucidated. In the lytic cycle of other temperate phages such as the k phage of Escherichia coli, the prophage is excised from the bacterial chromosome, forms a circular DNA and subsequently replicates through rolling-circle amplification to give rise to tandemly repeated phage genomic DNA, which is excised into a number of mature k linear DNA genomes (Campbell 00). In the process of lytic replication of WO, it is expected that circular DNA is produced by the ligation of both ends of the WO prophage to form the attp sequence (Tanaka et al. 009). The attp sequences detected in this study are possibly derived from the mature phage genomes, the circular DNA after prophage excision, and other forms that might be produced during lytic replication. The production patterns of the four attp sequences in E. kuehniella differed from those in insect cell lines: the most abundant attp in E. kuehniella was WOCauB (Fig. 4) and that in the cell lines was WOCauB-a (Fig. ). These observations suggest that the intracellular environment in which Wolbachia is harbored affects the lytic activity of WO, although the conditions that up- and downregulate the lytic pathway are unclear. Temperate phages often enter the lytic cycle when the hosts encounter stresses such as starvation, heat shock, and DNA damage (Young 99). Wolbachia is known to be excluded from spermatids during sperm maturation (Binnington and Hoffmann 989). Exclusion from the host cells may also occur during pupal development when larval tissues are degenerated, following which adult tissues are formed. We thus hypothesized that the lytic cycle of WO phages could be activated in testes and pupae in which Wolbachia released from the host cell possibly encounters stress. However, no increase of the attp was observed either at the pupal stage or in the testes of adult males. A recent study demonstrated that temperature changes have a marked effect on the induction of WO phage in N. vitripennis, in which both cold and heat treatments activated the lytic cycle (Bordenstein and Bordenstein 0). Such effects of temperature shifts, however, were not observed in wcaub-infected insect cell lines, although the attp titer of WOcauB-a in Sf9 cells was slightly higher at 8 C than at 8 C (Fig. ). This might be due to differences between insects and cell lines. The temperature changes may affect the lytic activity of WO indirectly rather than directly through the physiology of the host insect. It is also possible that the responses of WO phages to temperature shifts vary between WO-Wolbachia associations. The amount of each attp sequence relative to that of attl was \ in all measurements in the present study. Considering that multiple copies of phage genome are produced by a single prophage, the number of prophages that entered the lytic cycle must be even lower than that of the attp sequences detected. The weak lytic activity of these WO phages appears to be relevant to the fact that their host is an intracellular bacterium. A phage associated with free-living bacteria can spread its infection to new hosts by repeating lytic cycles. Conversely, the spread of WO phages is limited by the intracellular life of Wolbachia. Even if the WO particles are released from their host bacterial cells, they encounter other Wolbachia cells only in the same insect individual or possibly even in the same insect cell. Frequent repeats of lytic events of WO within a limited population of Wolbachia may extinguish the host population, which in turn results in extinction of WO itself. Under such conditions, being latent as a prophage would be a preferable strategy, in agreement with a general view that infectious elements that have less opportunity of finding new hosts express weak virulence and tend to establish a long-term association with their hosts. However, there are selective pressures that cause prophage degradation. Prophages are fragmented through recombination and/or deletion and are transformed into cryptic prophages incapable of producing particles (Lawrence et al. 00). Indeed, putative cryptic prophages have

7 Appl Entomol Zool (0) 47: been found in several Wolbachia strains (Wu et al. 004; Klasson et al. 008, 009; Kent et al. 0a). Horizontal transmission through the lytic pathway would therefore be necessary for the survival of inducible WO phages. Although Wolbachia is basically transmitted vertically from a female host to her offspring, it occasionally moves horizontally between different host species (Vavre et al. 999; Jamnongluk et al. 00). In addition, many insects carry multiple Wolbachia strains (Werren et al. 99; Werren and Windsor 000; Kikuchi and Fukatsu 00). Co-infections within a single host probably provide an opportunity for the lateral transfer of WO phages between Wolbachia strains. Analysis of orf7 encoding a minor capsid protein of WO has suggested the exchange of WO phages between co-infecting Wolbachia strains in several insect species (Masui et al. 000; Bordenstein and Wernegreen 004; Chafeeetal.009). Furthermore, a recent study using high-throughput sequencing indicated a transfer of the whole WO prophage between Wolbachia strains co-infecting N. vitripennis (Kentetal.0b). A new tripartite association of WO, Wolbachia, andahost insect would be established by lateral transfer of WO between co-infecting Wolbachia strains followed by horizontal infection of the Wolbachia between insect species. In this study, we quantified attp sequences using primers that hybridize to both ends of the prophage in an outward direction. This method may be applicable to other prophages with determined full-length sequences. In addition to WOCauB and WOCauB, evidence for the particle formation of particular WO prophages has been provided only for WOVitA of wvita in N. vitripennis (Bordenstein et al. 006) and WORiC of wri in the Riverside strain of Drosophila simulans (Sturtevant) (Diptera: Drosophilidae) (Biliske et al. 0). The method of detecting attp would be helpful for determining which of the diverse prophage elements are capable of producing particles, measuring their lytic activities, and also further investigating the phage density model of Wolbachia-mediated reproductive phenotypes. Acknowledgments This work was supported by the Grand Challenges in Global Health program Modifying mosquito population age structure to eliminate dengue transmission from the National Institutes of Health, USA. References Abedon ST, LeJeune JT (00) Why bacteriophage encode exotoxins and other virulence factors. Evol Bioinforma Online :97 0 Biliske JA, Batista PD, Grant CL, Harris HL (0) The bacteriophage WORiC is the active phage element in wri of Drosophila simulans and represents a conserved class of WO phages. BMC Microbiol : Binnington KC, Hoffmann AA (989) Wolbachia-like organisms and cytoplasmic incompatibility in Drosophila simulans. J Invertebr Pathol 4:44 Bordenstein SR, Bordenstein SR (0) Temperature affects the tripartite interactions between bacteriophage WO, Wolbachia, and cytoplasmic incompatibility. PLoS ONE 6:e906 Bordenstein SR, Wernegreen JJ (004) Bacteriophage flux in endosymbionts (Wolbachia): infection frequency, lateral transfer, and recombination rates. Mol Biol Evol :98 99 Bordenstein SR, Marshall ML, Fry AJ, Kim U, Wernegreen JJ (006) The tripartite associations between bacteriophage, Wolbachia, and arthropods. PLoS Pathog :e4 Brüssow H, Canchaya C, Hardt WD (004) Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev 68:60 60 Campbell A (00) The future of bacteriophage biology. Nat Rev Genet 4: Chafee ME, Funk DJ, Harrison RG, Bordenstein SR (009) Evolutionary genomics of a temperate bacteriophage in an obligate intracellular bacteria (Wolbachia). PLoS ONE 6:e4984 Dobson SL, Marsland EJ, Veneti Z, Bourtzis K, O Neill SL (00) Characterization of Wolbachia host cell range via the in vitro establishment of infections. Appl Environ Microbiol 68: Frank AC, Amiri H, Andersson SGE (00) Genome deterioration: loss of repeated sequences and accumulation of junk DNA. Genetica : Fujii Y, Kubo T, Ishikawa H, Sasaki T (004) Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont. Biochem Biophys Res Commun 7:8 88 Furukawa S, Tanaka K, Fukatsu T, Sasaki T (008) In vitro infection of Wolbachia in insect cell lines. Appl Entomol Zool 4:9 Gavotte L, Henri H, Stouthamer R, Charif D, Charlat S, Boulétreau M, Vavre F (007) A survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia. Mol Biol Evol 4:47 4 Iturbe-Ormaetxe I, Burke GR, Riegler M, O Neill SL (00) Distribution, expression, and motif variability of ankyrin domain genes in Wolbachia pipientis. J Bacteriol 87:6 4 Jamnongluk W, Kittayapong P, Baimai V, O Neill SL (00) Wolbachia infections of tephritid fruit flies: molecular evidence for five distinct strains in a single host species. Curr Microbiol 4: 60 Kent BN, Bordenstein SR (00) Phage WO of Wolbachia: lambda of the endosymbiont world. Trends Microbiol 8:7 8 Kent BN, Funkhouser LJ, Setia S, Bordenstein SR (0a) Evolutionary genomics of a temperate bacteriophage in an obligate intracellular bacteria (Wolbachia). PLoS ONE 6:e4984 Kent BN, Salichos L, Gibbons JG, Rokas A, Newton IL, Clark ME, Bordenstein SR (0b) Complete bacteriophage transfer in a bacterial endosymbiont (Wolbachia) determined by targeted genome capture. Genome Biol Evol :09 8 Kikuchi Y, Fukatsu T (00) Diversity of Wolbachia endosymbionts in heteropteran bugs. Appl Environ Microbiol 69: Klasson L, Walker T, Sebaihia M, Sanders MJ, Quail MA, Lord A, Sanders S, Earl J, O Neill SL, Thomson N, Sinkins SP, Parkhill J (008) Genome evolution of Wolbachia strain wpip from the Culex pipiens group. Mol Biol Evol : Klasson L, Westberg J, Sapountzis P, Näslund K, Lutnaes Y, Darby AC, Veneti Z, Chen L, Braig HR, Garrett R, Bourtzis K, Andersson SG (009) The mosaic genome structure of the Wolbachia wri strain infecting Drosophila simulans. Proc Natl Acad Sci USA 06:7 70 Lawrence JG, Hendrix RW, Casjens S (00) Where are the pseudogenes in bacterial genomes? Trends Microbiol 9: 40 Masui S, Kamoda S, Sasaki T, Ishikawa H (000) Distribution and evolution of bacteriophage WO in Wolbachia, the endosymbiont causing sexual alterations in arthropods. J Mol Evol :49 497

8 46 Appl Entomol Zool (0) 47: Sasaki T, Ishikawa H (999) Wolbachia infections and cytoplasmic incompatibility in the almond moth and the Mediterranean flour moth. Zool Sci 6: Sasaki T, Kubo T, Ishikawa H (00) Interspecific transfer of Wolbachia between two lepidopteran insects expressing cytoplasmic incompatibility: a Wolbachia variant naturally infecting Cadra cautella causes male killing in Ephestia kuehniella. Genetics 6: 9 Sinkins SP, Walker T, Lynd AR, Steven AR, Makepeace BL, Godfray HC, Parkhill J (00) Wolbachia variability and host effects on crossing type in Culex mosquitoes. Nature 46:7 60 Stouthamer R, Breeuwer JA, Hurst GD (999) Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu Rev Microbiol :7 0 Tamas I, Klasson L, Canbäck B, Näslund AK, Eriksson AS, Wernegreen JJ, Sandström JP, Moran NA, Andersson SG (00) 0 million years of genomic stasis in endosymbiotic bacteria. Science 96:76 79 Tanaka K, Furukawa S, Nikoh N, Sasaki T, Fukatsu T (009) Complete WO phage sequences reveal their dynamic evolutionary trajectories and putative functional elements required for integration into the Wolbachia genome. Appl Environ Microbiol 7: Vavre F, Fleury F, Lepetit D, Fouillet P, Boulétreau M (999) Phylogenetic evidence for horizontal transmission of Wolbachia in host parasitoid associations. Mol Biol Evol 6:7 7 Werren JH, Windsor D (000) Wolbachia infection frequencies in insects: evidence of a global equilibrium? Proc R Soc Lond B 67:77 8 Werren JH, Windsor D, Guo L (99) Distribution of Wolbachia among neotropical arthropods. Proc R Soc Lond B 6:97 04 Wommack KE, Colwell RR (000) Virioplankton: viruses in aquatic ecosystems. Microbiol Mol Biol Rev 64:69 4 Wu M, Sun LV, Vamathevan J, Riegler M, Deboy R, Brownlie JC, McGraw EA, Martin W, Esser C, Ahmadinejad N, Wiegand C, Madupu R, Beanan MJ, Brinkac LM, Daugherty SC, Durkin AS, Kolonay JF, Nelson WC, Mohamoud Y, Lee P, Berry K, Young MB, Utterback T, Weidman J, Nierman WC, Paulsen IT, Nelson KE, Tettelin H, O Neill SL, Eisen JA (004) Phylogenomics of the reproductive parasite Wolbachia pipientis wmel: a streamlined genome overrun by mobile genetic elements. PLoS Biol :E69 Young R (99) Bacteriophage lysis: mechanism and regulation. Microbiol Rev 6:40 48 Zug R, Hammerstein P (0) Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS ONE 7:e844