Insect Molecular Biology bs_bs_banner Insect Molecular Biology (2015) 24(1), 1–12 doi: 10.1111/imb.12128 How hosts react to endosymbionts? A new insight into the molecular mechanisms underlying the Wolbachia–host association Y.-K Zhang, X.-L Ding, X Rong and X.-Y Hong Introduction Department of Entomology, Nanjing Agricultural University, Nanjing, China Bacterial intracellular symbiosis is widespread in invertebrates Many intracellular bacteria are known to influence host biological processes, including developmental programmes, reproduction and immunity (Duron et al., 2008; Vallet-Gely et al., 2008; Tsuchida et al., 2010; Ivanov & Littman, 2011) The bacterium Wolbachia is perhaps the most abundant vertically transmitted microbe worldwide, infecting an estimated 40% of terrestrial arthropods (Zug & Hammerstein, 2012) Interestingly, Wolbachia can induce a range of reproductive manipulations in arthropods that facilitate vertical transmission (Werren et al., 2008) In addition to their effects on host reproduction, Wolbachia have mutualistic relationships with nematodes (Taylor et al., 2013) and increase the resistance of mosquitoes and flies to various pathogens, such as dengue fever virus, Chikungunya virus and yellow fever virus (Hedges et al., 2008; van den Hurk et al., 2012) Many studies have examined the phenotypic effects of Wolbachia infection on host physiology and immunity Recent studies have begun to clarify the molecular mechanisms underlying these effects In Drosophila melanogaster, the finding that most of the genes in larval testes putatively associated with reproduction (especially spermatogenesis) were downregulated by Wolbachia may help elucidate the underlying mechanisms of Wolbachia-induced cytoplasmic incompatibility (CI) (Zheng et al., 2011) In the wasp Asobara tabida, Wolbachia is required for oogenesis (Dedeine et al., 2005), possibly because of its interference with the expression of ferritin (Kremer et al., 2009, 2012) In the fruit flies D melanogaster and Drosophila simulans, Wolbachia confers resistance against RNA viral infection (Hedges et al., 2008; Teixeira et al., 2008; Osborne et al., 2009) In the mosquito Aedes aegypti, Wolbachia confers resistance against various pathogens notably by priming the innate immune system (Moreira et al., 2009; Bian et al., 2010) and affects the expression of microRNAs Abstract Wolbachia is an intracellular bacterium that has aroused intense interest because of its ability to alter the biology of its host in diverse ways In the twospotted spider mite, Tetranychus urticae, Wolbachia can induce complex cytoplasmic incompatibility (CI) phenotypes and fitness changes, although little is known about the mechanisms In the present study, we selected a strain of T urticae, in which Wolbachia infection was associated with strong CI and enhanced female fecundity, to investigate changes in the transcriptome of T urticae in Wolbachia-infected vs uninfected lines The responses were found to be sexspecific, with the transcription of 251 genes being affected in females and 171 genes being affected in males Some of the more profoundly affected genes in both sexes were lipocalin genes and genes involved in oxidation reduction, digestion and detoxification Several of the differentially expressed genes have potential roles in reproduction Interestingly, unlike certain Wolbachia transinfections in novel hosts, the Wolbachia–host association in the present study showed no clear evidence of host immune priming by Wolbachia, although a few potential immune genes were affected Keywords: two-spotted spider mite, Wolbachia, transcriptome sequencing, gene expression First published online 15 September 2014 Correspondence: Prof X.-Y Hong, Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China Tel & fax: 0086 25 84395339; e-mail: xyhong@njau.edu.cn © 2014 The Royal Entomological Society Y.-K Zhang et al (Hussain et al., 2011; Zhang et al., 2013) Taken together, these findings clearly indicate that Wolbachia can influence host gene transcription in ways that increase its own survival Because the effects of Wolbachia on host biology are strain- and host-specific (Serbus et al., 2008), studies of additional invertebrate systems are needed to unravel the conserved and diverged mechanisms in host–Wolbachia interactions The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and strong pesticide resistance (Bolland et al., 1998) Its genome, at 90 megabases, is the smallest sequenced arthropod genome (Grbic´ et al., 2011) It is also the first complete genome among the Chelicerates, which diverged from other arthropod lineages more than 450 Mya (Dunlop, 2010) Wolbachia is widely distributed in T urticae, in which it can induce anything from no CI to complete CI (Breeuwer, 1997; Perrot-Minnot et al., 2002; Vala et al., 2002; Gotoh et al., 2007) Wolbachia can also affect the fitness of T urticae For example, in mites collected from cucumber plants, the offspring of Wolbachiainfected females had higher survival rates (Vala et al., 2003), and in some Chinese populations, Wolbachia infection increased female fecundity (Xie et al., 2011; Zhao et al., 2013) Although gene expressions in T urticae are affected by host plant transfer and diapause (Dermauw et al., 2012; Bryon et al., 2013), little is known about how they are affected by Wolbachia infection In the present study, we explored the effects of Wolbachia on the T urticae transcriptome by comparing gene expression profiles in infected and uninfected adult mites of a strain whose CI and fecundity are strongly affected by Wolbachia (Zhao et al., 2013) Wolbachia was found to affect the expressions of many genes, including genes involving oxidation reduction, digestion detoxification and reproduction These results provide new insights for an understanding of the complex interactions between arthropods and Wolbachia Results Sequence data processing We sequenced four transcriptome libraries (Turt_FI, Turt_FU, Turt_MI and Turt_MU), each with between 55 and 66 million reads About 79% of the reads in each group were uniquely mapped to the reference genome (Table 1) A total of 18 813 genes, including 18 204 known T urticae genes and 519 novel genes, were detected in the four libraries To better evaluate the transcriptional status between libraries, the gene expression levels were divided into five grades according to their reads per kilobase of exon model per million mapped reads (RPKM) values Results indicated that most genes were expressed at low levels in all libraries, samples from the same sex had a similar gene expression pattern at each RPKM interval (Fig 1) Transcriptional responses to Wolbachia infection A global view of gene expressions in the four libraries is presented in the hierarchical clustering heat map in Fig 2A As shown, gene expressions are sex-specific Comparison of Turt_MI and Turt_MU and comparison of Turt_FI and Turt_FU show that each library has unique transcriptional changes, suggesting that the expressions of many genes are affected by Wolbachia infection, although we cannot exclude non-Wolbachia related genetic differences between lines that may have accumulated during the antibiotic curing and selection process, or antibiotic-mediated changes in gut bacterial composition In females, 148 genes were observed to be upregulated and 103 downregulated in the Wolbachia-infected line compared with the Wolbachia-uninfected line, while in males, 96 genes were upregulated and 75 downregulated in the Wolbachia-infected line In addition, 82 genes were differentially expressed in both females and males compared with uninfected ones (Fig 2B, Table S2A, S2B) Table Summary of sequencing results of Tetranychus urticae transcriptome Sample Name Turt_FI (%) Turt_FU (%) Turt_MI (%) Turt_MU (%) Total reads Total mapped Multiple mapped Uniquely mapped Read-1 Read-2 Reads map to ‘+’ Reads map to ‘−’ Non-splice reads Splice reads Reads mapped in proper pairs 56393360 47826531 (84.81) 2896665 (5.14) 44929866 (79.67) 22621025 (40.11) 22308841 (39.56) 22474530 (39.85) 22455336 (39.82) 38018379 (67.42) 6911487 (12.26) 38205728 (67.75) 56578458 44722961 (79.05) 2475089 (4.37) 42247872 (74.67) 21268311 (37.59) 20979561 (37.08) 21157983 (37.4) 21089889 (37.28) 36076226 (63.76) 6171646 (10.91) 36316772 (64.19) 55414202 46128185 (83.24) 2538586 (4.58) 43589599 (78.66) 21929817 (39.57) 21659782 (39.09) 21803649 (39.35) 21785950 (39.31) 35940342 (64.86) 7649257 (13.8) 38504308 (69.48) 66380956 55360502 (83.4) 3241915 (4.88) 52118587 (78.51) 26218097 (39.5) 25900490 (39.02) 26084424 (39.3) 26034163 (39.22) 42856016 (64.56) 9262571 (13.95) 45548706 (68.62) Turt_FI, infected females; Turt_FU, uninfected females; Turt_MI, infected males; Turt_MU, uninfected males © 2014 The Royal Entomological Society, 24, 1–12 Wolbachia affect host gene expression Figure Distribution of Tetranychus urticae unigenes The y-axis represents gene number; the x-axis represents reads per kilobase of exon model per million mapped reads (RPKM) range of genes Turt_FI, infected females; Turt_FU, uninfected females; Turt_MI, infected males; Turt_MU, uninfected males Figure Tetranychus urticae transcriptional responses to Wolbachia infection A Hierarchical clustering heat map of the gene abundance in the four samples Colours from blue to red represent the gene expression abundance from poor to rich B Venn diagram showing significant gene expression change in response to Wolbachia infection in T urticae females and males Turt_FI, infected females; Turt_FU, uninfected females; Turt_MI, infected males; Turt_MU, uninfected males © 2014 The Royal Entomological Society, 24, 1–12 Y.-K Zhang et al In females, the strongest changes in the Wolbachiainfected line were in the biological process gene ontology (GO) categories of the oxidation-reduction process, the chlorophyll metabolic process, virus–host interaction, interaction with the host and pigment metabolic process (Fig 3A) and in the following molecular function GO categories: carboxylic ester hydrolase activity; iron cluster binding; sulphur cluster binding; and oxidoreductase activity In males, the strongest changes were in the following GO categories of the oxidation-reduction process: iron cluster binding; sulfur cluster binding; and oxidoreductase activity (Fig 3B) Interestingly, no GO categories related to host immune priming were found to be affected in the Wolbachia-infected line in adults of either sex, although a few potential immune genes were affected Other GO categories of differentially expressed genes in females and males are listed in Table S3A and S3B, respectively genes, tetur13g00510 and tetur03g01690, which are involved in chromatin assembly, were upregulated and downregulated, respectively Remarkably, seven genes encoding cuticular protein were all upregulated (Table 2), possibly reflecting disruption of embryogenesis The expressions of 64 putative immunity-related genes involved in the Toll, Imd/Jnk and JAK-STAT pathways were not significantly affected in the Wolbachia-infected line The GO enrichment analysis confirmed these results Three cystatin genes (tetur06g06620, tetur09g03670 and tetur09g04770) were differentially expressed in females The former was downregulated whereas the latter two were upregulated Another cystatin gene (tetur06g01060) was downregulated in males Two of the genes in the autophagy pathway (tetur07g07470 and tetur24700010) encode ATG4 autophagy related homologue A The former was upregulated in box sexes and the latter was downregulated (Table 2) Kyoto Encyclopaedia of Genes and Genomes pathway analysis Quantitative real-time PCR validation In females, genes that were differentially expressed in the Wolbachia-infected line were involved in 44 Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways, mainly involving lysosome process, phenylalanine metabolism and valine, leucine and isoleucine degradation (Table S4A) In males the differentially expressed genes were involved in 45 KEGG pathways, mainly involving fatty acid metabolism, β-alanine metabolism, propanoate metabolism and lysosome process (Table S4B) Differentially expressed genes of interest Tetranychus urticae and other spider mites are known to express several genes for detoxifying enzymes, including cytochrome P450 monooxygenases (CYPs), glutathioneS-transferases and ABC transporters Wolbachia affected the expression of seven CYPs in females and five CYPs in males (Table 2) Most were downregulated Two glutathione-S-transferase genes were downregulated and five ABC transporter genes were upregulated Lipocalins, which are small proteins capable of binding to hydrophobic molecules, were mostly downregulated (eight of ten in females, five of six in males; Table 2) All the differentially expressed ribosomal proteins were downregulated in females The expression of many genes of unknown function changed dramatically in females (Table S2A) and in males (Table S2B) The products of many of these genes are predicted to be secreted or conserved hypothetical proteins Among genes potentially associated with oogenesis or embryogenesis, the gene for LKAP32 limkain-b1 (tetur11g00940), which interferes with meiosis, and two genes encoding vitellogenin (tetur20g01230, tetur39g00740), were upregulated Two Eighteen differentially expressed genes (ten in females, eight in males) were randomly selected to validate the expression profiles obtained with the RNA-Seq analysis All of them yielded PCR products, whose sequences matched the RNA-Seq generated sequences perfectly The changes in expression of all but two of the genes (tetur11g00940 and tetur04g02680) were in good agreement with the RNA-Seq results Among these genes in females, tetur13g00510 showed the largest upregulation and tetur97g00020 manifested the largest downregulation, and tetur26g01450 showed the largest downregulation in males, which was consistent with the RNA-seq results (Fig 4) These results demonstrate the reliability of the RNA-Seq results Discussion Wolbachia widely infects arthropods and can have important consequences for the fitness of their hosts Wolbachia can interact with their invertebrate hosts at both the molecular and cellular levels (Xi et al., 2008; Kremer et al., 2009, 2012; Yamada et al., 2011) These findings, together with the sequencing of genomes of Wolbachia strains that induce various phenotypic effects (Wu et al., 2004; Klasson et al., 2008; Salzberg et al., 2009; Darby et al., 2012) have greatly clarified the evolving relationship between Wolbachia and their hosts Although Wolbachia has various phenotypic effects on T urticae, the mechanisms are not well understood In the present study, we selected a strain of T urticae in which Wolbachia induces strong CI and increases female fecundity (Zhao et al., 2013) to investigate its responses to Wolbachia infection Wolbachia increases the fecundity of © 2014 The Royal Entomological Society, 24, 1–12 Figure Gene ontology (GO) enrichment analysis of differentially expressed genes in females (A) and males (B) The 30 most enriched GO terms are shown Asterisks indicate significantly enriched GO terms (P < 0.05) Wolbachia affect host gene expression © 2014 The Royal Entomological Society, 24, 1–12 Y.-K Zhang et al Table Candidate genes differentially expressed in response to Wolbachia infection in Tetranychus urticae Female Male Gene category Gene ID Description log2 (FC) P value log2 (FC) P value Digestion or Detoxification tetur03g00830 tetur03g04990 tetur03g05030 tetur03g05100 tetur03g05110 tetur03g09941 tetur05g04000 tetur06g02400 tetur25g02060 tetur26g01470 tetur26g01450 tetur26g01460 tetur06g00360 tetur09g01950 tetur09g04610 tetur19g01710 tetur19g01730 tetur01g05740 tetur04g06010 tetur06g02130 tetur06g02140 tetur06g02940 tetur09g04720 tetur24g01030 tetur31g00680 tetur31g00710 tetur31g00900 tetur31g00920 tetur06g01060 tetur06g06620 tetur09g03670 tetur09g04770 tetur07g07470 tetur247g00010 tetur03g01690 tetur11g00940 tetur13g00510 tetur20g01230 tetur39g00740 tetur01g00130 tetur01g12840 tetur04g01580 tetur04g01610 tetur09g06230 tetur11g00600 tetur12g02060 tetur10g04620 Cytochrome P450-CYP392A12 Cytochrome P450-CYP392D2 Cytochrome P450-CYP392D6 Cytochrome P450-CYP392Dn Cytochrome P450-CYP392Dn Cytochrome P450-CYP392A15 Cytochrome P450-CYP385B1 Cytochrome P450-CYP392E2 Cytochrome P450-CYP389B1 Cytochrome P450-CYP385C1 Glutathione S-transferase class delta Glutathione S-transferase class delta ABC-transporter class C ABC-transporter class G ABC-transporter class C ABC-transporter G family member 23 ABC-transporter G family member 20 Apolipoprotein D precursor Apolipoprotein D precursor Apolipoprotein D Apolipoprotein D Apolipoprotein D related protein Apolipoprotein D precursor Apolipoprotein D Apolipoprotein D precursor Apolipoprotein D probably a pseudogene Apolipoprotein D precursor Apolipoprotein D precursor L-Cystatin Cystatin L-Cystatin L-Cystatin ATG autophagy related homolog A ATG autophagy related homolog A Hypothetical protein LKAP32 limkain-b1 Histone H2B Vitellogenin Vitellogenin1 Cuticle protein Cuticle protein Hypothetical Cuticular Protein Cuticle (secreted) protein, putative Cuticle protein Cuticle protein Cuticle protein Juvenile hormone binding protein −1.06 −1.64 1.21 −2.27 – −1.63 −3.54 2.54 – – −6.68 −5.9 1.05 2.1 – – – −1 −3.45 5.03 5.39 −1.71 – −1.6 −6.54 −2.34 −2.18 −2.22 – −2.16 2.06 1.27 4.33 −2.27 −2.81 6.24 5.63 1.33 2.20 1.23 1.93 1.95 1.79 2.74 1.16 1.39 – −1.71 – – −1.16 −1.03 – – – 4.89 −1.2 −7.63 −5.9 – – 1.09 4.88 1.9 – −3.26 5.32 – – −2.33 – −7.77 −2.83 −1.67 – −1.02 – – – 4.45 −2.34 – – – – 6.89 – – – – – – – −1.16 Lipocalins Humoral immunity Autophagy pathway Potential in reproduction Structural constituent of cuticle Other 9.03E-10 1.25E-13 6.75E-25 1.20E-04 – 1.66E-06 3.86E-06 1.29E-05 – – 1.93E-08 9.12E-09 4.62E-05 2.38E-07 – – – 9.07E-15 1.89E-22 3.81E-05 2.65E-06 4.59E-09 – 1.86E-09 2.92E-14 6.18E-10 1.39E-17 2.33E-06 – 1.31E-07 1.29E-06 1.18E-35 3.44E-43 4.09E-37 6.70E-43 2.64E-10 3.95E-33 4.83E-06 6.68E-22 7.66E-67 1.03E-146 9.52E-15 7.35E-98 1.86E-43 1.11E-17 1.80E-106 – 1.33E-35 – – 6.82E-05 4.22E-23 – – – 2.64E-12 3.33E-10 1.13E-28 1.74E-08 – – 1.08E-10 6.62E-05 2.43E-07 – 8.62E-07 2.62E-06 – – 5.12E-07 – 6.21E-41 1.64E-33 8.28E-14 – 3.34E-20 – – – 3.01E-05 6.24E-05 – – – – 1.86E-15 – – – – – – – 1.89E-06 Genes are ranked by biological process and/or molecular function Gene IDs and descriptions were compiled from the T urticae genome project FC, fold change D mauritiana and the mitotic activity of germline stem cells, as well as decreases programmed cell death in the germarium (Fast et al., 2011) The strength of CI induced by Wolbachia infection dramatically decreased with both male age (Reynolds & Hoffmann, 2002) and larval stage development (Yamada et al., 2007) As a result, we hypothesized that Wolbachia would strongly affect female fecundity and early spermatogenesis in T urticae In the present study, newly emerged adult females and 1-day-old adult virgin males were collected for transcriptome analysis to test this hypothesis Most of the transcriptome reads that we obtained could be mapped to the reference genome The transcripts corresponded to both known genes and some novel genes Our findings suggest that the northeast China strain used in the present study genetically differs from the Canadian strain used for the genome project (Grbic´ et al., 2011), which is not surprising because mite strains are known © 2014 The Royal Entomological Society, 24, 1–12 Wolbachia affect host gene expression Figure Validation of RNA-sequencing data by real-time quantitative-PCR analysis in females (A) and males (B) Fold differences in the expression of selected genes in response to Wolbachia infection The fold differences were calculated using the 2−ΔΔCt method Data are presented as mean ± SD values of triplicate reactions for each gene transcript to be genetically diverse (Grbic´ et al., 2011) In addition, novel transcripts can be detected with increasing sequencing depth and coverage (Sims et al., 2014) The main objective of the present study was to detect differentially expressed processes in response to Wolbachia infection The high depth sequencing made it possible to analyse the libraries at the gene level The expression patterns of males and females differed in the Wolbachia-infected vs the uninfected lines, suggesting that there are specific differences between the sexes The number of genes regulated by Wolbachia infection is probably related to the cellular tropism and virulence of the © 2014 The Royal Entomological Society, 24, 1–12 strain of Wolbachia as well as its density in the host (Walker et al., 2011; Rancès et al., 2012) A notable finding of the GO analysis was the enrichment of gene sets related to oxidoreductase activity in both sexes Oxidoreductase is involved in energy metabolism and redox homeostasis One consequence of disrupted redox homeostasis is DNA damage, which includes single- and double-stranded breaks, base and deoxyribose modifications, and DNA cross-linking (Valko et al., 2006; Brennan et al., 2012) Wolbachia has been shown to disturb the cellular physiology of its insect host especially via the generation of oxidative stress (Brennan et al., Y.-K Zhang et al 2008; Pan et al., 2012) Similarly, Wolbachia induces an increase in antioxidant expression in mosquito cells, which could be an adaptation to symbiosis Moreover, in A tabida, Wolbachia interferes with iron metabolism, which limits oxidative stress and cell death, thus promoting its survival within host cells (Kremer et al., 2010) Our finding that multiple genes are involved in oxidation reduction raises the possibility that Wolbachia regulates redox reactions to reduce reactive oxygen species levels and thus maintain the Wolbachia–host symbiotic relationship; however, in T urticae, oxidoreductase activity was found to be associated with host plant transfer and diapause within T urticae (Grbic´ et al., 2011; Bryon et al., 2013), which raises the possibility that spider mites respond to stress by regulating oxidoreductase activity T urticae is among the most polyphagous herbivores and harbours a large number of detoxification genes In the present study, a set of these specialized genes was profoundly affected by Wolbachia infection, suggesting that Wolbachia affects spider mite feeding and detoxification Many lipocalins genes were downregulated in response to Wolbachia infection Lipocalins are small extracellular proteins that typically bind hydrophobic molecules (Chudzinski-Tavassi et al., 2010) In spider mites, they may bind pesticides/allelochemicals, resulting in sequestration of these toxic, generally hydrophobic compounds The fact that Wolbachia is widely distributed in natural populations of T urticae (Breeuwer, 1997; Perrot-Minnot et al., 2002; Vala et al., 2002; Gotoh et al., 2007) raises the possibility that it has a role in T urticae resistance to diverse plant chemicals and pesticides Further studies are needed to check this possibility Many of the genes that were differentially transcribed in the Wolbachia-infected vs the uninfected lines encode proteins that are secreted Although the roles of these proteins are unclear, the finding that Wolbachia are mainly located in the gnathosoma in both sexes (Zhao et al., 2013) may indicate that the affected genes are involved in the digestion and detoxification of food Among the genes affected by Wolbachia, many have no known function For example, 71 of these genes were unique to female T urticae and 63 were unique to male T urticae Although Wolbachia induces strong CI and enhances female fecundity in T urticae, we didn’t identify any affected genes that were related to oogenesis or spermatogenesis; however, some of the genes may be related to female reproduction For instance, two genes encoding vitellogenin, were upregulated in infected females Vitellogenins are important for growth and differentiation of oocytes and transporting metallic ions, lipids and vitamins into the oocytes (Raikhel & Dhadialla, 1981); hence, these genes might have a role in enhancing female fecundity Three other genes may have roles in meiosis, as two of them are involved in chromatin assembly, and one is involved in meiosis arrest Another set of upregulated genes encoded cuticle proteins In the nematode Brugia malayi, removal of Wolbachia downregulated transcripts involved in cuticle biosynthesis, possibly reflecting a disruption of the normal embryogenic programme (Ghedin et al., 2009) Several genes that may participate in CI have been identified in D melanogaster (Xi et al., 2008; Zheng et al., 2011) and D simulans (Clark et al., 2006; Landmann et al., 2009) In D melanogaster, male development time was found to be inversely correlated with the strength of CI (Yamada et al., 2007) In the present study, a gene (tetur10g04620) that encodes juvenile hormone-binding protein was found to be downregulated in infected males The presumably higher expression of this protein in uninfected males would allow the testes to develop completely and produce fully mature sperm that would not be able to induce CI In support of this idea, Zheng et al (2011) found that, in D melanogaster, Wolbachia infection resulted in a ∼10-fold increase in the transcription of the gene for juvenile hormone-induced protein (JhI-26) in the testes of late-stage larvae The host’s immune system is pivotal to maintaining a balanced relationship with an endosymbiont There is growing evidence that the presence of a symbiont can dramatically affect host immunity (reviewed by Gross et al., 2009); however, we found that the expression levels of almost all of the putative immunity-related genes were stable during Wolbachia infection The expression of only a few genes involved in humoral immunity and the autophagy pathway was altered in the Wolbachia-infected vs the uninfected lines, which is strikingly different from what was found in other host–Wolbachia associations (Chevalier et al., 2012; Kremer et al., 2012; Rancès et al., 2012) Although the T urticae genome has genes that are involved in the Toll and Imd pathways, it lacks other components that are essential for effective immune signalling The genome also lacks extracellular serine proteases, putative phenoloxidase and most of the antimicrobial peptide effector gene orthologues (Grbic´ et al., 2011) The repertoire of immunity genes found in T urticae is consistent with a pattern emerging from comparative studies in invertebrate immunity, suggesting that invertebrates use diverse solutions to build an immune response, probably driven by specific life histories (Loker et al., 2004) It is also possible that Wolbachia adopts a strategy for escaping from the host immune system in the evolutionary relationship with T urticae Wolbachia, being intracellular, are surrounded by cell membranes that may protect them from the host immune system To date, immune priming by Wolbachia has mainly been observed in experiments on heterologous host systems The immune gene upregulation has been observed in novel laboratorygenerated transinfections of naturally Wolbachia-free © 2014 The Royal Entomological Society, 24, 1–12 Wolbachia affect host gene expression species, not in long-established natural associations such as the one studied here Identifying differences in host immune response induction among different Wolbachia strains will help to clarify the interactions between Wolbachia and their hosts (Bourtzis et al., 2000; Chevalier et al., 2012; Kremer et al., 2012; Rancès et al., 2012) In summary, Wolbachia infection affects numerous biological processes in T urticae, including oxidation reduction processes, digestion and detoxification, and processes involving lipocalins Unexpectedly, we found no evidence for strong effects of Wolbachia infection on spider mite reproduction and immunity As a study of the molecular mechanisms underlying the Chelicerata– Wolbachia association, this work provided new insights for understanding the complex interactions between arthropods and Wolbachia Experimental procedures Mite rearing and sample collection Mites used in the present study were originally collected from Hohhot, Inner Mongolia, northeast China in July 2010 and reared on leaves of the common bean (Phaseolus vulgaris L.) placed on a water-saturated sponge mat in Petri dishes at 25 ± 1°C, 60% relative humidity and under 16 h light: h dark conditions To establish 100% infected and 100% uninfected Wolbachia lines with identical genetic backgrounds, one female from the teleiochrysalis stage was allowed to lay eggs without being crossed with males The eggs were reared until adulthood (males) After the males had reached sexual maturity, they were backcrossed with the mother After the cross, the female adults were transferred to new leaf discs and were allowed to lay eggs for 3–5 days Females were each checked for Wolbachia infection by PCR amplification The eggs were reared separately on new leaf discs depending on the infection status of the mother The above process was continued for four generations until all members of the population were confirmed to be infected with Wolbachia (a 100% singly Wolbachia-infected population was obtained) The uninfected lines were established by treating lines singly infected with Wolbachia with tetracycline Small leaf discs (∼3 cm2) from the common bean were placed on a cotton bed soaked in tetracycline solution (0.1%, w/v) in Petri dishes (9 cm in diameter), and kept for 24 h before they were used for rearing the newly hatched larvae Distilled water was added daily to keep the cotton beds wet The cotton and the leaf discs were replaced every days Three generations later, mites were checked using PCR to confirm that the lines were free of Wolbachia These lines were maintained in a mass-rearing environment without antibiotics for approximately four generations (2 months) before use, to avoid the potential side effects of antibiotic treatment Through PCR assays, neither line was found to be infected with Cardinium (Primers: CLO-f1: 5′-GGAACCTTACCTGGGCTAGAATGTATT3′, CLO-r1: 5′-GCCACTGTCTTCAAGCTCTACCAAC-3′) or Rickettsia (Primers: R1: 5′-GCTCTTGCAACTTCTATGTT-3′, R2: 5′-CATTGTTCGTCAGGTTGGCG-3′) (Duron et al., 2008), which can manipulate host reproduction Within the two lines, 1–3-dayold adult females (Turt_FI and Turt_FU, ‘I’ indicates infection and ‘U’ indicates uninfection) and 1-day-old adult virgin males © 2014 The Royal Entomological Society, 24, 1–12 (Turt_MI and Turt_MU) were respectively collected The samples were stored in liquid nitrogen until required for RNA isolation Library construction and RNA sequencing Total RNA was extracted using the Trizol protocol (Invitrogen, Carlsbad, CA, USA) and RNA quality was determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s recommendations Poly (A) mRNA was isolated with oligo-dT beads and then treated with the fragmentation buffer The cleaved RNA fragments were then transcribed into first-strand cDNA using reverse transcriptase and random hexamer primers This was followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH The double-stranded cDNA was further subjected to end repair using T4 DNA polymerase, Klenow fragment DNA polymerase I, and T4 polynucleotide kinase, followed by a single A base addition using Klenow 3′ to 5′ exo-polymerase It was then ligated with an adapter or index adapter using T4 quick DNA ligase Adaptorligated fragments were selected according to the size and the desired range of cDNA fragments was excised from the gel PCR was performed to selectively enrich and amplify the fragments Finally, after validating the fragment quality on an Agilent 2100 Bioanalyzer (Agilent Technologies) and ABI Step One plus RealTime PCR System (Applied Biosystems, Foster City, CA, USA), the cDNA library was sequenced on a flow cell using Illumina HiSeq2000 (San Diego, CA, USA) Sequencing data quality control After Illumina sequencing, a sequence-filtering process was used to select clean reads First, Illumina’s Failed-Chastity filter software was used to remove raw reads that fell into the relation ‘failed-chastity ≤1’, with a chastity threshold of 0.6 on the first 25 cycles Second, all raw reads showing signs of adaptor contamination or ambiguous trace peaks (denoted with an ‘N’ in the sequence trace) were removed Finally, raw reads showing >10% of bases with a Phredscaled probability (Q) [...]... response to the targeting of the Wolbachia endosymbiont by tetracycline treatment Plos Neglect Trop D 3: e525 Gotoh, T. , Sugasawa, J., Noda, H and Kitashima, Y (2007) Wolbachia induced cytoplasmic incompatibility in Japanese populations of Tetranychus urticae (Acari: Tetranychidae) Exp Appl Acarol 42: 1–16 Grbic´, M., Van Leeuwen, T. , Clark, R.M., 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