Zhang 2015 how do hosts react t

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Insect Molecular Biology (2015) 24(1), 1–12

doi: 10.1111/imb.12128

How do 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:

© 2014 The Royal Entomological Society

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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 1. 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

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Figure 1. 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 2. 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


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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; 4 iron cluster
binding; 4 sulphur cluster binding; and oxidoreductase
activity. In males, the strongest changes were in the following GO categories of the oxidation-reduction process: 4 iron cluster binding; 4 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 4 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 3. 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

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Y.-K. Zhang et al.

Table 2. 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 4 autophagy related 4 homolog A
ATG 4 autophagy related 4 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

7

Figure 4. 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.,


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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: 8 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 4 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

9

(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) <20 were
discarded. All the downstream analyses were based on the clean
data with high quality.
Reads mapping to the reference genome
Reference genome and gene model annotation files were downloaded from the T. urticae genome website (http://bioinformatics
.psb.ugent.be/webtools/bogas/overview/Tetur) directly. An index
of the reference genome was built using BOWTIE v2.0.6
(Langmead & Salzberg, 2012) and paired-end clean reads were
aligned to the reference genome using TOPHAT v2. 0. 7 (Trapnell
et al., 2009). We selected TOPHAT as the mapping tool because it
can generate a database of splice junctions based on the gene
model annotation file, which other non-splice mapping tools
cannot do.

Quantification of gene expression level
HTSEQ v0.5.3 (Anders & Huber, 2011) was used to count the
reads mapped to each gene. In this study we used ‘reads per
kilobase of exon model per million mapped reads’ (RPKM), which
considers the effect of sequencing depth and gene length for the
reads count at the same time, and is currently the most commonly
used method for estimating gene expression levels (Mortazavi


10

Y.-K. Zhang et al.

et al., 2008). For each gene, the RPKM value was calculated
based on the length of the gene and the number of reads mapped
to the gene.

Differential expression analysis
For each sequenced library, the read counts were adjusted by the
EDGER program package (Robinson et al., 2010) through one
scaling normalized factor. Differential expression analysis of two
samples (Turt_FI and Turt_FU, Turt_MI and Turt_MU) was then
analysed using the DEGSeq R package 1.12.0 (Anders & Huber,
2012). The P values were adjusted using the Benjamini &
Hochberg (1995) method. A corrected P value of 0.005 and a log2
(fold change) of 1 were set as the threshold for significant differential expression.

Gene ontology and Kyoto Encyclopedia of Genes and
Genomes enrichment analysis of differentially expressed genes
Gene ontology enrichment analysis of differentially expressed
genes was implemented by the GOseq R package (Young et al.,
2010), in which gene length bias was corrected. GO terms with a
corrected P value less than 0.05 were considered significantly
enriched. KEGG (Kanehisa et al., 2008) ( />kegg/) is a database resource for understanding high-level functions and utilities of a biological system, such as a cell, an
organism or an ecosystem, from molecular-level information,
especially large-scale molecular datasets generated by genome
sequencing and other high-throughput experimental technologies. We used KOBAS software (Mao et al., 2005) to test the
statistical enrichment of differentially expressed genes in KEGG
pathways.

Quantitative real-time PCR

To confirm the results of RNA-Seq analysis, the expression levels
of randomly selected genes were measured by quantitative realtime (qRT)-PCR. The primer sequences are summarized in
Table S1. The qRT reaction was performed using 5 μg of total
RNA for each sample and a random 9-mer primer mix, used
according to the manufacturer’s instructions (Surcel Biotech,
China). The qRT-PCR reactions were performed on the Applied
Bio-systems 7300 Real-Time PCR System with the SYBR Premix
Ex Taq (Takara Bio, Kyoto, Japan) in eight connected tubes
(Takara). Each sample was analysed in triplicate in a 20-μl total
reaction volume, containing 5 pmol of each primer, 12.5 μl SYBR
Green and 2 μl diluted cDNA. The relative expression levels were
calculated using the 2-ΔΔCt method (Livak & Schmittgen, 2001).

Data accessibility
The transcriptome data used in the present study are available in
the ArrayExpress database ( />under accession number E-MTAB-2491.

Acknowledgements
We thank Ya-Ting Chen, Jia-Fei Ju and Peng-Yu Jin of the
Department of Entomology, Nanjing Agricultural University, for their help with the experiment. This study was

supported in part by a grant-in-aid from the Science and
Technology Programme of the National Public Welfare
Professional Fund (201103020) from the Ministry of Agriculture of China, and a grant-in-aid for Scientific Research
(31172131 and 30871635) from the National Natural
Science Foundation of China, and a grant-in-aid for Innovation Project (CXZZ13_0303) from Jiangsu Province,
China.

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Supporting Information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Table S1. Real-time quantitative PCR primers used in this study.
Table S2. Differentially expressed genes in response to Wolbachia infection in Tetranychus urticae females (A) and males (B).
Table S3. Gene ontology (GO) enrichment analysis of differentially
expressed genes in Tetranychus urticae females (A) and males (B).
Table S4. Kyoto Encyclopaedia of Genes and Genomes pathways analysis of differentially expressed genes in Tetranychus urticae females (A) and
males (B).

© 2014 The Royal Entomological Society, 24, 1–12