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Parasitization by Scleroderma guani influences protein expression
in Tenebrio molitor pupae
Jia-Ying Zhu
a,
⇑
, Guo-Xing Wu
b
, Sang-Zi Ze
c
, David W. Stanley
d
, Bin Yang
a
a
Key Laboratory of Forest Disaster Warning and Control of Yunnan Province, Southwest Forestry University, Kunming 650224, China
b
College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China
c
Yunnan Forestry Technological College, Kunming 650224, China
d
USDA/Agricultural Research Service, Biological Control of Insects Research Laboratory, Columbia, MO 65203, USA
article info
Article history:
Received 26 November 2013
Received in revised form 9 May 2014
Accepted 12 May 2014
Available online 19 May 2014
Keywords:
Ectoparasitoid
Hymenoptera
Immunity
Proteomics
Interaction
abstract
Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are
chemically complex and they exert substantial impact on hosts, including permanent or temporary paral-
ysis and developmental arrest. These visible venom effects are due to changes in expression of genes
encoding physiologically relevant proteins. While the influence of parasitization on gene expression in
several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain
mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic
ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm
beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and
parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially
expressed (32"- and 9;-regulated) in parasitized pupae. We assigned these proteins to functional catego-
ries, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, sig-
naling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected
proteins following parasitization. Our findings support our hypothesis by documenting changes in pro-
tein expression in parasitized hosts.
Ó2014 Elsevier Ltd. All rights reserved.
1. Introduction
Parasitoids, as a group, are among the most widely used biolog-
ical control agents. They lay eggs on or inside the body of juvenile
pest insects. The eggs hatch and larvae acquire resources by con-
suming the host tissues and hemolymph, leading to its death
(Colinet et al., 2012). Host/parasitoid relationships are biologically
intricate. Parasitization stimulates defense responses in hosts, par-
ticularly hemocytic encapsulation and generations of reactive oxy-
gen species (Strand, 2008). Endoparasitoids inject a cocktail of
maternal secretions including venom, calyx fluid, and polydnavirus
into their host during oviposition (Asgari, 2006; Asgari and Rivers,
2011). These virulence factors suppress the host’s immune system,
and alter host physiology and development to create a microenvi-
ronment that meets the needs of parasitoids (Beckage and Gelman,
2004). Ectoparasitoids inject venom into their hosts and then
deposit eggs onto the surface. The venom paralyzes the host and
inhibits its development. The larvae typically feed by piercing the
host cuticle and imbibing its hemolymph. The salivary secretions
of some ectoparasitic larvae include several proteins that influence
host defense systems (Richards, 2012).
The mechanisms of host-parasitoid interactions are driven by
parasitoid virulence factors associated with adult venom and with
larval salivary glands. Knowledge of an increasing number of bioac-
tive peptides, proteins, and enzymes from the venoms and salivary
secretions of parasitoids is emerging, along with considerable
progress in understanding the functions and structures of these
molecules (Moreau and Guillot, 2005; Asgari and Rivers, 2011;
Dorémus et al., 2013). Expression of a large number of host genes
is changed at the transcriptional and post-transcriptional levels fol-
lowing parasitization (Wertheim et al., 2005; Etebari et al., 2011;
Zhu et al., 2013). Investigations have characterized the influence
of parasitoids on protein expression in a few systems including
Macrosiphum euphorbiae/Aphidius nigripes or Aphidius ervi,Plutella
xylostella/Cotesia plutellae,Spodoptera littoralis/Chelonus inanitus,
and Pieris rapae or Papilio xuthus/Pteromalus puparum (Kaeslin
et al., 2005; Nguyen et al., 2008; Zhu et al., 2009, 2011). Nonethe-
less, with over 12,000 described species of braconid wasps
(Whitfield et al., 2004) and thousands of ichneumonids, the
http://dx.doi.org/10.1016/j.jinsphys.2014.05.011
0022-1910/Ó2014 Elsevier Ltd. All rights reserved.
⇑
Corresponding author. Tel./fax: +86 871 63863145.
E-mail address: jyzhu001@gmail.com (J.-Y. Zhu).
Journal of Insect Physiology 66 (2014) 37–44
Contents lists available at ScienceDirect
Journal of Insect Physiology
journal homepage: www.elsevier.com/locate/jinsphys
molecular strategies of many host/parasitoid interactions remain
unknown.
Beyond these many unexplored ichneumonid and braconid sys-
tems, the Bethylidae consists of about 2200 species of primitive
aculeate wasps, all of which are ectoparasites of juvenile lepidopt-
erans and coleopterans (Hawkins and Gordh, 1986). The ant-like
bethylid Scleroderma guani is a polyphagous ectoparasitoid with
over 50 host species (Chen and Cheng, 2000). Since its discovery
in 1973, S. guani has been used in biocontrol programs for longhor-
ned beetles in China (Li et al., 2011). As with the majority of hyme-
nopteran parasitoids, there is very little information relative to the
mechanisms of its interactions with hosts. Based on recent work
with other host/parasitoid systems, we posed the hypothesis that
envenomation by S. guani leads to changes in protein expression
in the yellow mealworm beetle Tenebrio molitor. In this paper,
we report on the outcomes of experiments designed to test our
hypothesis.
2. Materials and methods
2.1. Insects and parasitization
A laboratory colony of S. guani was maintained on T. molitor
pupae as described by Zhu et al. (2013). Adult wasps were main-
tained on a 20% honey–water solution. T. molitor pupae (3 h
post-pupation, n= 5 per treatment) were exposed to S. guani.
When a pupa was parasitized, it was immediately collected and
maintained at 25 °C. The non-parasitized pupae group was held
as a control. Samples were collected at 6 h intervals to 48 h post
parasitization (PP) and kept at 80 °C until protein and RNA
extraction.
2.2. Protein extraction
The samples (n= 5 pupae/treatment) were ground to fine pow-
der in liquid nitrogen and suspended in lysis buffer (50 mM Tris,
1 mM phenylmethylsulfonyl fluoride [PMSF], 2 mM EDTA, 2 mM
dithiothreitol [DTT], pH 7.4). The lysates were homogenized, incu-
bated on ice for 1 h and centrifuged at 25,000gfor 20 min at 4 °C.
The protein in the supernatant was removed to a new tube and
reduced with 10 mM DTT (56 °C for 1 h). Cysteine residues blocked
with 55 mM iodoacetamide (IAM) (room temperature for 45 min).
Protein was precipitated with ice cold acetone at 20 °C for 2 h.
The precipitated protein was suspended in 0.5 M tetrylammonium
bromide (TEAB) buffer. Protein concentrations were determined by
the protein–dye method of Bradford (1976) using bovine serum
albumin as a quantitative standard.
2.3. iTRAQ analysis
Equal amounts of protein (n= 5 pupae/sample), prepared sepa-
rately from control and parasitized pupae at 6, 12, 24 and 48 h PP,
were pooled together and the reported changes (Table 1) represent
changes in protein expression of the pooled samples. Each 100
l
g
sample was digested with trypsin (protein:enzyme ratio = 20:1) at
37 °C overnight. The control peptides were labeled with iTRAQ 118
and the treatment peptides with iTRAQ 121 tags in accordance
with the manufacture’s manual (Applied Biosystems). After 2 h of
incubation at room temperature, the labeled peptide mixtures
were pooled together and dried by vacuum centrifugation. The
peptides were separated by 2-dimensional liquid chromatography,
first via anion exchange and second via reverse-phase anion
exchange. Peptides were reconstituted with 4 ml buffer A
(25 mM NaH
2
PO
4
in 25% acetonitrile [ACN], pH 2.7) and loaded
onto a 4.6 250 mm Ultremex SCX column containing 5
l
m
particles (Phenomenex). Separation was performed using a linear
binary gradient at a flow rate of 1 ml/min with a gradient of buffer
A for 10 min, 5–35% buffer B (25 mM NaH
2
PO
4
, 1 M KCl in 25%
ACN, pH 2.7) for 11 min, and 35–80% buffer B for 1 min. Elution
was monitored by measuring the absorbance at 214 nm, and frac-
tions were collected at 1 min intervals. Twelve fractions were col-
lected and desalted with a Strata X C18 column (Phenomenex).
After vacuum-drying, peptides (5
l
g) were taken up into 10
l
l
2% ACN, 0.1% trifluoroacetic acid (TFA) solvent and injected onto
a 2 cm C18 trap column (inner diameter 200
l
m) connected a
resolving 10 cm analytical C18 column (inner diameter 75
l
m)
on a Shimadzu LC-20AD nanoHPLC. Each sample was loaded onto
the column at 15
l
l/min for 4 min. The peptides were eluted with
a linear gradient of 2–35% buffer B (98% ACN, 0.1% TFA) at a flow
rate of 400
l
l/min for 44 min. Buffer B was then changed to 80%
by a 2 min linear gradient and held at 80% for another 4 min.
Finally, the column was washed using buffer A for 1 min. The frac-
tions with absorbance peaks at 214 nm were subjected to nano-
electrospray ionization followed by tandem mass spectrometry
(MS/MS) in an LTQ Orbitrap Velos (Thermo). Mass spectrometric
analysis was carried out in a data dependent manner using the
Orbitrap mass analyzer at 60,000 mass resolution. Peptides were
selected for MS/MS using the high-energy collision dissociation
operating mode above a threshold ion count of 5000 with collision
energy of 45%. The other LTQ-Orbitrap settings are described by Su
et al. (2013).
2.4. Protein identification and quantification
The MS/MS data was used to interrogate the NCBI non-redun-
dant nr and Tribolium castaneum genome database plus a trans-
lated database of T. molitor transcriptome sequences (Zhu et al.,
2013) using Mascot 2.3.02. Search criteria specified: Enzyme, Tryp-
sin; Fragment mass tolerance, ±0.05 Da; Mass values, Monoiso-
topic; Variable modifications, Gln ?pyro-Glu (N-term Q),
oxidation (M), iTRAQ8plex (Y); Peptide mass tolerance, ±10 ppm;
Instrument type, Default, Max missed cleavages, 1; Fixed modifica-
tions, Carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K);
Protein mass, Unrestricted. Peptides were accepted only if they
met the false discovery rate (FDR) threshold of 1%. All identified
peptides had an ion score above the Mascot peptide identity
threshold, and a protein was considered identified if at least one
such unique peptide match was apparent for the protein. Proteins
with only one identifying peptide were determined directly from
MS/MS spectra. Intensities of the reporter ions from iTRAQ tags
(the ratio of 121/118) were used for quantification. Quantification
of iTRAQ signals was performed with the Q+ module of the Scaffold
software package and exported as TSV (tab-separated values) files.
Calculation of values and statistical evaluation was performed
using the software package R (V2.15). The thresholds for protein
differential expression were set at 1.5-fold change compared to
the control and a two-tailed P-value <0.05.
2.5. Bioinformatics
Because we pooled the samples following the iTRAQ protocol,
we do not separately analyze controls and experimental samples.
Proteins were analyzed by Gene Ontology (GO) annotation accord-
ing to molecular function, biological process and cellular compo-
nent ontologies (http://www.geneontology.org). The proteins
were also aligned to the Clusters of Orthologous Groups (COG)
database (http://www.ncbi.nlmnih.gov/COG/) to predict and clas-
sify possible functions. Pathway assignments were carried out
based on the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database (http://www.genome.jp/kegg).
38 J.-Y. Zhu et al. / Journal of Insect Physiology 66 (2014) 37–44
2.6. Quantitative real time PCR (qPCR)
Gene expression levels for selected proteins identified using the
T. molitor transcriptome database were determined with the rela-
tive standard curve method using threshold cycle values. The gene
sequences of the candidate proteins were derived from the tran-
scriptome database (Zhu et al., 2013) for primer design. The 18S
RNA gene was used as a reference. The primer pairs were designed
by Primer Premier 5 software (Lalitha, 2000). All the primers used
in qPCR are shown in Table S1. Total RNAs from control and para-
sitized T. molitor pupae (for each gene, n= 3 independent biological
replicates, 3 pupae/replicate) at 6, 12, 24 and 48 h PP were isolated
using Trizol
Ò
reagent (Invitrogen) according to instructions of the
manufacturer. For each sample, first-strand cDNAs were reverse-
transcribed from RNAs treated with DNaseI (Fermentas) using
RevertAid™ First Strand cDNA Synthesis Kit (Fermentas). qPCR
was carried out in a Qiagene real time PCR system (Qiagene) using
SYBR Premix Ex Taq™ (Takara). The amplification reaction condi-
tions were: 1 min at 95 °C, followed by 39 cycles of 10 s at 95 °C
and 20 s at 58 °C. Relative expression levels normalized against
those of 18S RNA were calculated using the 2
DD
CT
method
(Livak and Schmittgen, 2001). Results are expressed as means ± SD.
3. Results
3.1. Protein identification and classification
We identified proteins (1088 proteins identified from 3289
unique spectra [Table S2]) on the basis of having at least one dis-
tinct peptide with the threshold 61% FDR. Among these proteins,
we identified 382 proteins with peptides P2. We identified
approximately 20% of the predicted T. molitor proteome based on
its transcriptome database (Zhu et al., 2013).
We assorted the identified proteins into functional categories
(Fig. 1). We obtained 3588 annotation counts and assigned most
proteins to biological processes (1637, 45.62%), followed by cellu-
lar components (1142, 31.83%) and molecular functions (809,
22.55%). Within biological processes, the cellular process category
contributed the largest proportion of all annotations (21.01%), fol-
lowed by metabolic process (20.28%), localization (6.84%), cellular
component organization or biogenesis (6.84%) and establishment
of localization (6.41%). Among molecular components, cell
(28.90%) and cell parts (28.90%) were the most enriched. We anno-
tated 18.30% of proteins as organelle, 10.68% as macromolecular
complex and 10.33% as organelle component. Within molecular
Fig. 1. Gene ontology (GO) assignments for identified proteins. Proteins were annotated in three categories. (A) Biological processes; (B) cellular components; (C) molecular
functions.
J.-Y. Zhu et al. / Journal of Insect Physiology 66 (2014) 37–44 39
functions, we recorded proteins in binding (45.61%) and catalytic
activity (37.08%).
We searched the identified proteins in COG classifications,
assigning 532 proteins to COG classifications. Among the 22 COG
categories, the main represented clusters were post-translational
modification, protein turnover, chaperones (17.29%), general func-
tion prediction only (15.98%), and energy production and conver-
sion (12.03%), followed by translation, ribosomal structure and
biogenesis (8.65%), cytoskeleton (6.95%), and signal transduction
mechanisms (5.26%) (Fig. 2).
We mapped the identified proteins to the reference KEGG. The
pathway-based analysis assigned 675 proteins with significant
matches in the database to 216 KEGG pathways (Table S3). The
largest group, approximately 23% of mapped proteins, was
predicted to function in metabolic pathways. In addition to the
proteins involved in the metabolic pathways, the pathways with
main representation by the unique proteins were biosynthesis of
secondary metabolites (11.85%) and microbial metabolism in
diverse environments (7.85%). We recorded 69 pathways that con-
tained no more than three members.
3.2. Differentially expressed proteins associated with parasitization
For proteins prepared from parasitized pupae, we set proteins
with a ratio of >1.5-fold as up-regulated, and those with a ratio
of <0.667 as down-regulated (P< 0.05). Of the 1088 proteins, 41
(32"- and 9;-regulated) were differentially expressed between
control and parasitized pupae (Table 1). These included proteins
related to immunity (serine protease and serpin), stress and detox-
ification (glutathione S transferase [GST], superoxide dismutase
[SOD] and peroxiredoxin), energy metabolism (e.g. cyclohex-1-
ene-1-carboxyl-CoA hydratase, alcohol dehydrogenase and alpha-
amylase), development (tenebrin and take-out-like carrier protein
JHBP-1), cytoskeleton (paramyosin), signaling (translationally con-
trolled tumor protein), and others (e.g. glycine cleavage system h
protein, histone H2b, and 12 kDa hemolymph protein d precursor).
3.3. qPCR analysis
We selected 14 genes encoding differentially regulated proteins
for qPCR analysis (Fig. 3), all of them with parallel mRNA and pro-
tein expression patterns. For example, based on proteomic analy-
sis, serine protease, and take-out-like carrier protein JHBP-1 were
up-regulated 2.31- and 2.15-fold, we similarly recorded increased
expression levels of the cognate genes (Unigene70443 and Uni-
gene64547) following parasitization.
4. Discussion
The data reported here support our hypothesis that envenoma-
tion by S. guani leads to changes in protein expression in yellow
mealworm beetle pupae. Two key points apply. First, we recorded
significant changes in expression of 41 parasitism-specific proteins
associated with several functional classes including immunity,
stress and detoxification, energy metabolism, development, cyto-
skeleton, signaling and others. Second, we verified these expres-
sion changes by qPCR, which demonstrated the changes in
protein abundance were parallel with changes in mRNA tran-
scripts. It is reasonable to suppose changes in protein expression
cannot be completely confirmed by qPCR, however, the changes
in protein expression reported here are the bases of new hypothe-
ses, the testing of which will create entirely independent testing of
our results. Taken together, our results demonstrate that envenom-
ation by S. guani influences protein expression in T. molitor.
Our results differ from an earlier paper by Zhu et al. (2013), who
reported substantial changes in expression of 3431 T. molitor tran-
scripts following S. guani parasitization, considerably more than
the 41 parasitism-related proteins reported here. Global changes
in transcript abundances are not readily comparable to changes
in protein abundances and, in our view, the small number of para-
sitoid-induced proteins reported in this paper reflects a limited
number of inducible proteins acting in this particular host-parasit-
oid interaction. Our results align with several studies of other host-
parasitoid systems, all reporting changes in a limited number of
parasitism-specific proteins (Kaeslin et al., 2005; Nguyen et al.,
2008; Song et al., 2008; Zhu et al., 2009, 2011).
Our iTRAQ analysis did not detect venom proteins. This is due to
the very small size of the adult wasp and the even smaller size of
its venom apparatus. Indeed, in preliminary work we made an
effort to directly analyze proteins in isolated venom apparatus.
The negative results confirm the apparatus do not contain detect-
able amounts of individual proteins (unpublished results).
Expression of two major immune regulatory proteins (serine
protease and its inhibitor, serpin), which occur in a wide range of
species from insects to mammals, were increased following para-
sitism. Similarly, serpins were up-regulated in P. xylostella larvae
after parasitism by Diadegma semiclausum (Etebari et al., 2011).
Expression of the masquerade-like serine proteinase homolog in
P. rapae was significantly up-regulated by P. puparum parasitiza-
tion (Zhu et al., 2011). In contrast, serpin1 protein expression in
P. xylostella was suppressed during parasitism by C. plutellae
(Song et al., 2008). Four serine protease genes and their activities
were down-regulated after P. xylostella parasitized by C. vestalis
Fig. 2. Clusters of orthologous groups (COG) classification of identified proteins. Of 1088 proteins, 532 proteins have a COG classification among the 22 categories.
40 J.-Y. Zhu et al. / Journal of Insect Physiology 66 (2014) 37–44
Table 1
Identification of differentially expressed proteins in Tenebrio molitor pupae parasitized by Scleroderma guani.
Function Translated unigene ID Representive NCBI
Accession No.
Mascot score E-value Annotation (BlastX derived) Fold change
Immunity Unigene70443 EFA05743 58 4E75 Serine protease H164 2.31
n/a EFA09184 74 0 Serpin peptidase inhibitor 18 1.56
Stress and detoxification Unigene3200 XP_966787 131 6E43 Similar to glutathione S transferase E7 CG17531-PA 2.40
Unigene34431 XP_966787 168 9E17 Similar to glutathione S transferase E7 CG17531-PA 1.57
Unigene63116 XP_969146 257 4E54 Similar to putative glutathione s-transferase 2.23
Unigene64530 EGI68526 44 9E42 Superoxide dismutase 6.67
n/a XP_968419 92 1E129 Similar to peroxiredoxin 1.77
Energy metabolism Unigene62676 XP_971757 102 1E85 Similar to cyclohex-1-ene-1-carboxyl-CoA hydratase 1.56
Unigene66934 XP_966954 375 7E94 Similar to putative alcohol dehydrogenase 1.76
Unigene67086 P56634 144 4E137 Alpha-amylase 2.00
Unigene67881 XP_967875 189 2E135 Similar to 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP
cyclohydrolase
1.63
Unigene69471 XP_975007 212 6E180 Similar to glycerol-3-phosphate dehydrogenase 0.59
Development Unigene42984 AAR97872 80 3E17 Tenebrin 1.56
Unigene63990 AAR97872 116 4E53 Tenebrin 1.58
Unigene64547 XP_970920 171 2E47 Similar to take-out-like carrier protein JHBP-1 2.15
Cytoskeleton Unigene55248 EHJ72946 92 1E19 Putative paramyosin 1.83
Signaling n/a EGI63884 176 5E93 Translationally controlled tumor protein 1.61
Others Unigene63011 XP_969000 339 2E49 Similar to glycine cleavage system H protein 0.61
n/a CAA36808 77 2E44 Histone H2b 0.54
n/a XP_001648354 74 2E69 40S ribosomal protein S24 0.58
n/a XP_973469 56 1E64 Similar to S20e ribosomal protein 1.62
Unigene18628 XP_971514 174 2E09 Similar to cockroach allergen-like protein 0.43
n/a XP_967475 83 7E117 Similar to Bla g 5 allergen isoform 1 1.83
Unigene52073 AAO18180 144 6E26 12 kDa hemolymph protein d precursor (THP12) 11.81
Unigene12570 XP_967043 81 2E110 Similar to CG3244 CG3244-PA isoform 1 2.59
Unigene13875 XP_969988 67 2E167 Similar to GA18850-PA 1.56
Unigene52083 n/a 43 n/a n/a 2.69
Unigene54092 EFA11221 103 3E28 Hypothetical protein TcasGA2_TC005186 1.83
Unigene65616 EEZ97252 106 7E74 Hypothetical protein TcasGA2_TC011052 1.62
Unigene67981 EFA05023 95 2E104 Hypothetical protein TcasGA2_TC015109 2.64
n/a EFA10099 329 0 Hypothetical protein TcasGA2_TC012278 1.51
n/a EFA12363 53 0 Hypothetical protein TcasGA2_TC002069 1.93
n/a XP_002426862 50 6E78 Conserved hypothetical protein 1.56
n/a XP_972301 141 0 Similar to AGAP004793-PA 2.14
n/a XP_976350 72 2E25 Hypothetical protein 2.06
n/a AEE63560 384 2E139 Unknown 1.91
n/a EEZ98629 85 1E103 Hypothetical protein TcasGA2_TC001152 1.65
n/a AER92525 72 1E60 Hypothetical protein 0.61
n/a AEE62511 423 0 Unknown 0.65
n/a XP_970719 348 0 Similar to AGAP004877-PA 0.61
n/a EFA02063 98 0 Hypothetical protein TcasGA2_TC007694 0.62
J.-Y. Zhu et al. / Journal of Insect Physiology 66 (2014) 37–44 41
(Shi et al., 2013). Microarray analyses also indicated that serpin
A3K of Bemisia tabaci was one of the genes steeply down-regulated
following parasitization by Eretmocerus mundus (Mahadav et al.,
2008). Serine proteases and serpins act in the activation of immune
defenses, including hemolymph coagulation and melanization
(Kanost and Jiang, 1997; Gubb et al., 2010). Parasitization often
inhibits host hemolymph coagulation and melanization (Asgari,
2006). We interpret these effects on host immune functions in
terms of directly disabling at least some components of the host
immune system to promote parasitoid development.
GSTs are glutathione metabolism-related proteins, which play a
central role in the detoxification of endogenous and xenobiotic
compounds and they act in protection against oxidative stress
(Enayati et al., 2005). Interest in insect GSTs has primarily focused
on their role in insecticide resistance. Three forms of GST were up-
regulated following parasitization in this study. Parasitization ele-
vated detoxifying enzyme activities in P. xylostella (Takeda et al.,
2006). Consistent with their study, GST was up-regulated in P. xylo-
stella during the early stage of parasitization (Song et al., 2008).
These results suggest that GST acts in host resistance against par-
asitoid attack. SODs comprise a ubiquitous family of metalloen-
zymes that function to catalyze the dismutation of superoxide
anions, a defense against reactive oxygen species generated by oxi-
dative stress (Zelko et al., 2002). Peroxiredoxins are a family of
thiol peroxidases that act in reducing oxidative damage by decom-
posing H
2
O
2
and organic hydroperoxides (Immenschuh and
Baumgart-Vogt, 2005). In this study, SOD and peroxiredoxin were
up-regulated following parasitization. Nguyen et al. (2008)
recorded similar up-regulation of two forms of thioredoxin perox-
idase in parasitized M. euphorbiae. Two proteins belonging to per-
oxidases, thiol peroxiredoxin and peroxiredoxin, were strongly
induced in the plasma of P. xuthus after parasitization by P. pupa-
rum (Zhu et al., 2009). Overproduction of reactive oxygen species
is commonly associated with various infections and physical injury
in mammals and in insects (Nappi et al., 1995; Akaike, 2001). Pro-
ducing reactive oxygen species may be a host response to parasit-
ization. The up-regulated SOD and peroxiredoxin suggest to us an
effective parasitoid strategy to deal with the oxidative stress
induced during parasitism.
Four energy metabolism proteins, cyclohex-1-ene-1-carboxyl-
CoA hydratase, alcohol dehydrogenase, alpha-amylase, and 5-
aminoimidazole-4-carboxamide ribonucleotide formyltransferase/
IMP cyclohydrolase were up-regulated following parasitization
and another enzyme, glycerol-3-phosphate dehydrogenase, was
down-regulated. We take these changes to illustrate the complex-
ity of the T. molitor/S. guani relationship. Energy production pro-
teins play major roles in development (Zheng et al., 2011). In our
view, the effects of parasitism on host energy metabolism are asso-
ciated with increasing host energy reserves to benefit parasitoid
development (Beckage et al., 1997; Nakamatsu and Tanaka,
2004; Nakamatsu et al., 2006).
Parasitization by S. guani led to developmental arrest in T. mol-
itor (He et al., 2006). Here, we identified tenebrin and take-out-like
carrier protein JHBP-1 among the parasitization-specific proteins
related to development. Tenebrin is a component of the extracellu-
lar matrix in Tenebrio, regulated by 20-hydroxyecdysone and juve-
nile-hormone (JH) analog during metamorphosis (Royer et al.,
2004). Takeout/JHBP can bind JH or its precursors. These two pro-
teins are candidates for future hypotheses about their roles in
manipulating host development by parasitization (Pennacchio
and Strand, 2006).
Paramyosin, a major structural component of thick filaments in
invertebrates (Vinós et al., 1991), was up-regulated following par-
asitization. Expression of several other cytoskeleton proteins
including actin, actin depolymerisation, tropomyosin and tubulin
were up-regulated after parasitization (Nguyen et al., 2008; Zhu
et al., 2010). The dynamic expression of paramyosin in this study
suggests to us that cytoskeleton proteins act in T. molitor/S. guani
interactions. In insects, expression of these cytoskeleton proteins
is induced by bacterial fragments and physical wounds, that is,
they act in cellular immunity (Vierstraete et al., 2004; Paskewitz
and Shi, 2005). We infer that parasitoid-driven changes in expres-
sion of these proteins act in disabling host cellular immune
Fig. 3. qPCR analysis of gene expression patterns of selected differentially
expressed proteins in proteomic analysis. 18S RNA was used as the internal
reference gene. In each assay, the expression level was normalized to the lowest
expression level, which was arbitrarily set at one. Each treatment and control value
represents means of three independent biological replicates. The control values at
6 h also represent time zero following parasitization. The proteins corresponding to
unigenes are shown in Table 1.
42 J.-Y. Zhu et al. / Journal of Insect Physiology 66 (2014) 37–44
responses (Nguyen et al., 2008; Zhu et al., 2010). This is another
testable hypothesis for future work.
With respect to signaling proteins, expression of translationally
controlled tumor protein increased following parasitization. This
protein was originally cloned from cancer cells and described as
a growth-related protein in mouse ascites and erythroleukemia
cells (Yenofsky et al., 1983). It acts in mitotic cell division and in
response to a variety of stress conditions with chaperone-like
activity (Gnanasekar et al., 2009). Song et al. (2008) proposed that
translationally controlled tumor protein functions in cell growth
and proliferation signaling for modulating the physiology of the
parasitized P. xylostella. Up-regulation of this protein leads us to
the hypothesis that it may also act in the T. molitor/S. guani
relationship.
Expression of another 24 proteins was altered following
parasitization. Functions of most of these proteins are unknown.
We also note that similar proteins, such as ribosomal protein S24
and ribosomal protein S20e were changed in expression in oppo-
site ways. While understanding the detailed mechanism of such
changes is beyond the scope of this paper, similar opposite changes
in expression have been reported for other related proteins, such as
two serpins in P. xylostella. One was up-regulated and other
down-regulated at 2 days post parasitization (Song et al., 2008).
This also occurs at the transcriptional level (Wertheim et al.,
2005; Etebari et al., 2011). As is true for many explorations of
changes in protein expression, the findings reported in this paper
generate new hypotheses that will drive future research.
Acknowledgments
We thank Yu-Zhi Yang for her assistance in the qPCR. This study
was funded by the National Natural Science Foundation of China
(31260449), Key Project of Chinese Ministry of Education
(211171), and Fund of Reserve Talents for Young and Middle-Aged
Leaders of Yunnan Province (2013HB077). Mention of trade names
or commercial products in this article is solely for the purpose of
providing specific information and does not imply recommenda-
tion or endorsement by the U.S. Department of Agriculture. All pro-
grams and services of the U.S. Department of Agriculture are
offered on a nondiscriminatory basis without regard to race, color,
national origin, religion, sex, age, marital status, or handicap.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jinsphys. 2014.
05.011.
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