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Parasitization by Macrocentrus cingulum HymenopteraBraconidae influences expression of prophenoloxidase in Asian corn borer Ostrinia furnacalis.

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A r t i c l e
PARASITIZATION BY
Macrocentrus cingulum
(HYMENOPTERA: BRACONIDAE)
INFLUENCES EXPRESSION
OF PROPHENOLOXIDASE
IN ASIAN CORN BORER
Ostrinia furnacalis
Congjing Feng
Department of Plant Protection, College of Horticulture and Plant
Protection, Yangzhou University, Yangzhou, Jiangsu Province, China
Jianhua Huang
Institute of Plant Physiology and Ecology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Qisheng Song
Division of Plant Sciences, University of Missouri, Columbia, Missouri
David Stanley
USDA/Agriculatural Research Service, Biological Control of Insects
Research Laboratory, Columbia, Missouri
Wenjing Lü
Department of Plant Protection, College of Horticulture and Plant
Protection, Yangzhou University, Yangzhou, Jiangsu Province, China
Yong Zhang and Yongping Huang
Institute of Plant Physiology and Ecology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Grant sponsor: National Natural Science Foundation of China; Grant numbers: 30971963; 30771445; Grant
sponsor: National Basic Research Program of China (973 Program); Grant number: 2006CB102002.
Congjing Feng and Jianhua Huang contributed equally to this work.
Correspondence to: Yongping Huang, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. E-mail: yphuang@sibs.ac.cn
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 77, No. 3, 99–117 (2011)
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20425
100
Archives of Insect Biochemistry and Physiology, July 2011
A prophenoloxidase (PPO) cDNA (OfPPO) was cloned from the Asian
corn borer Ostrinia furnacalis. Sequence analysis revealed a full length
transcript of the OfPPO cDNA with 2,686 bp, containing a 2,079 bp
open reading frame (ORF), a 73-bp 50 -untranslated region, and
a 534-bp 30 -untranslated region with a poly(A) signal. The ORF encodes
a 693-amino acid polypeptide, containing two distinct copper-binding
regions, a plausible thiol ester site, two proteolytic activation sites, and
a conserved C-terminal region, but lacks a signal peptide sequence.
Expression of the OfPPO transcript in the plasma, hemocytes, fat body
and midgut was inhibited by Macrocentrus cingulum at 4 h postparasitization (pp). In situ hybridization analysis showed that the
hemocytes, especially the oenocytoids, hybridized strongly with the DNA
probes of the OfPPO gene. No signal was detected in the cuticular
epithelium or fat body of the parasitized larvae. Colloidal gold particles
were used to visualize the PPO by immunoelectron microscopy. The time
course study revealed a decrease in the labeling of the OfPPO at 4, 6, 8,
12, and 1 day pp in the larval integument and midgut parasitized by
M. cingulum. We infer from time course studies of OfPPO gene expression
and PO enzymatic activity that OfPPO in the integument is released from
hemocytes and that the OfPPO expression was influenced at the
transcriptional, translational, and then the post-translational level by
C 2011 Wiley Periodicals, Inc.
parasitization challenge. Keywords: prophenoloxidase; Ostrinia furnacalis; Macrocentrus cingulum;
insect immunity; real-time PCR; in situ hybridization; immunoelectron
microscopy
INTRODUCTION
The insect innate immune system consists of cellular and humoral components that
defend against pathogens and parasites. Phenoloxidase (PO) is an important component
of insect immunity (Cerenius and Söderhäll, 2004). PO is synthesized as a zymogen,
prophenoloxidase (PPO), that is activated following injury, infection or parasite invasion
by a serine protease (PPO activating enzyme, PPAE), which directly cleaves PPO to form
active PO. PO catalyzes the early steps of melanin formation by reducing monophenols
into o-diphenols and then oxidizing o-diphenols to quinone intermediates. Quinones are
subsequently converted to melanins, which act in the recognition and encapsulation of
invaders (Cerenius and Söderhäll, 2004). When insects are infected, PPO activation is
triggered by recognition of microbial surface component, such as lipopolysaccharide,
peptidoglycans, and b-1,3-glucose (Lee et al., 2000).
Endoparasitoids develop through their immature stages inside the hemocoel of
their hosts, which requires them to overcome or evade host immune responses,
including PO-dependent melanization (Doucet et al., 2008). Parasitoid wasps have
evolved several mechanisms to inhibit PO activity and other defense mechanisms
(Shelby et al., 2000). Many hymenopteran parasitoids suppress their host’s immune
system using maternal secretions, such as polydnaviruses (PDVs), ovarian proteins
(Madanagopal and Kim, 2006), and venom (Fang et al., 2010). Macrocentrus cingulum
Archives of Insect Biochemistry and Physiology
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101
(Hymenoptera: Braconidae) is a polyembryonic endoparasitoid. The egg begins to
divide into numerous embryos about 1.5 h after oviposition in the host body cavity.
The embryonic stage lasts about 7–9 days. Because the adults of this species do not
have PDVs (Hu et al., 2003), how the endoparasitoid overcomes the immune defenses
of its lepidopteran host, the Asian corn borer Ostrinia furnacalis remains unclear.
So far, we have purified a PPO from hemolymph of O. furnacalis and recorded PPO
protein in hemocytes (Feng and Fu, 2004), reported that M. cingulum parasitization
leads to reduced hemolymph melanization (Feng et al., 2004) and generated a
biochemical characterization of the purified PPO protein (Feng et al., 2008). The
purpose of the work reported in this article was to investigate the expression of PPO in
the O. furnacalis (host)/M. cingulum (parasite) system. We posed the hypothesis that
factors associated with parasitism inhibit expression of PPO in the host. Here we report
on the outcomes of experiments designed to test our hypothesis.
MATERIALS AND METHODS
Experimental Animals
M. cingulum was reared in a habitual host, O. furnacalis. O. furnacalis larvae were fed an
artificial medium (Table 1), as described by Zhou et al. (1980). Briefly, the listed
components were mixed, brought to boiling to dissolve the agar and cooled slightly.
Although still warm, the medium was poured into cups (250 ml). After cooling to room
temperature, eggs were placed on the medium for hatching and larval development as
described just below for parasitoids. The parasitoid wasps, M. cingulum, and host larvae
used in this study were originally collected from cornfields in Jiangsu province, China and
maintained in our laboratory since 1998. Adult parasitoids were fed a 20% honey solution
and maintained at 251C with a 16:8 L:D photoperiod. Parasitism was carried out by
exposing the late-stage fourth-instar larvae (defined as the head capsule clearly separated
from the prototum) to the female wasps 2 days after mating, at a ratio of 1 larva:2 wasps
for 4 h in the presence of fresh corn stems. Parasitization was determined either by
dissecting the host after collecting hemolymph or keeping the host until the adult wasps’
emergence. All solutions, glassware, and plastic materials used in the study were sterilized.
Collection of Hemolymph, Hemocytes, Fat Body, Midgut and Integument
Larvae were anesthetized on ice and surface sterilized by swabbing with EtOH. The
first abdominal proleg was cut, hemolymph was allowed to drip onto a parafilm, and
Table 1. The Artificial Diet Ingredients for O. furnacalis Larvaea
Main ingredients of artificial diet
Soybean meal
Corn meal
Brewer‘s yeast
Glucose with vitamin mixtures
Scorbic acid
Agar
H2O
a
Weight (g)
15.0
19.0
9.0
7.5
0.5
2.0
120.0
Formalin (0.2 ml) and scorbic acid were added directly into boiling agar solution as antibiotic reagents (Zhou et al., 1980).
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then transferred into 1.5 ml eppendorf tubes, each containing 0.5 ml sodium
cacodylate (CAC) buffer (100 mM CaCl2, 10 mM Na2CAC, pH 6.5) in an ice-bath.
After centrifugation (800 g, 10 min, 41C), the supernatant was transferred into
eppendorf tubes, vortexed, and kept at 41C. The pellet containing hemocytes was
kept on ice for later use. The fat body and midgut were isolated, and washed with CAC
buffer solution three times. The fat body and midgut were separately homogenized
using a Dounce homogenizer in an ice-bath. After centrifugation (10,000 g, 10 min,
41C), the supernatant was transferred into eppendorf tubes, and then continuously
centrifuged (12,000 g, 10 min, 41C) three times until no visible pellet was observed.
The supernatant was kept at 701C for later use. The epithelial layer was scraped from
the inner side of integument taken from abdominal segments and homogenized using
a Dounce homogenizer in an ice-bath. After centrifugation (10,000 g, 10 min, 41C), the
supernatant was transferred into eppendorf tubes and kept at 701C for later use.
Degenerate Primer Design and Strategy of PPO cDNA Cloning
Multiple alignments of PPO amino acid sequences from other lepidopterans were
performed using the ClustalW multiple sequence alignments program (http://www.
ebi.ac.uk/Tools/clustalw/). Based on the two highly conserved amino acid sequences in
PPOs, HHWHLVY and MGFPFDR, a pair of degenerate primers was designed:
forward primer 50 -CAYCAYTKBCAYYTNGT-30 and reverse primer 50 -CKRTCRAA
NGGRWANCCCAT-30 . Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA)
according to the manufacturer’s protocol. First-strand cDNA was synthesized using
the ReverTraAce-a kit (cat. no. FSK-100; Toyobo, Osaka, Japan). cDNA quality was
checked on an 1% agarose gel and quantified by spectrophometry at A260. PCR
amplification of the target cDNA was performed in a reaction mixture containing 1 ml of
the cDNA from the reverse transcription reaction. The PCR condition was as following:
5 min initial denaturation at 941C, then 35 cycles of denaturation at 941C for 30 sec,
primer annealing at 541C for 30 sec and extension at 721C for 1 min, followed by a
10 min final extension at 721C and cooling to 41C. Reaction products were purified on
agarose gels, a fragment of 1.3 kb was cloned into the T vector (Takara, Tokyo, Japan)
and then sequenced using an ABI 3730 sequencer (SeqGen Inc., Torrance, CA).
Rapid amplification of the 50 and 30 ends of the full length OfPPO was performed
using the GeneRacer kit (cat. no. L1500-01, Invitrogen) according to the manufacturer’s protocol. About 200 ng of RNA were treated with calf intestinal phosphatase
at 501C for 1 h to remove the 50 -phosphates. Tobacco acid pyrophosphatase was added
to the reaction and incubated at 371C for 1 h to remove the 50 -cap structure from
intact mRNAs. The RNA oligo was ligated to the decapped mRNA with T4 RNA ligase
at 371C for 1 h. The RNA oligo provides a known priming site for the 50 RACE
(sequence 50 -CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-30 ).
The cDNA was synthesized with a cloned AMV RT reverse transcriptase in the kit
(Invitrogen), using an oligo(dT) primer: 50 -GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)18-30 . The adaptor-ligated cDNAs were subjected to PCR for 50 RACE
under the following conditions: 951C for 5 min, followed by five cycles at 941C for 30 sec
and 721C for 3 min, and 25 cycles at 941C for 30 sec, 621C for 30 sec, and 681C for 3 min.
The reactions were maintained at 681C for 10 min after the last cycle. The forward primer
was 50 -CGACTGGAGCACGAGGACACTGA-30 , homologous to the RNA oligo, and the
reverse primer was 50 -GACGCTCACAGTTGTATCGG-30 , designed on the basis of the
1.3 kb fragment obtained as described above. The 30 -end was amplified using the forward
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primer 50 -GTCCGTCAGTCTACCGAGCCC-30 , designed on the basis of the 1.3 kb
fragment, and the reverse primer was 50 -CGCTACGTAACGGCATGACAGTG-30 . These
PCR products were ligated into the T vector plasmid and then sequenced using an ABI
3730 sequencer.
Sequence and Phylogenetic Analysis
The full-length cDNA sequence and the deduced amino acid sequence of OfPPO were
compared, with default settings, to the complete, nonredundant GenBank database,
using the BLASTP program (http://www.ncbi.nlm.nih.gov/blast/). The PPO amino acid
sequences of other species were downloaded from GenBank in FASTA format. Protein
motif features were predicted using the Simple Modular Architecture Research Tool
(http://smart.embl-heidelberg.de/). Alignment of multiple PPO sequences was performed using the ClustalW program, with default parameters. The alignments of the
conserved copper A- and copper B-binding site histidines were verified. SignalP 3.0
(http://www.cbs.dtu.dk/services/SignalP/) was used to predict the signal peptide.
Phylogenetic trees were constructed on the basis of the amino acid differences
(p-distance) of PPO by the neighbor-joining method, using the MEGA4 software.
The freshwater crayfish Pacifastacus leniusculus PPO sequence was used as an outgroup.
For construction of the phylogenetic tree, indels were removed from the multiple
alignments. The reliability of the branching was assessed by a bootstrap re-sampling
method, using 1,000 bootstrap replications.
Expression of the OfPPO Gene in Tissues
For collection of hemocytes, fat body, midgut, and integument, ten unparasitized and
parasitized larvae were anesthetized on ice and tissues were dissected with a pair of
sterile forceps in diethyl pyrocarbonate (DEPC)-treated phosphate-buffered saline
(PBS). First, hemocytes from larvae were prepared as just described and washed in
DEPC-treated PBS. Midgut, integument, and fat bodies were isolated. And after not
longer than 5 min washing, the tissues were transferred to sterile, ice-chilled microfuge
tubes, containing 0.2 ml DEPC-treated PBS and vortexed vigorously. Isolated tissues
were blotted and weighed, and then homogenized in TRIzol reagent (Invitrogen),
using a plastic homogenizer. Total RNA was extracted as described above. The
extracted and purified total RNA was digested with RNAse-free DNAse, to ensure
that any contaminating DNA was not amplified. The RNA was quantified by measuring
the absorbance at 260 nm. Total RNA (5 mg) and a first-strand cDNA synthesis kit
(ReverTraAce-a, Toyobo) were used to synthesize the first-strand cDNA. The SYBR
Green I quantitative real-time PCR assay was carried out in a Rotor-Gene RG-3000
(Corbett Research, Mortlake, Australia). The amplifications were performed using
a LightCycler FastStart DNA Master SYBR Green I Kit (Roche, Indianapolis, IN) in
an individual sample capillary, with a final volume of 25 ml. The reaction cocktail
components were as the followings: thermoscript reaction mixture (22 ml), forward
primer 50 -GGCATTGGCATCTCGTCTATC-30 (1 ml; 0.5 mM), reverse primer
50 -ATGTTGGACTGCCGTGGTGG-30 (1 ml; 0.5 mM), and cDNA (1 ml). The reaction
conditions were an initial denaturing step for 10 sec at 951C, followed by 40 cycles of
denaturing (951C for 10 sec) and annealing (601C for 20 sec). Fluorescence detection
was performed after each extension step. A 141-bp fragment was amplified, using
O. furnacalis ribosomal protein L8 (RPL8) primers (RPL8-F 50 -ACGGAGGTGGTAACCATCAACA-30 and RPL8-R 50 -ACGCCTCCTTCTTGGTGTCG-30 ) to calibrate the
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cDNA template as an internal control. To confirm that only one PCR product was
amplified and detected, dissociation curve analysis of the amplification products was
performed at the end of each PCR reaction. Fluorescence values were measured and
amplification plots were generated in real-time by the Rotor-Gene RG-3000 program.
Data were analyzed with standard software of Rotor-Gene RG-3000 program.
Quantitative analysis of the OfPPO transcript followed the DCT method. Three
independent biological replicates were run and all data are given in terms of relative
mRNA expression, as means7SDs.
Phenoloxidase Assay
Because PPO is an inactive zymogen, it was activated before assessing PO activity using
the method described by Jiang et al. (1997). Briefly, a reaction mixture (1 ml)
containing 2 mM L -DOPA, 50 mM sodium phosphate buffer (pH 6.0), and enzyme
protein (5–10 mg) was incubated at 301C, and the increase in absorbance at 490 nm,
associated with the production of dopaminechrome, was continuously monitored after
activating the PPO by the addition of 10 ml 10% cetylpyridinium chloride. One unit
was defined as a 0.001 absorbance increase per min at 490 nm. Three independent
biological replicates were run.
In situ Hybridization (ISH)
Hemocytes from nonparasitized and parasitized larvae were prepared as described
above. Paraformaldehyde-fixed hemocytes were allowed to attach to glass slides
treated with 0.1% poly-L -lysine. The fixed hemocytes were dehydrated for 5 min each
in 70, 90, and 100% ethanol, air dried, and hybridized with the probes. For probe
synthesis, plasmid DNA containing a full-length cDNA of OfPPO was used as a
template, with primers 50 -GGCATTGGCATCTCGTCTATC-30 and 50 -ATGTTGGACTGCCGTGGTGG-30 . Plasmid DNA (30 pg), 200 nM of each primer, and 2.5 U
Taq polymerase per 20 ml reaction were used. Thirty cycles were performed at 941C
and 601C for 30 sec each, and at 721C for 1 min. After removal of free nucleotides
using the QIA Quick PCR Purification Kit (Qiagen, Hilden, Germany), the products
were analyzed on a 1% agarose gel. A 293 bp fragment was amplified and used as a
template DNA for probe labeling, using the DIG High Prime DNA labeling and
Detection Starter Kit (Roche). The amount of DNA containing DIG was determined by
dotting several dilutions on a nylon membrane. The spot intensity was compared with
a DIG-labeled standard DNA, and the probes were used only if the concentration of
the labeled DNA was at least 0.2 pg/ml. To detect the DIG, the slides were blocked with
a blocking solution supplied with the DIG High Prime DNA labeling and Detection
Starter Kit according to the manufacturer’s instructions, and then incubated with 5 ml
hybridization solution containing the digoxigenin-labeled DNA probe (1 ng/ml) at 651C
overnight. After several washes with washing buffer (1 PBS, 0.1% Tween-20), the
slides were incubated with antidigoxigenin Fab fragments, conjugated with alkaline
phosphatase (1:100 dilution) at 41C overnight. After washing with washing buffer to
remove the excess Fab fragments, an alkaline phosphatase substrate solution [11%
(v/v) DMSO, 3.13 mg/ml nitroblue tetrazolium chloride, 1.57 mg/ml 5-bromo-4-chloro3-indolyl-phosphate, 100 mM Tris-HCI (pH 9.5), 100 mM NaCl] was added to the slide
for color development at room temperature for 30 min. The reaction was stopped by
washing with 50 mM ethylenediaminetetraacetic acid (EDTA) and 0.1% Tween-20 in
PBS (PBST). Six independent biological replicates were run.
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105
Integument, midgut, and fat body from unparasitized and parasitized larvae were
fixed in 4% (w/v) paraformaldehyde in PBS overnight at 41C. After fixing, the samples
were washed twice in PBS and dehydrated in an ascending series of ethanol from 70 to
100%. The samples were embedded in paraffin wax at 601C and sectioned at 7 mm.
The sections were spread on poly-L -lysine-treated slides and dried completely at 371C
overnight. The sections were dewaxed and washed in two changes of fresh DEPCtreated PBS. For protelysis, the slides were incubated at 371C for 30 min with Trisbuffered saline containing 15 mg/ml Proteinase K (Roche). The slides were rinsed with
distilled water and rehydrated in a descending ethanol series, from 100% ethanol to
DEPC-treated water before ISH. After two washes with DEPC-treated PBS, cover slips
were placed on the slides. Prehybridization was done with 5 ml hybridization solution
[50% formamide, 10% dextran sulfate, 2 SSC (1 SSC is 0.15 M NaCl, 15 mM
sodium citrate, pH 7.0), 50 mM sodium phosphate buffer, 250 ng/ml salmon sperm
DNA)] on one slide with no probe at 421C for at least 2 h in a box saturated with
2 SSC. For hybridization, cover slips were placed upside down on 5 ml hybridization
solution with the DIG-labeled probe (5 mg/ml). Hybridization was carried out in a box
saturated with 2 SSC for 36 h at 421C. Tissues were washed first with 50% formamide
in 2 SSC (three times for 5 min at 421C), then with 2 SSC (three times for 5 min at
421C), and finally with 4 SSC, 0.1% Tween 20 (once for 5 min at 421C). For detection,
the samples were first washed with washing buffer for 5 min and then blocked for
30 min in 10 ml of blocking solution (Roche) at room temperature. After blocking,
2 ml of a sheep antidigoxigenin antibody conjugated with alkaline phosphatase (AntiDigoxigenin-AP, Fab fragments, Roche) diluted 1:5,000 in the blocking solution were
added to each well. Incubation was either for 30 min at room temperature or
overnight at 41C. The antibody solution was removed and samples were washed twice
with 10 ml of washing buffer for 15 min at room tmperature. Ten milliliter of detection
buffer were added for equilibration for 5 min, and 10 ml of color-substrate solution
were added in the dark for 10 min. The extent of staining was regularly checked under
microscope and was stopped by two washes with TE (10 mM Tris, 1 mM Na2EDTA,
pH 8.0). The coverslips were mounted with Glycergel (Dako, Glostrup, Denmark) on
Superfrost slides. The slides were observed with an Eclipse E600 (Nikon, Tokyo,
Japan) microscope, connected to a digital camera (DXM1200, Nikon). Pictures of the
experimental samples and controls were taken under the same conditions. The data
from six independent biological replicates were analyzed.
Immunoelectron Miroscopy Detection of PPO
Integuments, midguts, and fat bodies were dissected from fourth-instar unparasitized
and parasitized O. furnacalis larvae at 4 h, 6 h, 8 h, 12 h, 1 day (1d), 2 day(2d), and 3 day
(3d) pp. Tissues were immediately fixed in 4% paraformaldehyde in 0.1 M PBS (pH
7.2) at 41C, then rinsed in PBST for three times, 5 min each. Following a series of
ethanol dehydration steps, the samples were incubated in acetone for 30 min, and then
embedded in Epon 812 for 2 h. Ultra-thin sections were made using a diamond knife
on several integument and midgut samples, and then immunolabeled for PPO using
the mouse anti-OfPPO IgG (1:50 dilution) as a primiary antibody. PPO was isolated
from the hemolymph of O. furnacalis larvae and purified to homogeneity by employing
ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography, and Phenyl
Sepharose CL-4B chromatography (Feng et al., 2008), and the purified PPO was
injected into Balb/C mouse to prepare polyclonal antibodies (Feng et al., 2009). A goat
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Archives of Insect Biochemistry and Physiology, July 2011
antimouse IgG conjugated to 10 nm colloidal gold (1:20 dilution) was used to reveal
the primary antibody. The negative control experiments were conducted in the same
fashion except that anti-OfPPO IgG was substituted by the nonimmune mouse IgG at
the same concentration. The sections were viewed and photographed at 80 KV on
Tecnai12 electron microscope. The number of the gold labels was determined by
counting the gold particles in 10 random fields on the micrographs per sections.
Statistics
All data were analyzed using SPSS software (version 11.5.0, SPSS Inc., Chicago, IL).
Differences between means were analyzed by independent sample t-tests and one-way
analysis of variance. Where appropriate, data were analyzed by Duncan’s multiple
range test. Significance was set at Po0.05.
RESULTS
Sequence of OfPPO cDNA
The complete cDNA sequence and the deduced amino acid sequence of OfPPO are
shown in Figure 1. The full-length cDNA of OfPPO consists of 2,686 bp, containing
an 2,079 bp open reading frame (ORF), a 73 bp 50 -untranslated region, and a 534 bp
30 -untranslated region with a poly(A) signal. The ORF begins at nucleotide 74 and
ends at nucleotide 2152. Based on the deduced polypeptide sequence, the ORF of
OfPPO consists of 693 amino acids (Fig. 1). The calculated molecular mass of the
OfPPO protein is 79.8 kDa, with an estimated pI of 5.72. The OfPPO cDNA sequence
and deduced amino acid sequence have been submitted to the NCBI GenBank under
accession number DQ333883. The OfPPO does not contain a typical secretion signal
peptide at its N-terminal region.
BLASTP analysis revealed that OfPPO exhibits high amino acid sequence identity
(50–79%) with those of other insect species, Helicoverpa armigera, Spodoptera litura,
Bombyx mori, Manduca sexta, and Galleria mellonella. OfPPO contains a conserved
sequence (Arg-Phe-Gly) around the first activation site. A second possible proteolytic
cleavage site, Arg-Glu-Met (indicated by an arrow), is conserved in the other insect
PPOs compared. The conserved two copper-binding sites, Cu A (IGVNLHHWHWHLVYPFTATDRSIVAKDRRGELFFYMHQQIIARY) and Cu B (SLHNNGHSFTAYMHDPNHRYLESFGVMADEATTMRDPFFYRWHAFIDDIFQKHK) show extensive
homology (Fig. 1). The six histidine residues (H 213, H 217, H 243, H 366, H 370,
H 406), thought to bind two copper ions, are present at the conserved sites in OfPPO.
Additionally, a previously characterized possible thiol ester site (CGCGWPQHML) and
a conserved motif MGFPFDR at the C-terminal end are conserved in OfPPO. A NJ
phylogenic tree was constructed based on the PPO amino acid sequences, using the
MEGA4 software. The in silico PPO sequences of the freshwater crayfish P. leniusculus
were used as the outgroup, because they are outside the insect phylogeny (Fig. 2).
There are five distinct sister groups in the phylogenetic tree, including the subfamilies
of two lepidopteran PPOs, two dipteran PPOs, and a coleopteran PPO.
Quantitative Analysis of OfPPO mRNA Level
The OfPPO mRNA transcript was detected in hemocytes, midgut, and fat body at
differing levels, but not in integument (Fig. 3). In the unparasitized, control larvae, the
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107
Figure 1. Nucleotide sequence (above) and deduced amino acid sequence (below) of O. furnacalis PPO
cDNA. The sequence was deposited in GenBank (NCBI accession number: DQ333883). Nucleotides are
numbered from the first base at the 50 -end. Amino acids are numbered from the initiating methionine. Both
start and stop codons are marked with asterisks (). The polyadenylation signal (AATAAA) and poly(A) tail
are underlined. Conserved motifs are doubly underlined and numbered. Note in region I: copper binding
site A (Cu A), region II: copper binding site B (Cu B), region III: the putative thiol ester sites and in region
IV: a conserved motif MGFPFDR at the C-terminal end. The six histidine residues within the Cu A (213, 217,
243) and Cu B (366, 370, 406) binding sites are shown in bold with solid lines ( ).
highest level of OfPPO transcript was recorded in the hemocytes. The hemocyte level
of OfPPO transcript at 4 h pp was reduced to undetectable, then increased by 1 d pp
and was again undetectable at 2 d pp. A similar pattern of inhibition, recovery and
sudden dropping was also observed in fat body and midgut although the abundance of
OfPPO transcript from the midgut and fat body were significantly lower compared
with hemocytes in the parasitized and unparasitized larvae.
PO Activity in Selected Tissues Post-parasitization
PO activity was detected in hemocytes, plasma, integument, midgut, and fat body.
At over 30 U/mg, the highest specific activity was recorded in plasma, significantly
higher compared with the other tissues (Table 2). Inhibition of the PO activity
occurred over the 72 h time course in hemocytes, plasma and integument of
parasitized larvae. Hemocyte PO activity was unchanged until 12 h pp; plasma and
integument activity remained high until 1 d pp; Midgut activity was reduced at 1 d and
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Archives of Insect Biochemistry and Physiology, July 2011
Figure 1. Continued.
2 d pp and then recovered to normal levels at 3 days. Parasitization did not influence
fat body PO activity during the 72 h time course pp.
In situ Hybridization
The results of the in situ hybridization analysis from the six experimental animals
were generally similar. In the unparasitized control group, hemocytes, especially the
oenocytoids, hybridized strongly with several DNA probes (Fig. 4, D1). Signals were
detected in some hemocytes at 4 h pp, after which only faint signals were seen from 6 h
to 3 d pp. In the midgut tissues, the OfPPO transcript decreased at 6 h and 8 h pp
(Fig. 4, C3–C4). No signal was seen in the endothelial cells surrounding the lumen and
the epithelial cells of the midgut at 2 d pp (Fig. 4, C7–C8), and there was no
hybridization signal in the integument (Fig. 4, A2–A8) or fat body tissue (Fig. 4, B2–B8)
of the parasitized larvae.
Quantitative Analysis and Spatial Localization of OfPPO Immunogold Particles
Ultrathin sections of integument and midgut of fourth-instar parasitized larvae were
used for immunogold labeling of OfPPO with the polyclonal antibodies. Immunogold
label for PPO was observed predominantly in the integument and midgut of the
Archives of Insect Biochemistry and Physiology
Prophenoloxidase Expression in Parasitized O. furnacalis
109
Figure 1. Continued.
unparasitized control group (Fig. 5, A2 and B2). Integument labeling decreased at 4 h
pp (Fig. 5, A3), but was not changed at 6 h and 8 h pp (Fig. 5, A4 and A5). A very few
gold particles at 1 d pp, which significantly increased at 2 d and 3 d pp (Fig. 5, A7–A9).
No gold particles were recorded in the negative control group in which the primary
antibody was replaced by a nonimmune serum (Fig. 5, A1). Figures 5, B1–B9 display
the PPO labeling seen in midguts.
Quantitative analysis of immunogold labeling is presented in Table 3. Labeling in
the integument declined steadily from control values to 1 d pp, then increased at 2 d
and 3 d. We recorded similar declines in midgut labeling to 12 h pp, followed by
increased labeling over the following 60 h.
DISCUSSION
The data reported in this article strongly support our hypothesis that factors associated
with parasitism inhibit expression of PPO in the host, as seen in several arguments.
First, the cloning and phylogenetic analysis demonstrate the presence of at least one
PPO gene in O. furnacalis. Second, quantitative PCR shows expression of PPO mRNA
in hemocytes (the source of PPO) declined sharply following parasitization. Third,
activity of the hemocyte PO enzyme decreased following parasitization. Fourth,
immunogold labeling documents the presence of the PPO gene predominantly in
oenocytoids, the main source of immune-related PPO. The labeling declined following
parasitization. Taken together, these points provide solid support for our hypothesis.
The primary PO activity in insects, shrimp, and crayfish is due to a PPO protein in
expressed in hemocytes. In insects, PPO occurs primarily in hemocytes and plasma,
Archives of Insect Biochemistry and Physiology
110
Archives of Insect Biochemistry and Physiology, July 2011
100
100
75
Md-PPO
Sb-PPO1
Ag-PPO3
Dipteran insect
Hd-PPO1
Hd-PPO2
Tm-PPO
98
80
Am-PPO
Hc-PPO1
92
Bm-PPO1
100
Ms-PPO1
53
Gm-PPO1
52
Pi-PPO
82
Of-PPO
57
Bm-PPO2
Ms-PPO
100
Hc-PPO2
80
Gm-PPO2
22
Ha-PPO2
56
Sl-PPO
100
Se-PPO
99
Sb-PPO2
100
Ag-PPO4
100
Ag-PPO5
90
Ars-PPO4
Ag-PPO7
100
67
Ag-PPO8
100
Ag-PPO9
46
Ars-PPO3
98
Ars-PPO6
Ag-PPO1
93
98
Ag-PPO2
Ag-PPO6
51
Ars-PPO2
90
Aa-PPO1
99
83
Ars-PPO1
Aa-PPO2
61
Ars-PPO5
98
90
Coleopteran insect
97
Lepidopteran insect
PPO 1
Lepidopteran insect
PPO 2
Dipteran insect
Pl-PPO
0.1
Figure 2. Neighbor-joining phylogenetic tree of PPO amino acid sequences from different species of
Crustacea and Insecta, based on Poisson-corrected protein distances. Crustacea: Pl-PPO Pacifastacus
leniusculus (X83494); Insecta: Aa-PPO1 and Aa-PPO2 Aedes aegypti (AF292114 and AF292113); Ag-PPO1, AgPPO2, Ag-PPO3, Ag-PPO4, Ag-PPO5, Ag-PPO6, Ag-PPO7, Ag-PPO8, and Ag-PPO9 Anopheles gambiae
(AF004915, AF004916, L76038, AJ010193, AJ010194, AJ010195, AJ459960, AJ459961, and AJ459962);
Am-PPO Apis mellifera (AY242387); Ars-PPO1, Ars-PPO2, Ars-PPO3, Ars-PPO4, Ars-PPO5, and Ars-PPO6
Armigeres subalbatus (AF260567, AF286468, AY487171, AY487172, DQ862064, and DQ862065); Bm-PPO1,
Bm-PPO2, Bombyx mori (D49370, and D49371); Gm-PPO1 and Gm-PPO2 Galleria mellonella (AF336289 and
AY371489); Ha-PPO2 Helicoverpa armigera (DQ114946); Hc-PPO1 and Hc-PPO2 Hyphantria cunea (U86875
and AF020391); Hd-PPO1 and Hd-PPO2 Holotrichia diomphalia (AB079664 and AB079665); Md-PPO Musca
domestica (AY494738); Ms-PPO and Ms-PPO1 Manduca sexta (L42556 and AF003253); Of-PPO Ostrinia
furnacalis (DQ333883); Pi-PPO Plodia interpunctella (AY665397); Sb-PPO1 and Sb-PPO2 Sarcophaga bullata
(AF161260 and AF161261); Se-PPO Spodoptera exigua (EF684939); Sl-PPO1 Spodoptera litura (AY703825),
and Tm-PPO Tenebrio molitor (AB020738). Numbers at tree nodes refer to bootstrap values from 1,000
replications. Scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site.
and the latter has been used as a source to isolate PPO (Jiang et al., 1997). To date,
PPO has been purified and characterized from a number of insect species (Durrant
et al., 1993; Chase et al., 2000; Feng et al., 2008). The tissue distribution and temporal
expression of PPO vary among taxa (Gillespie et al., 1997; Sidjanski et al., 1997), but its
function in immunity is believed to be the same. PO activity occurs in hemocytes,
epidermis, cuticle, intestine and venom (Parkinson et al., 2001). The most general
Archives of Insect Biochemistry and Physiology
Prophenoloxidase Expression in Parasitized O. furnacalis
111
Figure 3. Expression of OfPPO mRNA in four O. furnacalis tissues. (A) Gels showing mRNA in each tissue
as a function of time post-parasitization. (B) Relative expression of PPO mRNA. Each point is an average
of three assays. RPL8 was used as a control in all tissues. CK, unparasitized larvae; larvae at 4 h, 6 h, 8 h, 12 h,
1 day, 2 days and 3 days post-parasitization.
finding is that insects certainly express genes encoding PPO; however, the mechanisms
responsible for expressing the genes and activating the zymogens are not yet
understood in detail.
PPO cDNAs from species representing Lepidoptera, Diptera, and Coleoptera have
been cloned and sequenced (Hall et al., 1995; Kawabata et al., 1995; Jiang et al., 1997;
Chase et al., 2000; Cui et al., 2000; Hartzer et al., 2005; Lourenc- o et al., 2005; Doucet
et al., 2008; Shelby and Pophom, 2008). The OfPPO cDNA has a strong homology
with the known PPOs from other lepidopteran species. The general topology of the
phylogenetic tree is similar to previously published molecular phylogenies based on
PPO (Lourenc- o et al., 2005). The results of the phylogenetic analysis are consistent
with traditional concepts of taxonomy.
The temporal profiles of OfPPO expression pp, both at the mRNA and protein
activity levels, are instructive in understanding the roles of PPO in immune
mechanisms. Inhibition of OfPPO transcript in hemocytes at 4 h pp is apparently
due to maternal injection, along with parasitoid eggs, of physiologically active factors
such as calyx fluid, venom, and ovarian protein into hosts. These factors operate to
suppress host innate defense mechanisms. The following gradual recovery by 12 h is
consistent with the idea that a single injection of inhibitory factors is not sufficient to
Archives of Insect Biochemistry and Physiology
25.672.6
24.374.6
22.374.1
22.175.2
24.372.1
23.274.1
24.675.7
a(c)
a(c)
a(b)
a(c)
a(de)
a(d)
a(c)
Control
hemocytes
23.472.7
22.673.4
21.172.9
12.375.1
11.570.8
8.271.5
7.571.3
a(bc)
a(bc)
a(b)
b(ab)
b(ab)
c(ab)
c(a)
Test
hemocytes
37.875.6
37.574.8
38.471.9
36.477.2
38.675.6
35.371.5
35.974.2
a(d)
a(d)
a(d)
a(d)
a(f)
a(e)
a(d)
Control
plasma
34.377.1
33.572.6
32.676.2
32.473.5
27.871.5
20.374.3
18.672.8
a(d)
a(d)
a(c)
a(d)
b(e)
c(cd)
c(b)
Test
plasma
20.471.5
20.974.6
19.671.9
20.372.5
19.473.4
18.372.9
20.372.1
a(bc)
a(bc)
a(b)
a(c)
a(cd)
a(c)
a(bc)
Control
integument
18.774.6
17.573.2
17.675.2
16.473.8
16.274.9
10.371.5
9.472.5
a(b)
a(b)
a(b)
a(bc)
b(bc)
c(b)
c(a)
Test
integument
16.972.5
17.673.6
17.272.5
18.372.7
19.473.1
16.771.5
17.372.4
a(b)
a(b)
a(b)
a(bc)
a(cd)
a(c)
a(b)
Control
midgut
9.170.5
8.671.6
7.472.4
7.770.7
6.671.6
5.371.9
8.772.5
a(a)
a(a)
a(a)
a(a)
b(a)
c(a)
a(a)
Test
midgut
9.670.2
8.871.3
8.671.6
7.971.5
8.372.4
7.472.2
8.471.7
a(a)
a(a)
a(a)
a(a)
a(a)
a(ab)
a(a)
Control
fat body
9.471.3
9.870.5
8.670.9
8.571.5
7.271.2
7.370.7
6.470.2
a(a)
a(a)
a(a)
a(a)
a(a)
a(ab)
a(a)
Test
fat body
Values represent mean enzyme activity (U/mg)7SD values within a column followed by different letters are significantly different, and those followed by different letters in
parentheses indicate significant difference among tissues.
4h
6h
8h
12 h
1d
2d
3d
Time
postchallenge
Table 2. Phenoloxidase Activity in Fifth-Instar O. furnacalis Larval Tissues at the Indicated Time Points Following Parasitization by M. cingulum
112
Archives of Insect Biochemistry and Physiology, July 2011
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Prophenoloxidase Expression in Parasitized O. furnacalis
113
Figure 4. Prophenoloxidase mRNA expression in integument (A), fat body (B), midgut (C), and hemocytes
(D) of O. furnacalis larvae post-parasitization by M. cingulum at 4 h (column 2), 6 h (column 3), 8 h (column 4),
12 h (column 5), 1 d (column 6), 2 d (column 7), and 3 d (column 8) pp. Unparasitized, control larvae
are shown in column 1. Transcripts were detected as described in M&Ms. E, epithelium; Ep, epithelial cell;
Ed, endothelial cell; M, muscles; Oe, oenocytoids; Gr, granular hemocytes; Pl, plasmatocytes. Scale bars:
A, B, C 5 100 mm and D 5 40 mm.
Figure 5. Localizaiton of PPO in the integument (A) and midgut (B) of O. furnacalis larvae parasitized
by M. cingulum at 4 h (A3, B3), 6 h (A4, B4), 8 h (A5, B5), 12 h (A6, B6), 1 d (A7, B7), 2 d (A8, B8), and 3 d
(A9, B9) pp. The negative control experiments (A1, B1) were conducted in the same fashion except anti-PPO
IgG was replaced by the nonimmune mouse IgG at the same concentration. Unparasitized, control larvae are
shown in A2 and B2. Immunolabeling was carried out as described in M&Ms. Space bars: A1, B2, B4, B7,
and B8 5 2 mm; and A2, A3, A4, A5, A6, A7, A8, A9, B1, B3, B5, B6, and B9 5 5 mm.
permanently bar expression of PPO mRNA. Subsequently, OfPPO transcript level
declined at 2 d pp. We infer that other mechanisms, possibly nutritional ones related to
the presence of parasitoid larvae within the body, lead to reduced mRNA expression.
Parasitization of lepidopteran larvae by endoparasitoids usually leads to suppression of host cellular and humoral immunity. In this study, inhibition of PO activity
occurred over time in the hemocytes, plasma, integument, and midgut of the larvae
following parasitization. Immunoelectron labeling showed that the lowest number of
gold particles in integument and midgut in the parasitized larvae was detected at 1 d
and 12 h pp, respectively. Our previous study also showed that PO activity and
melaninization in larval hemolymph were inhibited by injection of calyx fluid or the
mixture of M. cingulum calyx fluid plus venom (Feng et al., 2004). We hypothesized the
calyx fluid was the main factor responsible for inhibiting host immunity. Reduced
hemolymph melanization in the parasitized host accompanied by reduced PO activity
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Archives of Insect Biochemistry and Physiology, July 2011
Table 3. Localization of PPO Within Integument and Midgut of O. furnacalis Following
Parasitization by M. cingulum
Number of PPO-gold particles
Time post-challenge
Control
4h
6h
8h
12 h
1d
2d
3d
Integument
Midgut
294.3719.6 a
264.776.8 b
180.377.4 c
172.076.2 c
88.0710.5 d
25.074.6 e
65.776.0 f
91.078.5 d
218.0710.5 A
146.776.1 B
127.076.6 C
110.774.0 D
47.074.6 G
59.073.6 F
78.074.6 E
128.373.5 C
Values represent mean numbers of immunolabeled gold particles7SD, n 5 10. Tissue sections were prepared as
described in M&Ms. Values within a column followed by different letters are significantly different.
has been recorded in other insects. For example, M. sexta larvae, parasitized by Cotesia
congregata, exhibited reduced rates of hemolymph melanization and monophenoloxidase activity (Beckage et al., 1990). Stoltz and Cook (1983) reported similar results in
Trichoplusia ni, parasitized by Hyposoter exiguae. In some host/parasite systems, the
functions of PDV (Beckage et al., 1990) or teratocytes (Kitano et al., 1990) have been
studied. Stoltz and Guzo (1986) found markedly slower rates of melanization in
hemolymph from the host Malacosoma disstria parasitized by H. fugitivus, and attributed
this to the presence of PDV and to the enhanced stability of the normally labile
oenocytoids, hemocytes involved in the synthesis, storage and release of PPO.
However, there is no PDV in the maternal substances of M. cingulum (Hu et al., 2003).
We infer that this parasitoid has evolved any of several alternative strategies to reduce
host PO activity. These may include transcriptional, translational, or post-translational
interference of PO or depletion of the phenolic substrates used by the enzyme (Shelby
et al., 2000). The transcription level of PPO may be inhibited directly by physiological
factors of parasitic wasps. Ovarian calyx fluid of the ichneumonid endoparasitoid
Venturia canescens can alter host hemocyte spreading and inhibit host hemolymph
melanization due to the presence of a serine proteinase inhibitor (Beck et al., 2000).
POs have been studied primarily in hemolymph. Their presence in the cuticle was
first suggested by Lai-Fook (1966). Since then, cuticular POs have been extracted and
partially purified from some insect species, and they are similar to the hemolymph
POs. Cuticle also contains the PPO activating cascade. Asano and Ashida (2001) did not
detect PPO transcripts in epidermal cells by Northern blot analysis of total RNA; they
thought the cuticular PPO was synthesized in hemocytes and the PO isoforms were
transported from the hemolymph to the cuticle via the epidermis. The detection of
PPO mRNAs in the epidermis of Anopheles stephensi (Cui et al., 2000), in the midgut of
A. gambiae (Müller et al., 1999) and in the fat body of A. stephensi (Cui et al., 2000)
suggest additional sites of PPO synthesis. Working with the malaria mosquito
A. stephensi, Cui et al. (2000) reported low levels of PPO expression in epidermal
and ovarian tissues, as detected by ISH. Ashida and Brey (1995) demonstrated the
presence of PPO in the cuticle matrix of the silkworm, but did not detect PPO mRNA
in the epidermis by Northern blot analysis. In B. mori, immunolocalization assays
shows PPO in the hemocytes, and electron micrographs of hemocyte sections reveal
Archives of Insect Biochemistry and Physiology
Prophenoloxidase Expression in Parasitized O. furnacalis
115
that PPO is present in both the cytoplasm and nucleus of plasmtocytes and oenocytoids
(Ashida et al., 1988). In O. furnacalis, PPO is present on the cell membrane of larval
granulocytes and oenocytoids (Feng and Fu, 2004). In this, the OfPPO transcripts were
detected in hemocytes and midgut of O. furnacalis by ISH and by qPCR. Although PO
activity was detected in the integument, the OfPPO transcript was not found in the
integument of O. furnacalis by real time-PCR, from which we infer that PPO is
synthsized from other cells and transported to the integument.
We investigated the presence of PPO in the integument and midgut of O. furnacalis
larvae parasitized by M. cingulum using immunoelectron microscopy. In M. sexta, PPO
binds to the surface of hemocytes (Ling and Yu, 2005). Protein sequence analysis
showed that the OfPPO protein does not contain a signal secretion sequence or
transmembrane domains. Thus, PPO may bind to hemocyte surface via direct
interaction with hemocyte surface molecules or with the help of other hemolymph
proteins (Ai et al., 2009). Jiang et al. (1997) concluded that PPO of M. sexta does not
contain a signal secretion sequence, and may be released from hemocytes into the
hemolymph by rupture. This was confirmed by Shrestha and Kim (2009) in their
report that eicosanoids mediate oenocytoid rupture to release PPO from oenocytoids
in S. exigua. Although there may be a transport mechanism for PPO, it is more likely
that these tissues express PPO mRNA and provide PPO to satisfy the defense needs of
the lepidopteran larva. Some hemolymph proteins are transported from the hemolymph to the cuticle and vice versa in lepidopterans (Csikós et al., 1999). The cuticular
epidermal cells did not contain gene transcripts encoding PPO subunits, from which it
was inferred that cuticular PPO is transepithelially transported from hemolymph.
Asano and Ashida (2001) found a similar result in B. mori. The transportation of
hemolymph PPO to the cuticle has been suggested (Ashida and Brey, 1995). PPO in
O. furnacalis hemolymph seems to move to the cuticle by an unknown mechanism.
As suggested by Csikós et al.. (1999), insect epithelium exports and imports proteins
in a continuous and dynamic manner.
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Archives of Insect Biochemistry and Physiology
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expressions, hymenopterabraconidae, cingulum, ostrinia, parasitization, corn, macrocentrus, furnacalis, prophenoloxidase, borel, influence, asia
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