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Exogenous JH and ecdysteroid applications alter initiation of
polydnaviral replication in an endoparasitoid wasp, Cotesia
plutellae (Braconidae: Hymenoptera)
Bokri Park & Yonggyun Kim*
Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea
Polydnaviruses are a group of double-stranded DNA viruses
and are symbiotically associated with some ichneumonoid
wasps. As proviruses, the replication of polydnaviruses occurs
in the female reproductive organ at the pupal stage. This study
analyzed the effects of two developmental hormones, juvenile
hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site (‘AGCTTT’)
from their proviral segments. Using quantitative real-time PCR
based on this excision/rejoining site marker, initiation of CpBV
replication was determined to have occurred on day 4 on the
pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited
adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had
no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays
against viral coat proteins support the effects of the hormone
agonists on viral replication. [BMB reports 2011; 44(6): 393398]
INTRODUCTION
Polydnaviruses (PDVs) are a group of unique double-stranded
DNA viruses symbiotic with some endoparasitoid wasps (1). It
comprises two genera, Ichnovirus (IV) and Bracovirus (BV), depending on the host wasp family, Ichneumonidae or Braconidae (2). These two PDV taxa are also characterized by viral
morphology and serological independence or DNA hybridization analyses, suggesting their independent origin (3). IV virions are relatively uniform in size and contain biconvex nucleocapsids surrounded by two unit membranes, whereas BVs
are highly variable in length and have cylindrical nucleocap*Corresponding author. Tel: 82-54-820-5638; Fax: 82-54-820-6320;
E-mail: [email protected]
DOI 10.5483/BMBRep.2011.44.6.393
Received 3 January 2011, Accepted 11 April 2011
Keywords: Cotesia plutellae, CpBV, Ecdysteroid, JH, Polydnavirus,
Replication
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sids surrounded by a single envelope (4). The outer membranes of IVs are acquired by budding off from calyx cells. In
comparison, the single envelope of BV suggests its release into
the oviduct lumen by cell lysis (5).
PDVs are also unique in their viral transmission mode. Their
full genomes are located on their host chromosome(s) and
maintain vertical transmission along with wasp generation (6).
However, their replication into viral particles for horizontal
transmission to their specific lepidopteran hosts occurs only in
the ovarian calyx region during the host pupal stage (7, 8).
Cotesia plutellae (Braconidae: Hymenoptera) is a solitary
endoparasitoid wasp that parasitizes the diamondback moth
Plutella xylostella (9). C. plutellae bracovirus (CpBV) has been
previously identified and is known to be replicated in the ovarian calyx during the late pupal stage (10). It is regarded as a
major factor in reducing host cellular immune capacity in parasitized P. xylostella (11, 12). For example, CpBV15β effectively inhibits hemocyte-spreading behavior in P. xylostella
(13). CpBV-lectin exhibits early expression during parasitization and has been suspected to inhibit non-self recognition in P. xylostella (14, 15). CpBV-PTPs impair cellular immune processes such as phagocytosis and encapsulation probably by altering the phosphorylation status of target proteins
within hemocytes (16, 17). CpBV-H4, CpBV-E94K, and CpBVELP1 are speculated to inhibit host immune capacity (18, 19).
Recently, CpBV-RNase T2 was shown to inhibit both the humoral and cellular immune responses of P. xylostella (20).
Despite significant roles of PDVs in the parasitism of endoparasitoids, little has been shown about endocrine signaling in
PDV replication. Juvenile hormone (JH) and ecdysteroid are
key endocrine signals in insect development during the immature stages (21). In Campoletis sonorensis IV, ecdysteroid
was shown to stimulate PDV replication in an in vitro organ
culture assay and a thoracic ligation assay (7). In Chelonus inanitus BV, however, ecdysteroid may be not a direct endocrine signal for the initation of viral replication based on the
titer measurements during pupal development (22).
In this study, we performed direct application of hormonal
analogs during the adult development of C. plutellae. To determine CpBV replication, two molecular processes were
monitored. One monitored a specific stage of CpBV genome
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Polydnavirus replication in Cotesia plutellae
Bokri Park and Yonggyun Kim
amplification by quantitative polymerase chain reaction
(qPCR), whereas the other used PCR to analyze a specific stage
of CpBV genome excision/rejoining at the viral excision/rejoining DNA site, where the proviral CpBV was replicated into its
episomal viral form. Using these molecular monitoring techniques, this study analyzed the effects of JH and ecdysteroid on
initiation of CpBV replication within the host C. plutellae.
RESULTS
Excision/rejoining sites of different CpBV segments are
conserved
Molecular processes of PDV replication include excision of
amplified PDV segments from wasp chromosome(s) (8). The
excision sites appear to be conserved among different segments and even different PDV species (23). The conserved sequence ('AGCTTT') was searched in the CpBV segments (Fig.
1). All 23 known CpBV segments contained this putative excision/rejoining site sequence as well as consensus sequences
around the excision sites.
To test the putative excision/rejoining sites, CpBV-S3 was
chosen and used to design two segment-specific primer sets:
rFP1/rRP1 for the non-excision site and rFP2/rRP2 for the excision site (Fig. 2A). PCR using non-excision site primers resulted in a consistent predicted product during the different developmental stages of C. plutellae. However, PCR using excision site primers showed the predicted PCR products only in
the late pupal periods (P4 and P5) and adult stage, during
which viral replication occurred in the ovary of the abdomen.
This suggests that CpBV replication began 4 days after pupal
ecdysis. TEM study showed that the pupal female ovary on day
5 was actively producing virus, as detected from virogenic
stroma in the nucleus (Fig. 2B).
show any effect at any pupal stages on the adult development
of C. plutellae (data not shown). When the hormone agonists
were treated on day 1 of the pupae stage, pyriproxyfen had a
significant inhibitory effect at concentrations above 0.01 μg.
On the other hand, RH5992 did not have any effect on the
adult development at concentrations in the 0.0001-1 μg range
(data not shown). In response to 0.1 μg hormonal treatments,
the pupal developmental rate was completely inhibited by the
JH analog but appeared to be slightly accelerated by the ecdysteroid agonist, especially 5 days after treatment (data not shown).
The effects of both hormones on CpBV replication were analyzed in terms of CpBV segment amplification, excision, and
viral capsid formation (Fig. 3). Using rFP1/rRP1 primers with
qPCR, CpBV segment amplification was initiated in 4-day-old
control pupae (left panel in Fig. 3A). However, JH agonist
CpBV replication is acclerated by ecdysteroid, but delayed by
JH
To analyze the effects of endocrine signaling on CpBV replication, we began by testing the effects on adult development
of C. plutellae. We topically applied both JH and ecdysteroid
agonists to wasp pupae. Pyriproxyfen, a JH agonist, clearly inhibited adult development, especially during the young pupae
stage. However, RH5992, an ecdysteroid agonist, did not
Fig. 1. A putative conserved rejoining site of 23 different CpBV
segments. Alignment of nucleotide sequences showing a conserved 'AGCTTT' sequence.
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Fig. 2. Replication of CpBV. (A) A molecular marker using inverse PCR with rFP2 and rRP2 primers around the rejoining site
of CpBV segment #3 (CpBV-S3). In contrast, a non-rejoining site
was amplified using rFP1 and rRP1 primers. Larva (L), 1 to 5-dayold pupa (P1-P5), and male and female adults were analyzed using these primer sites. Further, in female adults, different body
parts were analyzed by PCR. (B) TEM structure of the ovary of
5-day-old pupa. ⓐ A nucleoplasm structure (30,000x) showing a
virogenic stroma (‘vs’) ⓑ a structure (50,000x) around ‘vs’. ⓒ release of viral particles (95,000x) from ‘vs’ ⓓ nucleocapsids (‘nc’)
in the nucleoplasm (70,000x) near ‘vs’. Size of scale bars are denoted in each Figure.
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Polydnavirus replication in Cotesia plutellae
Bokri Park and Yonggyun Kim
Fig. 3. Effects of two hormone agonists on CpBV replication during the pupal period (day 1-day 5: D1-D5) of Cotesia plutellae.
Pyriproxyfen (PYR, a JH agonist) and RH5992 (an ecdysteroid agonist) were topically applied on 1-day-old pupae at 0.1 μg. (A)
Measurement of CpBV-S3 amplification using rFP1 and rRP1 primers or excision using rFP2 and rRP2 primers by quantitative real-time PCR. Each treatment was replicated three times. The PCR
products were visualized on agarose gel by staining with ethidium bromide. (B) Immunoblotting of CpBV coat proteins (left
lane, Coomassie staining) using CpBV polyclonal antibody. Two
coat proteins (right lane, immunoblotting) were detected, as indicated by thick, stained bands at approximately 30 and 35 kDa.
(C) Effects of two hormones on synthesis of viral coat proteins.
treatment delayed the process by 1 day. Using rFP2/rRP2 primers, viral segment excision also began in 4-day-old control
pupae (right panel in Fig. 3A). The ecdysteroid agonist accelerated the amplification process by almost 1 day compared to
the control. These hormonal effects on viral DNA replication
were further supported by the formation of viral coat proteins,
as detected by immunoblotting assay. Two coat proteins were
detected using polyclonal antibody, in which a dark staining
band was located at approximately 30 (clear band) and 35 kDa
(faint band) (Fig. 3B). Control pupae synthesized coat proteins
on day 4 (Fig. 3C). PYR treatment delayed the synthesis of coat
proteins, whereas RH5992 treatment accelerated coat protein
production.
DISCUSSION
The genomes of PDVs, as proviruses, are present in host wasp
chromosome(s) (24). In BVs, viral integration of an ancestral
nudivirus type was estimated to have occurred 100 MYA ago
(25). The integrated viral genome segments are amplified, excised, and then encapsulated during replication without reentry back into wasp host chromosomes (26). Thus, viral replication occurs for the horizontal transfer of viral particles to the
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parasitized lepidopteran host, whereas the integrated form of
the viral genome is vertically transmitted along with wasp generation (1). This study showed a virogenic stroma in the nuclei
of pupal ovarian follicles of C. plutellae around the calyx area.
The virogenic stroma, which possesses viral particles undergoing viral assembly and release processes, looks similar to
those of other PDVs (27).
Viral production during the pupal stage of C. plutellae was
supported by the excision period of CpBV segments. All 23
CpBV segments shared a putative excision site ('AGCTTT'),
which has been reported in other PDVs (23). In CpBV, PCR
analysis around the excision site clearly showed the products
on P4 or later, indicating initiation of viral segment replication.
Our qPCR and immunoblotting data also show that segment
amplification of CpBV as well as viral coat protein synthesis
began on P4. All previously analyzed PDVs begin their replication during the pupal stage (8, 28-31). For PDV replication,
including CpBV, a viral DNA polymerase or any associated
factor(s) that use host DNA polymerase are necessary. However,
no PDV-possessing DNA polymerase has been identified since
its gene is not likely to be encapsulated in viral particles (32).
Current approaches for identifying PDV genomes using ovarian EST or analysis of BAC clones containing PDV segments
clearly suggest that viral coat protein genes, which are not encapsulated, are actively expressed during viral replication (33,
34).
Viral DNA amplification during replication has not been observed for PDVs. A study on CiBV prevoiusly proposed two
hypothetical models of viral DNA amplification: amplification
of the clustered viral genome area followed by excision of
each segment, and alternatively, a rolling circle model after excision of the clustered area (22). Partial amplification of the viral genome area in the wasp chromosome may be understood
by the molecular process of chorion gene amplification during
choriogenesis of Drosophila melanogaster (35). Upon amplification signaling, DNA replication occurs at multiple origins,
followed by repetitive amplification (36). Our qPCR data indicate that amplification and excision began on the same day
(day 4 pupal stage, 'P4'). Moreover, immunoassay against the
viral particles indicated that P4 pupae contained viral coat
proteins. Considering that viral coat protein genes are not encapsidated in other PDVs, including CpBV, the viral coat protein genes must have been actively expressed when the CpBV
was amplified and excised.
RH5992 accelerated CpBV replication while pyriproxyfen
delayed it. RH5992, an ecdysteroid analog, did not affect the
adult emergence rate, but it did significantly accelerate CpBV
production, including amplification, excision, and viral coat
protein formation. By measuring ecdysteroid titers during the
pupal stages of C. inanitus, ecdysteroid peaks in the early pupal stages were found to occur during intensive cell proliferation and differentiation of the ovarian calyx (22). Thus,
the effect of ecdysteroid on PDV replication, including CpBV
replication, in this study may be understood in terms of early
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Polydnavirus replication in Cotesia plutellae
Bokri Park and Yonggyun Kim
ovarian morphogenesis for preparation of subsequent massive
DNA replication. Alternatively, the ecdysteroid peak may be
directly associated with viral replication only in the ovarian calyx, as other tissues in wasp also possess the CpBV genome. In
choriogenesis of D. melanogaster, only the follicle cells in the
terminal oocytes undergoing DNA amplification of the chorion
gene express Broad-Complex by the EcR-USP complex in response to ecdysteroid signaling (37). The Broad-Complex induces expression of endoreplication genes, which in turn recognize the replication origin and induce gene amplification
(38). The inhibitory action of the JH agonist against viral replication may be explained based on its interference of Broad
gene expression as well demonstrated in tissue remodeling
during metamorphosis (39). Ecdysteroid signaling during cellular processes may occur in the ovarian calyx cells of C. plutellae to amplify the CpBV genome, followed by excision and
viral particle formation. This hypothesis needs to be explored
in a future study.
MATERIALS AND METHODS
Insect rearing
P. xylostella larvae were reared on cabbage leaves at 25 ± 1oC
under a photoperiod of 16：8 (L：D) h. Adults were fed 10%
sucrose solution. Late second instar P. xylostella larvae (4 days
o
after oviposition at 25 ± 1 C ) were parasitized by C. plutellae
at a 1：2 (wasp: P. xylostella) ratio for 24 h. Adults emerged
o
from their cocoons (11 days after parasitization at 25 ± 1 C), were
collected, and allowed to mate for 24 h before parasitization.
Hormone treatment
Pyriproxyfen (95% technical, Dongbang-Agro, Seoul, Korea)
and RH5992 (96% technical, Kyungnong, Seoul, Korea) were
used as agonists of JH and ecdysteroid, respectively. Different
concentrations of both analogs were dissolved in acetone and
topically applied to pupae of C. plutellae in a 5 μl volume.
Genomic DNA (gDNA) extraction
From the different developmental stages of C. plutellae, gDNAs
were extracted using extraction buffer (10 mM Tris, 0.1 M
EDTA, 20 μg/ml RNase, 0.5% SDS, pH 8.0), in which each developmental sample consisted of 100 larvae, 100 pupae, or 30
adults. Larvae were 7-8 days old after parasitization. Pupae of
different ages (from 1 to 5 days) were chosen after pupation.
One-day-old adults after emergence were separated into male
and females. After proteinase K treatment, gDNAs were purified by phenol extraction and ethanol precipitation.
Test PCR for excision/rejoining site and quantitative real-time
PCR (qPCR)
For analysis of excision process of CpBV DNA replication,
qPCR was designed using primers (rFP2: 5'-TCG GTC CAA
ATA CGG TGT AG-3' and rRP2: 5'-GGA GAG AGA AGC ATA
TGC AGA G-3') specific to CpBV DNA segment #3 (CpBV-S3)
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(see Fig. 2A). Using gDNAs isolated from the different developmental stages of C. plutellae, multiple PCR reactions were
o
performed consisting of 35 cycles of 30 s at 94 C, 30 s at
o
o
53 C, and 1 min at 72 C.
To monitor amplification and excision rates of CpBV-S3
within the ovary, qPCR reactions were performed with primers
specific to the non-excision site (rFP1: 5'-GAC GTC TTA GTG
TGA ACG AT-3' and rRP: 15'-CAT TCC TTC CAG CTT CAC
TG-3') or with rFP2 and rRP2 primers specific to the excision
site (see Fig. 2A) using a Real-Time PCR ABI Prism 7500
(Prism 7500, Applied Biosystems, Foster City, CA, USA) along
TM
with SYBR green chemistry of Accupower Greenstar PCR
premix (Bioneer) and real-time fluorescence measurements.
TM
Each 20 μl reaction mixture consisted of 1x Greenstar PCR
Master Mix, 10 mM MgCl2, 0.5 mM of primers, and 90 ng of
o
DNA. Initial incubation at 95 C for 15 min was carried out to
activate Hotstart Taq DNA polymerase. qPCRs consisted of 40
cycles of 30 s at 94oC, 30 s at 50oC (amplification site)/53oC
o
(excision site), and 1 min at 72 C. β-Actin gene was used as a
control with primers: 5'-TGG CAC CAC ACC TTC TAC-3' and
5'-CAT GAT CTG GGT CAT CTT CT-3'. Each cycle was scanned to quantify the PCR products of each treatment with three
replications. Amplification plots in real-time were constructed
Ⓡ
using ABI PRISM 7500. Quantitative analysis of DNA amplification or excision frequencies was carried out using the comparative CT (ΔΔCT) method (40).
Ultrastructure of ovarian calyx cells using transmission
electron microscope (TEM)
Ovaries were isolated from 5-day-old pupae and fixed in 2.5%
glutaraldehyde in 0.01 M phosphate buffer (pH 7.2) for 2 h at
o
4 C. After several washes in phosphate buffered saline (PBS,
100 mM phosphate, 0.7% NaCl, pH 7.4), the ovaries were
o
post-fixed with 1% osmium tetroxide for 30 min at 4 C and
then washed again with PBS. The ovary samples were then dehydrated through a graded ethanol series, substituted with propylene oxide, and then embedded in Epon 812 for 48 h at
o
60 C. Sections (＜90 nm) were cut on a Leica Ultracut UCT ultramicrotome using a diamond knife (Ultracut UCT, Leica
Microsystems, Wetzlar, Germany). Ultrathin sections were
transferred onto 200 mesh copper grids and stained with uranyl acetate for 20 min, and then with lead citrate for another
10 min. The sections were examined by TEM (H-7650, Hitachi,
Tokyo, Japan) at 80 kV.
Immunoblotting analysis of CpBV coat proteins
Proteins were extracted from pupae and adults of different
ages using PBS, in which each sample consisted of 100 pupae
or 100 adults. The extracted protein samples were diluted with
PBS and mixed with the same volume of denaturing buffer
(4% SDS, 20% glycerol, 10% β-mercaptoethanol in 62.5 mM
o
Tris-HC1, pH 6.8). After boiling for 5 min at 95 C, the samples
(50 μg per lane) were separated by 10% SDS-PAGE. Electrophoresis was performed under denaturing conditions (41) until
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Polydnavirus replication in Cotesia plutellae
Bokri Park and Yonggyun Kim
tracking dyes migrated to the end of the gel. The separated
proteins on the gel were transferred onto nitrocellulose paper
by the method of Towbin et al. (1979) (42). Non-specific sites
were blocked by 5% skim milk for 1 h at room temperature.
After three washes with PBS, the membrane was incubated for
2 h at room temperature with polyclonal antibody recognizing
coat proteins of CpBV (43). The polyclonal antibody was raised
in rabbit using an antigen sample of CpBV viral particles isolated from the ovarian calyx of C. plutellae. After three washes
with PBS, the membrane was incubated for 1 h at room temperature with secondary antibody (1/2,000 dilution) conjugated
with alkaline phosphatase. After three washes with PBS, the
membrane was stained with an alkaline phosphatase substrate
solution containing nitro blue tetrazolium/5-mono-4-chloro-3indolyl phosphate (NBT/BCIP, Sigma-Aldrich Korea, Seoul,
Korea) in 10 mM phosphate buffer (pH 9.5).
Sequence analysis of CpBV segment excision site
The conserved PDV excision site sequence obtained from previous related PDVs (8, 44) was used to locate sites in different
CpBV segments (NCBI accession number): S2 (DQ075354), S3
(DQ075355), S4 (DQ075356), S5 (DQ075357), S8 (DQ0753
58), S9 (DQ075359), S10 (EF067319), S11 (DQ075360), S14
(EF067320), S16 (EF067321), S21 (EF067322), S22 (EF067323),
S27 (DQ067324), S28 (AY651829), S30 (AY651828), S33 (AY
651830), S35 (EF067325), S36 (EF067326), S37 (EF067327),
S38 (EF067328), S41 (EF067329), S48 (EF067330), S50 (EF06
7331), and S51 (EF067332). Multiple alignment of these terminal CpBV repeats was performed using the DNAstar program
(Version 5.02, DNAstar Inc., Madison, WI, USA).
Statistical analysis
The means were compared by a least squared difference (LSD)
test of one way ANOVA using PROC GLM of SAS program
(45) and discriminated at Type I error = 0.05.
Acknowledgements
This study was funded by the AGENDA 2010 program of the
Rural Development Administration, Suwon, Korea. Y. Kim and
B. Park were supported by the second stage BK21 program of
the Ministry of Education, Science and Technology, Korea.
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