An ovarian protein involved in passive avoidance of an endoparasitoid

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An ovarian protein involved in passive avoidance of an endoparasitoid to evade its host immune response Ziwen Teng, Huizi Wu, Xinhai Ye, Shijiao Xiong, Gang Xu, Fang Wang, Qi Fang, and Gong-yin Ye J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00824 • Publication Date (Web): 06 Jun 2019 Downloaded from http://pubs.acs.org on June 6, 2019

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Journal of Proteome Research

An ovarian protein involved in passive avoidance of an endoparasitoid to evade its host immune response Ziwen Teng#, Huizi Wu#, Xinhai Ye#, Shijiao Xiong#, Gang Xu#, Fang Wang#, Qi Fang#, Gongyin Ye*# #State

Key Laboratory of Rice Biology & Ministry of Agriculture Key Laboratory of

Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China

Corresponding Author

* E-mail: [email protected]; Phone: (+86)-571-88982696; fax: (+86)-571-88982988.

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ABSTRACT. Through a combination of transcriptomic and proteomic analyses, we identified 817 secreted ovarian proteins from an endoparasitoid wasp, Cotesia chilonis, of which five proteins are probably involved in passive evasion. The results of an encapsulation assay revealed that one of these passive evasion-associated proteins (Crp32B), a homologue of a 32-kDa protein (Crp32) from C. rubecula, could protect resin beads from being encapsulated by host hemocytes in a dose-dependent manner. Crp32B is transcribed in ovarian cells, nurse cells, follicular cells and oocytes, and the protein located throughout the ovary and on the egg surface. Moreover, Crp32B has antigenic similarity to several host components. These results indicate that C. chilonis may use molecular mimicry as a mechanism to avoid host cellular immune response.

KEYWORDS. parasitoid wasp, ovarian protein, passive strategy, Crp32, encapsulation, mimicry Introduction To successfully develop in the host hemocoel, endoparasitoids have evolved diverse strategies in different parasitoid-host systems that can be categorized as “active” or “passive”.1,2 For the “active” strategy, parasitism factors include venoms,3 polydnaviruses (PDVs),4 teratocytes,5 virus-like particles (VLPs)6 and ovarian proteins7 that can suppress host immune responses. Numerous intensive investigations have been performed to identify and characterize PDV gene products,8,9 venom proteins,3,10 teratocyte secretory products5,11 and VLP components6,12 by classical


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approaches and high-throughput sequencing. However, compared to other parasitism factors, ovarian proteins have been paid little attention. In contrast to “active” strategies, endoparasitoids also have evolved ‘‘passive strategies” in which endoparasitoids develop in the host hemocoel but passively avoid encapsulation by the host because of their progeny surface features. The results showed that the parasitoid progeny were not recognized as non-self by host hemocytes, resulting in host hemocytes being unable to attach to the progeny. In two braconid wasps, Toxoneuron nigriceps and Macrocentrus cingulum, a surface fibrous layer of the eggs has been shown to play an important role in protecting eggs from host immune responses.13,14 In the ichneumonid wasp V. canescens, egg-covering VLP proteins were shown to be antigenically related to a host 42kDa protein, suggesting that the particles may not be recognized as a foreign substance.15 In addition to VLPs, Venturia hemomucin, a component of the mucinous layer on the egg and larval surface, can form a complex with host lipophorin and other hemolymph components. This protective mucinous layer protects parasitoid eggs from host immune responses.16 In Cotesia kariyai, an immunoevasive protein (IEP) from the ovary has been reported to protect parasitoid eggs from host cellular reaction.17,18, 19 Similarly, C. rubecula eggs are protected by a 32-kDa surface protein (Crp32). However, the molecular mechanism of Crp32-mediated protection is not well known.20 In contrast, the passive avoidance of host encapsulation in other parasitoids is mediated by molecules of extraembryonic membrane. A 97-kDa transmembrane hemomucin homologue from M. cingulum containing 51 potential O3 ACS Paragon Plus Environment


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glycosylation sites has been found to protect embryos from being encapsulated by their host, an activity in which its sugar chains play an important role.21 A polyembryonic encyrtid Copidosoma floridanum, remains enveloped by an extraembryonic membrane throughout development, which is required for protection from the host immune system.22 Cotesia chilonis (Hymenoptera: Braconidae) is an obligate larval endoparasitoid that effectively regulates the population density of the striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae), one of the most economically important rice pests in Asia, Northern Africa, and Southern Europe.23,24 In our previous studies, C. chilonis was observed to use a combination of active and passive strategies to escape from host cellular immune responses.25 Additionally, we identified two venomassociated passive avoidance-related proteins, IEP-2A (Accession No. KU663635) and IEP-2B (Accession No. KU663636) through a combination of transcriptomic and proteomic analyses.10 In the current study, we identify secreted ovarian proteins by C. chilonis through a combination of transcriptomic and proteomic approaches, resulting in the identification of five proteins that are likely involved in passive evasion. Moreover, we present evidence that Crp32B, one of the identified passive avoidancerelated proteins, may be used as a molecular mimic involved to promote the passive evasion of C. chilonis eggs from the host immune response. Our results will provide insight into a more comprehensive understanding of the secreted ovarian components and the functions of ovarian proteins associated with passive strategy to evade host immune response for parasitoid wasps. 4 ACS Paragon Plus Environment

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Experimental Section Transcriptomic Analysis of Ovaries The details of insect rearing are presented in supplementary methods. Transcripts originated from ovaries of female wasps at the later pupal (4 or 5 days after pupation, FPOvary) and adult (1 or 2 days after eclosion, FAOvary) stage, each with three biological replicates. RNA sequencing was performed by Novogene Bioinformatics Institute (Beijing, China) according to a previous study.10 Detailed procedures are presented in supplementary methods. Proteomic Analysis of the Secreted Ovarian Proteins Ovaries of female adult wasps, aged 2 days, were carefully dissected and squeezed to release the fluids from the lateral oviducts in Pringle’s phosphate-buffered saline (PBS, pH  7.4) supplemented with 1 mM phenylmethanesulfonyl fluoride (Sigma, St. Louis, MO, USA). After centrifugation at 8000 g for 10 min at 4C, the supernatant was filtered through a 0.22 m Millipore filter. The filtrate was quantified with the BCA Protein Assay kit (Bio-Rad, USA) and then was subsequently stored at -80C until use. The proteomic analysis was performed by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China). The filter-aided sample preparation method was used,26 detailed procedures for which are presented in supplementary methods. The resulting peptides as a filtrate were desalted for each sample and loaded on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma). The peptides were concentrated by vacuum centrifugation and 5 ACS Paragon Plus Environment


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reconstituted in 40 µl of 0.1 (v/v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm using an extinctions coefficient of 1.1 of 0.1 (g/l) solution that was calculated based on the frequency of tryptophan and tyrosine in vertebrate proteins. Each fraction was analyzed on a nano LC-MS/MS (liquid chromatography−mass spectrometry/mass spectrometry) system. 3 g peptide mixture was loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 m2 cm, nanoViper C18) connected to a C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 m inner diameter, 3 m resin) in buffer A (0.1 formic acid) and separated with a linear gradient of buffer B (84 acetonitrile and 0.1 formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology. The linear gradient was as follows: 0-55 buffer B for 110 min, 55100 buffer B for 5 min, followed by a hold in 100 buffer B for 5 min. LC-MS/MS analysis was performed on a Q exactive mass spectrometer (Thermo Scientific) that was coupled to an Easy nLC instrument (Proxeon Biosystems, now Thermo Fisher Scientific) for 120 min. The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300– 1800 m/z) for high-energy collisional dissociation (HCD) fragmentation. The settings used were as follows: gain control target: 1e6; maximum inject time: 50 ms; dynamic exclusion duration: 60.0 s; resolution of survey scans acquired: 70,000 at 200 m/z; 6 ACS Paragon Plus Environment

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resolution for HCD spectra: 17,500 at 200 m/z; isolation width: 2 m/z; normalized collision energy: 30 eV; and underfill ratio: 0.1%. The MS data were analyzed using MaxQuant version 1.5.3.17 (Max Planck Institute of Biochemistry in Martinsried, Germany) 27 and were searched against the translated FPOvary and FAOvary transcriptomes. We set an initial search at a precursor mass window of 6 ppm, and then performed an enzymatic cleavage rule of trypsin. The following parameters were set: MS/MS tolerance: 20 ppm; missed cleavage: 2; fixed modification: carbamidomethyl; variable modification: oxidation; and database pattern: reverse. The cutoff of the global false discovery rate (FDR) for peptide and protein identification was set to 0.01. The transcript sequences of C. chilonis genes encoding the secreted ovarian proteins were submitted to the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and deposited under the accession numbers MH365478-MH366300. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository28 with the dataset identifier PXD012914 Expression and Purification of Recombinant Proteins Single-strand cDNA was synthesized from 1 g RNA and used as template for polymerase chain reaction (PCR). The 5’ ends of cDNA sequences of C. chilonis IEP1 and IEP-2B were identified by rapid amplification of cDNA ends (RACE) using a 5’ RACE kit (Takara-Clontech, CA, USA). At least one gene specific primer was designed for each partial IEP sequence (Table S1). All amplified PCR products were 7 ACS Paragon Plus Environment


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cloned into a pGEM®-T Easy Vector (Promega Biotech Co., Ltd. Beijing, China) and sequenced. Recombinant Crp32B (rCrp32B) and C. suppressalis -tubulin (rCs-tubulin) were both expressed in Escherichia coli. The softwares and websites used for sequence analysis and detailed procedures for protein expression, purification and dialysis are presented in supplementary methods. The purified rCrp32B was submitted to GenScript Co., Ltd. (Nanjing, China) as an antigen for immunization in rabbits. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblot Analysis Protein samples were subjected to the PAGE or polyacrylamide gradient gel electrophoresis (GenScript Co., Ltd., Nanjing, China) and stained with Coomassie Brilliant Blue R-250. The proteins separated by electrophoresis were transferred to a polyvinylidene difluoride membrane (Sigma, St. Louis, MO, USA). For Crp32B detection, we used rabbit anti-rCrp32B as the primary antibody (diluted 1:5000) and goat anti-rabbit IgG (HL) (HuaAn Biotechnology, Hangzhou, China) as the secondary antibody (diluted 1:5000). For -tubulin detection, we used a -tubulin monoclonal antibody (Invitrogen, Carlsbad, CA, USA) as the primary antibody (diluted 1:5000) and goat anti-mouse IgG (HL) (GenScript Co., Ltd., Nanjing, China) or 6 His-Tag HRP (HuaAn Biotechnology, Hangzhou, China) as the secondary antibody (diluted 1:5000). The immunoblot signal was detected by


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tetramethylbenzidine stabilized substrate for horseradish peroxidase (Promega, Madison, WI, USA). Quantitative real-time PCR (qPCR) Specific primers were designed using Primer3 Input (version 0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/) (Table S1). The detailed protocol is presented in supplementary methods. 28S rRNA was used as a reference gene.10 The relative quantification in each tissue was calculated using the comparative 2CT method following the guidelines of Bustin et al..29,30 The results are presented as relative mRNA expression of three independent biological replicates. RNA fluorescence in situ hybridization (RNA-FISH) and Immunofluorescence RNA-FISH, immunohistochemistry and immunocytochemistry procedures were performed essentially as described previously,31,32 with detailed procedures presented in supplementary methods. The primary antibody was a rabbit anti-rCrp32B polyclonal antibody or a -tubulin monoclonal antibody. The secondary antibody used was an Alexa Fluor 594 conjugated goat anti-rabbit IgG (Proteintech, USA), or an Alexa Fluor 568 conjugated goat anti-mouse IgG (Thermo Fisher Scientific, USA). Actin filaments and nuclei were stained with 1  Alexa Fluor™ 488 Phalloidin (Thermo Fisher Scientific, Wilmington, DE, USA) and 1 g/ml 4’-6-diamidino-2phenylindole (DAPI, Sigma, St. Louis, MO, USA) respectively. Paraffin embedding and sectioning of ovarian calyx were performed by Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). 9 ACS Paragon Plus Environment


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In Vivo Encapsulation Assay We injected cOmplete His-Tag purification resin beads (Roche, IN, USA) or newly laid eggs of C. chilonis into C. suppressalis larvae to observe in vivo encapsulation. The beads were incubated with PBS, anti-rCrp32B serum (1: 500), rCrp32B (50, 100 and 200 ng/l) and eGFP (200 ng/l) expressed in the same bacterial expression system with rCrp32B at 4C for 2 h. To mask the rCrp32B-covered beads with antibodies, the beads were also incubated with anti-rCrp32B serum (1: 500) at 4C for another 2 h. Newly laid C. chilonis eggs were dissected out of the hemocoel of parasitized host larvae and washed with PBS at least three times. To mask the Crp32B on the egg surface, we incubated the eggs with anti-rCrp32B serum (1:500) at 4C for 2 h. All beads and eggs were washed three times in sterilized PBS before being injected into the hosts. Approximately thirty beads or newly laid eggs were injected into a host recipient, with five recipient larvae used for one treatment. Samples were recollected from recipient larvae 4 h after injection and observed under a phase contrast microscope (Nikon eclipse TS-100, Japan). To compare the extent of encapsulation, capsules of beads were classified into six classes (0–5) as described by Teng et al. (2016). The encapsulation index ()    the number of beads with a defined encapsulated grade  its corresponding grade number / total number of beads observed  5   100. Immunoprecipitation, Pull-Down Assay and LC-MS/MS


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Immunoprecipitation and pull-down assay were performed using Pierce™ Classic Magnetic IP/Co-IP kit (Thermo Fisher Scientific, Wilmington, DE, USA) and Pierce Pull-Down PolyHis Protein: Protein Interaction kit (Thermo Scientific, Rockford, Illinois, USA) according to the manufacturer’s protocols, respectively. Detailed procedures are presented in supplementary methods. After SDS-PAGE analysis, five protein bands were cut out from the gel and sent to be commercially analyzed by mass spectrometry (Sangon Biotech, Shanghai, China). Briefly, each gel slice was digested with trypsin and lyophilized. An online Nano-RPLC was used on the Eksigent nanoLC-Ultra 2D System (AB SCIEX, USA). The samples were loaded onto a C18 nanoLC trap column (100 m x 3 cm, C18, 3 m, 150 Å) and washed with Nano-RPLC Buffer A (0.1 formic acid, 2 acetonitrile) at 2 l /min for 10 mins. An elution gradient of 5-35 acetonitrile (0.1 formic acid) in 90 mins gradient was used on an analytical ChromXP C18 column (75 m x 15 cm, C18, 3 m, 120 Å) with a spray tip. Data acquisition was performed with a Triple TOF 5600 System (AB SCIEX, USA) fitted with a Nanospray III source (AB SCIEX, USA) and a pulled quartz tip as the emitter (New Objectives, USA). Data were acquired using an ion spray voltage of 2.5 kV, a curtain gas pressure of 30 PSI, a nebulizer gas pressure of 5 PSI, and an interface heater temperature of 150C. For information-dependent acquisition, survey scans were acquired in 250 ms, and as many as 35 product ion scans were collected if they exceeded a threshold of 150 counts per second (counts/s) with a 2 to 5 charge-state. The total cycle time was fixed to 2.5 s. A rolling collision energy setting was applied to all precursor ions for collision-induced 11 ACS Paragon Plus Environment


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dissociation. Dynamic exclusion was set for 1/2 of the peak width (18 s), and the precursor was then refreshed off the exclusion list. The resulting MS and MS/MS spectra were searched against the C. suppressalis transcriptome33 which has been deposited at DDBJ/EMBL/GenBank under the accession number GAJS00000000 in the MASCOT V2.3 search engine (Matrix Science Ltd., London, U.K.). The proteins were successfully identified based on 95 or higher confidence interval of their scores. The following search parameters were used: up to two missed cleavages allowed, carbamidomethyl cysteine as fixed modification, acetyl (protein N-term), deamidated, dioxidation and oxidation as variable modifications, 30ppm for precursor ion tolerance and 0.15Da for fragment ion tolerance. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository28 with the dataset identifier PXD012937. Statistical Analysis The differences between the means in qPCR analysis were compared using one-way analysis of variance (ANOVA) and Tukey’s test with differences considered significant at P  0.05. The mean results for different treatments in the encapsulation assay were analyzed by Student’s t-test with differences considered significant at P  0.05, P  0.01 or P  0.001. All statistical calculations were performed by Data Processing System (DPS) package (Version 9.5).34 Results 12 ACS Paragon Plus Environment

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Identification of Passive Avoidance-Related Proteins For the six libraries, 49.1-58.2 million clean reads were generated (Table S2), with 3,607 FAOvary genes upregulated and 3,625 downregulated compared to FPOvary genes. KEGG pathway analysis revealed that upregulated FPOvary genes mapped significantly to the ribosome, spliceosome, and proteins processing, which are related to gene transcription and translation. Compared to the FPOvary sample, cytokinecytokine receptor interaction genes were overrepresented in the FAOvary sample, with these genes being involved in innate immunity. We inferred that the adult wasps need more effective immune defenses, since they may be much more exposed to pathogens compared to pupal wasps (Table S3). We identified 817 proteins as secreted ovarian proteins (SOPs) matched the FPOvary or FAOvary by LC-MS/MS (Table S4), with 346 genes (42.35) annotated in GO. At level 2, SOP genes were classified into ten molecular functional categories (Table S5). Ovarian proteins containing conserved enzymatic domains constituted a large proportion of secreted ovarian components. From the SOPs, we identified five proteins that are likely involved in passive evasion: IEP-1, IEP-2A, Crp32A, Crp32B and Crp32C (Table 1). In our previous study, we identified two passive avoidancerelated proteins, IEP-2A and IEP-2B, which were specifically expressed in the ovary and venom gland, respectively.10


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Table 1. Proteins Involved in Cotesia chilonis Passive Avoidance

protein name/ gene ID IEP-1/

molecular weight (kDa)

sequence length (signal peptide)

7.51/34.72

325 (Y)

isoelectric point /

No. of No. of

BLAST information unique

peptides peptides 3

3

Cluster-8110.29162

immunoevasive protein-1

MH365478

[C. kariyai] (6e33; BAB72014.1)

Cluster-8110.25416 IEP-2A/

accession No. (E-Value; GenBank No.)

7.67/

298 (Y)

9

9

30.52


MH365479

[C. kariyai] (1e69; BAB72015.1)

APD15629


MH365480

[C. kariyai] (4e41; BAB72015.1)

APD15630

32 kDa protein Crp32

MH365481

Comp39249_c0* IEP-2B/ Comp42303_c0* Crp32A/ Cluster-8110.21682 Crp32B/ Cluster-8110.27662 Crp32C/ Cluster-8110.14141

7.94/

324 (Y)

34.47 8.24/

242 (N)

8

8

[C. rubecula] (1e17; AAC31393.1)

26.70 8.24/

251 (N)

25

25

27.08

MH365482

[C. rubecula] (1e70; AAC31393.1)

27.60 8.98/


246 (N)

12

12


MH365483

[C. rubecula] (4e18; AAC31393.1)

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*indicates a gene from reference.10 Y represents the deduced amino acid sequences with predicted signal peptides, N represents the deduced amino acid sequences without predicted signal peptides.


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The full-length cDNA sequences of IEP-1 and IEP-2B were obtained by 5’ RACE, which showed 100% identity to the sequences obtained from the transcriptomic data. Each IEP homologue has a predicted signal peptide at N-terminus, and different numbers of epidermal growth factor-like domains were identified (Figure S1A). These three Crp32 homologues have no predicted signal peptides, and only one motif at N-terminus with high identity values was detected (Figure S1B). Expression Profiles of Crp32B qPCR results showed that IEP-1 and three Crp32 homologue mRNA were all expressed at significantly higher levels in the ovary than other female adult tissues (Figure 1A and Figure S2A-S2C). After pupation of C. chilonis larvae, the mRNA levels of IEP-2A, IEP-2B and three Crp32 homologs increased in the ovary starting at day 2, peaking at day 3 or 4 and then decreasing at day 5 and remained at a low level at day 2 post-eclosion (Figure 1B and Figure S2E-S2H). The level of IEP-1 mRNA peaked at day 1 after eclosion (Figure S2D). We expressed rCrp32B in E. coli and highly enriched it using His-Tag resin beads for the production of the rabbit polyclonal antibody (Figure S3B). Western blot results showed that Crp32B was present in the ovary but not in other tissues (Figure 1C), which is different from the qPCR-based expression profiles (Figure 1A). This result is likely due to the low expression level of Crp32B. Similarly, although Crp32B was transcribed at day 2 post-pupation (Figure 1B), Crp32B protein was not detected at this timepoint. Crp32B accumulated during the following days and peaked at day 2 post-eclosion (Figure 1D). RNA-FISH results suggested that Crp32B was transcribed throughout the ovary, especially in the ovarian calyx. Crp32B transcripts were also expressed in nurse cells, follicular cells and oocytes (Figure 2). In addition,


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immunofluorescent staining revealed that Crp32B protein was present in all parts of the ovary and on the egg surface (Figure 3).


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Figure 1. Expression profiles of Cotesia chilonis Crp32B. (A) Crp32B mRNA expression profile in different tissues of C. chilonis female adults. (B) Developmental expression profile of Crp32B mRNA in C. chilonis ovaries. Total RNAs (1g each) of different tissues and ovaries from different C. chilonis stages were reversed transcribed to cDNAs and used as templates for qPCR analysis, with the C. chilonis 28S rRNA gene used as an internal control. Data are presented as the means ± standard deviation. The different letters are significantly different based on one-way analysis of variance (ANOVA) and Tukey’s test with differences considered significant at P  0.05. (C) The Crp32B expression profile in C. chilonis adults. Total proteins (0.5 g each) from male adult, female adult different tissues were analyzed by 12 SDS-PAGE. (D) Developmental expression profile of Crp32B protein in C. chilonis ovaries. Total ovarian proteins (0.5 g each) from different C. chilonis stages were analyzed by 12 SDS-PAGE. Crp32B was detected by immunoblotting using an anti-rCrp32B rabbit polyclonal antibody. -actin was used as an internal control.


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Figure 2. RNA-FISH analysis of Crp32B transcripts. (A-C) An ovary and (J-L) immature egg from female pupa were incubated with DEPC-treated water as controls; (D-I) An ovary and (MO) immature egg from female pupa were hybridized with Cy3-labeled Crp32B mRNA probes (red). (G-I) Detailed views of the ovarian calyx of (D-F). The nuclei were stained with DAPI (blue).


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Figure 3. Immunohistochemical visualization of endogenous Crp32B in the ovaries and on the egg surfaces of Cotesia chilonis adults. (A-C) Immunolocalization of Crp32B in a mature ovary. C is the detailed view of the box in B. (D) Immunolocalization of Crp32B in the ovarian calyx


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and on egg surfaces. Immunolocalization of Crp32B on the surfaces of immature eggs (E and F) and newly laid eggs (G and H). A rabbit anti-rCrp32B polyclonal antibody was used as the primary antibody (B, C, D, F and H) and the preimmune rabbit serum was used as the control (A, E and G). Alexa Fluor 594-conjugated goat anti-rabbit IgG (red) was used as the secondary antibody. The nuclei were stained with DAPI (blue). Effect of Crp32B on the Host Encapsulation Response The rCrp32B protein significantly prevented the beads from being encapsulated by host hemocytes dose-dependently as compared to the control beads that lacked rCrp32B or were covered with GFP. When rCrp32B-covered resin beads were masked with an anti-rCrp32B antibody, the encapsulation was significantly increased (Figure 4A). Moreover, the encapsulation extent of mature eggs was significantly enhanced when they were pre-incubated with the antirCrp32B antibody (Figure 4B).


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Figure 4. In vivo encapsulation analysis of resin beads (A) and mature Cotesia chilonis eggs (B). Data are presented as means  standard deviation. Significant differences are indicated by Student’s t-test (***, P  0.001; NS, no significantly different). Identification of Proteins Antigenically Related to Crp32B in C. suppressalis Western blot and immunofluorescence results indicated that antibodies against rCrp32B could cross detect several components in different areas of C. suppressalis larva, and two protein components with molecular weights of 70 and 55 kDa were labeled in all C. suppressalis tissues tested (Figure 5A and Figure S4). Immunoprecipitation followed by proteomics analysis was performed to identify host proteins having antigenic similarity to Crp32B. The proteins that were solely present in the anti-rCrp32B serum group (band1, 2 and 3) but absent in the IgG group (band4 and 5) were identified as components antigenically related to Crp32B (Figure 5B and Table S6). The peptides identified by MS/MS in at least one of the bands 1, 2 and 3 with an ion


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score of  100 but absent in bands 4 and 5 are shown in Table 2. More than half of the components listed in Table 2 are proteins containing conserved enzymatic domains, with tubulin being the most abundant component among these proteins. -tubulin (a single protein component with a molecular weight of 55 kDa) of C. suppressalis could be detected using a commercially purchased -tubulin monoclonal antibody (Figure S5). The molecular weight of rCs--tubulin was higher than 55 kDa for an unknown reason. However, it could be recognized by the -tubulin monoclonal antibody (Figure S6). The pulldown assay showed that rCs--tubulin could capture the anti-rCrp32B antibody (Figure 5C), and rCrp32B could cross react with the -tubulin monoclonal antibody (Figure 5D). We predicted continuous (linear) epitopes of Crp32B and Cs--tubulin with a window length of 10 using ABCpred (Table S7), which resulted in the identification of two groups of similar continuous epitopes (Figure 5E).


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Figure 5. Analysis of Chilo suppressalis proteins antigenically related to Crp32B. (A) Immunodetection of the proteins antigenically related to Crp32B in different tissues of C. suppressalis. Proteins (3 g each) from the whole body or different tissues of C. suppressalis were analyzed on a 4-12 gradient gel. Rabbit anti-rCrp32B antibody was used as a primary antibody. (B) 4-12 gradient gel of immunoprecipitated proteins antigenically related to Crp32B in C. suppressalis. The five sections of the gel that were cut out are indicated by the red boxes. Tryptic peptides extracted from each section were excised and analyzed by LC-MS/MS. IgG was


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used as a control. (C) 8 SDS-PAGE showing the interactions of recombinant C. suppressalis tubulin (rCs--tubulin) (bait) with the anti-rCrp32B antibody (prey) by pull-down assay. Lane 1: input (anti-rCrp32B-antibody). Lane 2: non-treated gel control (minus bait, plus prey). Lane 3: immobilized bait control (plus polyhistidine-tagged eGFP, minus prey). Lane 4: polyhistidinetagged eGFP: anti-rCrp32B antibody interaction. Lane 5: immobilized bait control (plus rCs-tubulin, minus prey). Lane 6: rCs--tubulin: anti-rCrp32B antibody interaction. (D) Western blot analysis showing rCrp32B (3 g) that was detected with the -tubulin monoclonal antibody. (E) Alignment of similar predicted continuous epitopes of Crp32B and C. suppressalis -tubulin.


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Table 2. Immunoprecipitated Chilo suppressalis Proteins Identified by mass spectrometry No. of no.

GenBank ID

molecular weight

isoelectric point

No. of unique

Description

band1 score

band2 score

band3 score

peptides peptides

1

GAJS01059437

30404

4.78

43 (33)

9 (9)

-tubulin

661

1006

953

2

GAJS01063451

8296

7.74

12 (8)

5 (4)

-tubulin

161

204

232

3

GAJS01070710

51461

5.41

25 (7)

16 (5)

protein disulfide-isomerase

153

60

36

4

GAJS01025742

18815

8.62

14 (6)

3 (2)

tripeptidyl peptidase II

134

N

N

5

GAJS01011104

47085

8.2

20 (7)

12 (7)

tripeptidylpeptidase II

128

35

15

6

GAJS01022830

53166

6.22

15 (6)

12 (6)

signal recognition particle

119

38

N

7

GAJS01021389

17589

9.19

5 (2)

4 (2)

dihydrolipoamide dehydrogenase

116

N

N

8

GAJS01024731

10644

6.44

9 (4)

6 (3)

mitochondrial aldehyde dehydrogenase

103

43

123

9

GAJS01022263

49123

6.55

14 (7)

10 (6)

6-phosphofructo-2-kinase/fructose-2,6bisphosphatase short form

102

106

62

10

GAJS01068019

11565

10

5 (3)

4 (3)

ATP synthase

101

N

27

11

GAJS01023271

48235

5.81

31 (16)

23 (15)

regulatory particle triple-A ATPase 1

N

248

80

12

GAJS01017921

48096

6.66

24 (8)

20 (8)

methionine-rich storage protein

26

192

86

13

GAJS01011382

38708

7.23

16 (9)

13 (8)

rho-associated protein kinase 1

N

177

59

14

GAJS01070161

20105

5.66

10 (5)

7 (5)

strand H transporting ATP synthase beta subunit

N

177

N

15

GAJS01024516

46187

5.31

20 (9)

11 (5)

protease regulatory subunit 6A

N

161

36

16

GAJS01019375

23092

7.15

13 (6)

8 (5)

similar to AGAP001884-PA

N

161

N


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Page 28 of 40

17

GAJS01013427

10668

6.75

11 (6)

7 (5)

6-phosphogluconate dehydrogenase

N

160

62

18

GAJS01000632

46540

8.39

13 (5)

6 (1)

similar to GA18629-PA

N

154

54

19

GAJS01011252

39925

5.34

10 (9)

8 (8)

tripeptidylpeptidase II

54

150

72

20

GAJS01070552

33437

7.74

18 (10)

10 (7)

heat shock protein 90

33

145

N

21

GAJS01017121

20768

4.47

5 (3)

4 (3)

protein disulfide-isomerase A6 precursor

N

143

N

22

GAJS01020835

49556

8.68

21 (8)

15 (7)

Ribophorin

N

135

22

23

GAJS01070219

22261

5.12

14 (8)

9 (7)

eukaryotic translation initiation factor 4A

N

133

72

24

GAJS01011491

34122

8.66

7 (4)

6 (3)

AMP dependent CoA ligase

N

132

N

25

GAJS01070376

26131

6.01

9 (6)

5 (4)

V-type proton ATPase subunit H

N

129

54

26

GAJS01070500

27524

5.43

13 (4)

11 (4)

Enolase

31

128

137

27

GAJS01070699

48562

8.81

11 (6)

8 (6)

nucleolar protein 56

N

119

30

28

GAJS01000076

30886

8.93

10 (5)

9 (5)

succinic semialdehyde dehydrogenase

N

116

N

29

GAJS01070436

28276

8.74

10 (5)

8 (4)

microtubule-associated protein futsch-like

77

113

120

30

GAJS01005495

113672

9.13

8 (4)

6 (4)

bifunctional aminoacyl-tRNA synthetas

N

111

55

31

GAJS01021408

29404

8.77

11 (5)

7 (4)

similar to CG6904-PA

N

103

21

32

GAJS01064328

8715

5.3

11 (2)

5 (1)

seminal fluid protein HACP059

40

100

84

33

GAJS01024993

44178

8.16

16 (6)

11 (4)

phosphoglycerate kinase

N

22

135

34

GAJS01009609

19700

6.28

4 (2)

2 (1)

similar to ENSANGP00000015662

N

92

129

35

GAJS01070621

36696

4.94

7 (6)

4 (3)

flocculation protein FLO11 isoform X1

34

85

105

36

GAJS01022971

32333

6.84

5 (4)

5 (4)

dolichyl-diphosphooligosaccharide protein glycotransferase

N

68

105

The peptides identified by MS/MS in at least one of the bands 1, 2 and 3 with an ion score of  100 but were absent in bands 4 and 5 are shown. N indicates that the protein was not identified in the corresponding band.


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Discussion Using high-throughput sequencing, molecular biological and physiological approaches, we identified 817 secreted ovarian proteins of C. chilonis and presented evidence that one of five avoidance-related proteins, Crp32B, played a role in the passive evasion of parasitoid eggs. These results are supported by those of our previous studies at the physiological level in which C. chilonis was shown to use a combination of passive and active strategies to escape from the host cellular immune response.25 In previous studies, the ovary connected to the venom apparatus was chosen as the comparative tissue, because it clearly performs a distinct function in wasp reproduction.35 Burke and Strand identified differentially upregulated transcripts in the venom gland, teratocyte and larval transcriptomes compared to those observed in ovaries.35 However, in some parasitic wasp species, secreted ovarian proteins play important roles in regulating host immune responses.7,20,36 Therefore, in this study, we carefully dissected and squeezed C. chilonis ovaries to collect the fluids from the lateral oviducts instead of pooling the samples from the entire ovaries, eliminating the effects of egg/cellular proteins to the greatest extent possible. Genes that were significantly upregulated in the ovary of Nasonia vitripennis showed GO enrichment for terms related to cell division and transcription, characteristic of the reproductive functions of the ovary.37 However, our results indicated that proteins containing conserved enzymatic domains constitute a large proportion of secreted ovarian components in molecular functional categories of the GO analysis, similar to that observed in parasitoid venoms.10,38 Thus, the results in this study may more accurately reflect the true components of ovarian fluids injected into host hemocoel by an endoparasitoid. We speculate that ovarian enzymes may be involved in host regulation or ovary metabolism in the endoparasitoid, although the exact role of these enzymes


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remains unknown. This is the first report in which a combination of transcriptomic and proteomic approach was used to identify the secreted ovarian proteins of a parasitoid wasp. This dataset may provide further opportunities to investigate novel features of the secreted ovarian proteins. Endoparasitoid wasps use a mimicry strategy in the passive evasion of host immunity. For example, CkIEP-1 protects parasitoid eggs from encapsulation by host Pseudaletia separata hemocytes but not from hemocytes of larval cutworms, Spodoptera litura, an incompatible host for C. kariyai.19 Plasma proteins that cross-reacted with IEP antiserum are present in P. separata, but not in S. litura. However, the specific components that parasitoids mimic remain unclear. In the current study, among the 36 proteins identified in immunoprecipitates from C. suppressalis, Cs--tubulin is the most abundant component and it is antigenically related to Crp32B. To identify the site of the protein exposed to the host immune system, we performed multiple alignments of the predicted continuous epitopes in Cs--tubulin and Crp32B, although the whole amino acid sequences of these two proteins showed low similarity. We identified two groups of similar continuous epitopes exhibited the same or very similar five-continuous amino acid regions that are likely involved in the cross detection. In future work, these epitope peptides should be synthesized as immunogens to generate antibodies that could aid in determining whether the predicted continuous epitopes are recognized by the antibodies. However, most antigenic determinants of proteins are discontinuous, and without 3D structural data of Crp32 homologues, it is difficult to predict the discontinuous (conformational) epitopes.39 Besides tubulin, it is unclear whether the 35 other proteins identified in the immunoprecipitates could also share similar epitopes with Crp32B, and this possibility should be further studied in the future.


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Although the extent of encapsulation of mature eggs was enhanced significantly when they were pre-incubated with the anti-rCrp32B antibody, some eggs remained unencapsulated. In contrast, in other parasitoid/ host systems, most of the wasp eggs were encapsulated when the related protein was blocked by its antibody20 or knocked down by RNA interference.21 Thus, it appeared that one passive-related protein was enough to protect the eggs from encapsulation in other systems. However, our results regarding egg encapsulation indicated that there were likely other functional components involved in protecting C. chilonis eggs from host immune responses. In addition to Crp32B, the functions of other known passive-related proteins and components on the egg surface remain to be studied. Conclusions In this study, we described the comprehensive transcriptome and proteome profiling of secreted ovarian proteins from a parasitoid wasp and identified proteins involved in passive evasion against the host immune response as well as host antigens exhibiting molecular mimicry with one of the passive avoidance-related proteins. The bioinformatic, molecular and physiological data in this study provided valuable clues for understanding the functions of secreted ovarian proteins in successful parasitism and the interaction between parasitoids and their hosts. These results will be useful for improving parasitoid efficacy for the future management of rice pests. ASSOCIATED CONTENT Supporting Information Supplementary Methods: insects rearing, transcriptomic analysis, sample preparation for proteomic analysis, expression and purification of recombinant proteins, quantitative real-time


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PCR (qPCR), RNA fluorescence in situ hybridization (RNA-FISH) and immunofluorescence, immunoprecipitation (IP), pull-down assay and associated references. (PDF) Figure S1. Multiple alignment of amino acid sequences of passive evasion-related proteins. Figure S2. The mRNA expression profiles of Cotesia chilonis passive evasion-related proteins. Figure S3. Prokaryotic expression of recombinant Cotesia chilonis Crp32B. Figure S4. Immunofluorescence of proteins antigenically related to Crp32B in host Chilo suppressalis. Figure S5. Immunodetection of -tubulin in different Chilo suppressalis tissues. Figure S6. Prokaryotic expression of recombinant Chilo suppressalis -tubulin (rCs--tubulin). (PDF) Supplementary Table S1: Primers used in this study. Supplementary Table S2: Summary statistics of the analysis of the Cotesia chilonis ovary reads. Supplementary Table S3: The most enriched KEGG pathways of differentially expressed genes (DEGs) from female later pupal (FPOvary) and adult ovaries (FAOvary). Supplementary Table S4: The 817 proteins matched to the FPOvary or FAOvary cDNA libraries. Supplementary Table S5: Functional characterization of secreted protein encoding genes from the Cotesia chilonis ovary. Supplementary Table S6: Immunoprecipitated Chilo suppressalis proteins identified by mass spectrometry. Supplementary Table S7: Predicted continuous epitopes of Crp32B and Chilo suppressalis -tubulin. (XLSX)

AUTHOR INFORMATION Corresponding Author *Tel.: 86-571-88982696. fax: 86-571-88982988. E-mail address: [email protected] Author Contributions


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Z.-W.T., Q.F., and G.-Y.Y. conceived and designed the experimental plan. Z.-W.T., and X.-H.Y. analyzed and interpreted the sequence and experimental data. Z.-W.T., H.-Z.W., S.-J.X., G.X., and F.W. performed the experiments. Z.-W.T. wrote the manuscript. Q.F., and G.-Y.Y. provided valuable suggestions regarding this work. All authors read and approved the final manuscript. Funding Sources Financial support was provided by National Natural Science Foundation of China (Grant No. 31801796, http://www.nsfc.gov.cn/), Major International (Regional) Joint Research Project of National Natural Science Foundation (Grant No. 31620103915, http://www.nsfc.gov.cn/), National Key R&D Program of China (No. 2017YFD0200400), China Postdoctoral Science Foundation (2018M632482), The Program for Chinese Innovation Team in Key Areas of Science and Technology of Ministry of Science and Technology of the People's Republic of China (2016RA4008), Program for Chinese Outstanding Talents in Agricultural Scientific Research of Ministry of Agriculture and Rural Affairs of the People's Republic of China. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support was provided by National Natural Science Foundation of China (Grant No. 31801796, http://www.nsfc.gov.cn/), Major International (Regional) Joint Research Project of National Natural Science Foundation (Grant No. 31620103915, http://www.nsfc.gov.cn/), National Key R&D Program of China (No. 2017YFD0200400), China Postdoctoral Science Foundation (2018M632482), The Program for Chinese Innovation Team in Key Areas of


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Science and Technology of Ministry of Science and Technology of the People's Republic of China (2016RA4008), Program for Chinese Outstanding Talents in Agricultural Scientific Research of Ministry of Agriculture and Rural Affairs of the People's Republic of China. In addition, we would like to thank Prof. Fei Li (Institute of Insect Sciences, Zhejiang University) for kindly providing us the complete sequence of -tubulin from the Chilo suppressalis genome. ABBREVIATIONS CkIEP, Cotesia kariyai IEP; Crp32, Cotesia rubecula protein 32; DAPI, 4’-6-diamidino-2phenylindole; DEG, differentially expressed gene; DPS, Data Processing System; FDR, false discovery rate; FPOvary, transcripts originated from ovaries of later pupal female wasps (4 or 5 days after pupation); FAOvary, transcripts originated from ovaries of adult stage female wasps (1 or 2 days after eclosion); GO, Gene Ontology; IEP, immunoevasive protein; kDa, kiloDalton; LC−MS/MS, liquid chromatography−mass spectrometry/mass spectrometry; PBS, Pringle’s phosphate-buffered saline; PCR, polymerase chain reaction; PDV, polydnaviruses; pI, isoelectric point; RACE, rapid amplification of cDNA ends; rCrp32B, recombinant Crp32B was expressed in E. coli; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SOP, secreted ovarian proteins; VLPs, virus-like particles REFERENCES (1)

Schmidt, O.; Theopold, U.; Strand, M. Innate immunity and its evasion and suppression by

hymenopteran endoparasitoids. Bioessays 2001, 23 (4), 344-351. (2)

Dorémus, T.; Jouan, V.; Urbach, S.; Cousserans, F.; Wincker, P.; Ravallec, M.; Wajnberg,

E.; Volkoff, A. N. Hyposoter didymator uses a combination of passive and active strategies to escape from the Spodoptera frugiperda cellular immune response. J. Insect Physiol. 2013, 59 (4), 500-508.


34

Page 35 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(3)

Asgari, S.; Rivers, D. B. Venom proteins from endoparasitoid wasps and their role in host-

parasite interactions. Annu. Rev. Entomol. 2011, 56, 313-335. (4)

Strand, M. R.; Burke, G. R. Polydnavirus-wasp associations: evolution, genome

organization, and function. Curr. Opin. Virol. 2013, 3 (5), 587-594. (5)

Strand, M. R. Teratocytes and their functions in parasitoids. Curr. Opin. Insect Sci. 2014, 6,

68-73. (6)

Heavner, M. E.; Ramroop, J.; Gueguen, G.; Ramrattan, G.; Dolios, G.; Scarpati, M.; Kwiat,

J.; Bhattacharya, S.; Wang, R.; Singh, S.; Govind, S. Novel organelles with elements of bacterial and eukaryotic secretion systems weaponize parasites of Drosophila. Curr. Biol. 2017, 27 (18), 2869-2877. (7)

Webb, B. A.; Luckhart, S. Factors mediating short- and long-term immune suppression in a

parasitized insect. J. Insect Physiol. 1996, 42 (1), 33-40. (8)

Chevignon, G.; Theze, J.; Cambier, S.; Poulain, J.; Da Silva, C.; Bezier, A.; Musset, K.;

Moreau, S. J. M.; Drezen, J. M.; Huguet, E. Functional annotation of Cotesia congregata bracovirus: Identification of viral genes expressed in parasitized host immune tissues. J. Virol. 2014, 88 (16), 8795-8812. (9)

Ye, X. Q.; Shi, M.; Huang, J. H.; Chen, X. X. Parasitoid polydnaviruses and immune

interaction with secondary hosts. Dev. Comp. Immunol. 2018, doi:10.1016/j.dci.2018.01.007. (10)

Teng, Z. W.; Xiong, S. J.; Xu, G.; Gan, S. Y.; Chen, X.; Stanley, D.; Yan, Z. C.; Ye, G.

Y.; Fang, Q. Protein discovery: combined transcriptomic and proteomic analyses of venom from the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae). Toxins 2017, 9 (4), 135.


35


(11)

Page 36 of 40

Ali, M. R.; Seo, J.; Lee, D.; Kim, Y. Teratocyte-secreting proteins of an endoparasitoid

wasp, Cotesia plutellae, prevent host metamorphosis by altering endocrine signals. Comp. Biochem. Phys. A 2013, 166 (2), 251-262. (12)

Morales, J.; Chiu, H.; Oo, T.; Plaza, R.; Hoskins, S.; Govind, S. Biogenesis, structure, and

immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae. J. Insect Physiol. 2005, 51 (2), 181-195. (13)

Hu, J.; Zhu, X. X.; Fu, W. J. Passive evasion of encapsulation in Macrocentrus cingulum

Brischke (Hymenoptera : Braconidae), a polyembryonic parasitoid of Ostrinia furnacalis Guenee (Lepidoptera : Pyralidae). J. Insect Physiol. 2003, 49 (4), 367-375. (14)

Davies, D. H.; Vinson, S. B. Passive evasion by eggs of braconid parasitoid Cardiochiles

nigriceps of encapsulation in vitro by hemocytes of host Heliothis virescens. Possible role for fibrous layer in immunity. J. Insect Physiol. 1986, 32 (12), 1003-1010. (15)

Berg, R.; Schuchmann-feddersen, I.; Schmidt, O. Bacterial infection induces a moth

(Ephestia kuhniella) protein which has antigenic similarity to virus-like particle proteins of a parasitoid wasp (Venturia canescens). J. Insect Physiol. 1988, 34 (6), 473-480. (16)

Kinuthia, W.; Li, D. M.; Schmidt, O.; Theopold, U. Is the surface of endoparasitic wasp

eggs and larvae covered by a limited coagulation reaction? J. Insect Physiol. 1999, 45 (5), 501506. (17)

Tanaka, T. Morphology and functions of calyx fluid filaments in the reproductive tracts of

endoparasitoid, Microplitis mediator (Hym.: Braconidae). Entomophaga 1987, 32 (1), 9-17. (18)

Tanaka, K.; Matsumoto, H.; Hayakawa, Y. Detailed characterization of polydnavirus

immunoevasive proteins in an endoparasitoid wasp. Eur. J. Biochem. 2002, 269 (10), 2557-2566.


36

Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(19)

Furihata, S.; Tanaka, K.; Ryuda, M.; Ochiai, M.; Matsumoto, H.; Csikos, G.; Hayakawa,

Y. Immunoevasive protein (IEP)-containing surface layer covering polydnavirus particles is essential for viral infection. J. Invertebr. Pathol. 2014, 115, 26-32. (20)

Asgari, S.; Theopold, U.; Wellby, C.; Schmidt, O. A protein with protective properties

against the cellular defense reactions in insects. Proc. Natl. Acad. Sci. USA 1998, 95 (7), 36903695. (21)

Hu, J.; Xu, Q. Y.; Hu, S. F.; Yu, X. Q.; Liang, Z. K.; Zhang, W. Q. Hemomucin, an O-

glycosylated protein on embryos of the wasp Macrocentrus cingulum that protects It against encapsulation by hemocytes of the host Ostrinia furnacalis. J. Innate Immun. 2014, 6 (5), 663675. (22)

Nishikawa, H.; Yoshimura, J.; Iwabuchi, K. Sex differences in the protection of host

immune systems by a polyembryonic parasitoid. Biology Lett. 2013, 9 (6), 20130839. (23)

Lou, Y. G.; Zhang, G. R.; Zhang, W. Q.; Hu, Y.; Zhang, J. Reprint of: biological control

of rice insect pests in China. Biol. Control. 2014, 68, 103-116. (24)

Qi, Y. X.; Teng, Z. W.; Gao, L. F.; Wu, S. F.; Huang, J.; Ye, G. Y.; Fang, Q.

Transcriptome analysis of an endoparasitoid wasp Cotesia chilonis (Hymenoptera: Braconidae) reveals genes involved in successful parasitism. Arch. Insect Biochem. Physiol. 2014, 88 (4), 203-221. (25)

Teng, Z. W.; Xu, G.; Gan, S. Y.; Chen, X.; Fang, Q.; Ye, G. Y. Effects of the

endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism, venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae. J. Insect Physiol. 2016, 85, 46-56.


37


(26)

Page 38 of 40

Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal sample preparation

method for proteome analysis. Nature Methods 2009, 6 (5), 359-360. (27)

Cox, J.; Mann, M. MaxQuant enables high peptide identification rates, individualized

p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008, 26 (12), 1367-1372. (28)

Ma, J.; Chen, T.; Wu, S.; Yang, C.; Bai, M.; Shu, K.; Li, K.; Zhang, G.; Jin, Z.; He, F.;

Hermjakob, H.; Zhu, Y. iProX: an integrated proteome resource. Nucleic Acids Res. 2019, 47 (D1), D1211-D1217. (29)

Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time

quantitative PCR and the 2-  CT method. Methods 2001, 25 (4), 402-408. (30)

Bustin, S. A.; Benes, V.; Garson, J. A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller,

R.; Nolan, T.; Pfaffl, M. W.; Shipley, G. L.; Vandesompele, J.; Wittwer, C. T. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55 (4), 611-622. (31)

Lécuyer, E.; Parthasarathy, N.; Krause, H. M., Fluorescent in situ hybridization protocols

in Drosophila embryos and tissues. In Methods in Molecular Biology, 1st ed.; Dahmann, C., Ed.; Humana Press: New Jersey, 2008; pp 289-302. (32)

Wu, J. S.; Luo, L. Q. A protocol for dissecting Drosophila melanogaster brains for live

imaging or immunostaining. Nat. Protoc. 2006, 1 (4), 2110-2115. (33)

Wu, S. F.; Sun, F. D.; Qi, Y. X.; Yao, Y.; Fang, Q.; Huang, J.; Stanley, D.; Ye, G. Y.

Parasitization by Cotesia chilonis influences gene expression in fatbody and hemocytes of Chilo suppressalis. PLoS ONE. 2013, 8 (9), e74309.


38

Page 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34)

Tang, Q. Y.; Zhang, C. X. Data Processing System (DPS) software with experimental

design, statistical analysis and data mining developed for use in entomological research. Insect Sci. 2013, 20 (2), 254-260. (35)

Burke, G. R.; Strand, M. R. Systematic analysis of a wasp parasitism arsenal. Mol. Ecol.

2014, 23 (4), 890-901. (36)

Li, Y.; Lu, J. F.; Feng, C. J.; Ke, X.; Fu, W. J. Role of venom and ovarian proteins in

immune suppression of Ostrinia furnacalis (Lepidoptera : Pyralidae) larvae parasitized by Macrocentrus cingulum (Hymenoptera : Braconidae), a polyembryonic parasitoid. Insect Sci. 2007, 14 (2), 93-100. (37)

Sim, A. D.; Wheeler, D. The venom gland transcriptome of the parasitoid wasp Nasonia

vitripennis highlights the importance of novel genes in venom function. BMC Genomics 2016, 17, 571. (38)

Dorémus, T.; Urbach, S.; Jouan, V.; Cousserans, F.; Ravallec, M.; Demettre, E.;

Wajnberg, E.; Poulain, J.; Azema-Dossat, C.; Darboux, I.; Escoubas, J. M.; Colinet, D.; Gatti, J. L.; Poirie, M.; Volkoff, A. N. Venom gland extract is not required for successful parasitism in the polydnavirus-associated endoparasitoid Hyposoter didymator (Hym. Ichneumonidae) despite the presence of numerous novel and conserved venom proteins. Insect Biochem. Molec. 2013, 43 (3), 292-307. (39)

Saha, S.; Raghava, G. P. S. Prediction of continuous B-cell epitopes in an antigen using

recurrent neural network. Proteins 2006, 65 (1), 40-48.


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An ovarian protein involved in passive avoidance of an endoparasitoid

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