The genomic and transcriptomic analyses of serine proteases and their homologs in an endoparasitoid, Pteromalus puparum

Accepted Manuscript The genomic and transcriptomic analyses of serine proteases and their homologs in an endoparasitoid, Pteromalus puparum Lei Yang, Zhe Lin, Qi Fang, Jiale Wang, Zhichao Yan, Zhen Zou, Qisheng Song, Gongyin Ye PII:

S0145-305X(17)30164-7

DOI:

10.1016/j.dci.2017.07.014

Reference:

DCI 2942

To appear in:

Developmental and Comparative Immunology

Received Date: 25 April 2017 Revised Date:

12 July 2017

Accepted Date: 12 July 2017

Please cite this article as: Yang, L., Lin, Z., Fang, Q., Wang, J., Yan, Z., Zou, Z., Song, Q., Ye, G., The genomic and transcriptomic analyses of serine proteases and their homologs in an endoparasitoid, Pteromalus puparum, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.07.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

The genomic and transcriptomic analyses of serine proteases and

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their homologs in an endoparasitoid, Pteromalus puparum

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Lei Yang1, Zhe Lin2, Qi Fang1, Jiale Wang1, Zhichao Yan1, Zhen

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Zou2, Qisheng Song3, Gongyin Ye1*

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Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects, Institute of Insect

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Sciences, Zhejiang University, Hangzhou, China

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Chinese Academy of Sciences, Beijing, 100101, China

State Key Laboratory of Rice Biology & Ministry of

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Missouri, Columbia, Missouri, USA

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Division of Plant Sciences, College of Agriculture, Food and Natural Resources, University of

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State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology,

*Corresponding author: GY Ye, e-mail: [email protected].

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Highlights

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Abstract

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In insects, serine proteases (SPs) and serine protease homologs (SPHs) constitute a

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large family of proteins involved in multiple physiological processes such as digestion,

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development, and immunity. Here we identified 145 SPs and 38 SPHs in the genome

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of an endoparasitoid, Pteromalus puparum. Gene duplication and tandem repeats

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were observed in this large SPs/SPHs family. We then analyzed the expression

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profiles of SP/SPH genes in response to different microbial infections (Gram-positive

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bacterium Micrococcus luteus, Gram-negative bacterium Escherichia coli, and

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entomopathogenic fungus Beauveria bassiana), as well as in different developmental

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stages and tissues. Some SPs/SPHs also displayed distinct expression patterns in

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venom gland, suggesting their specific physiological functions as venom proteins. Our

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finding lays groundwork for further research of SPs and SPHs expressed in the venom

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glands.

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Key words

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Pteromalus puparum; Serine protease; Serine protease homolog; Expression profile; Venom gland

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1. Introduction

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One hundred and eighty-three serine proteases (SPs) and serine protease homologs (SPHs) were identified in the genome of an endoparasitoid, Pteromalus puparum. Detailed information of SPs and SPHs on the gene structure, distributions in scaffolds was revealed by genome scale analysis and the abundant levels of SPs/SPHs in different developmental stages and tissues were also obtained. Tandem repeats of SPs and SPHs with similar genomic structures indicated gene duplication events happened during evolution. Six SPs and two SPHs were predicted as venom proteins.

ACCEPTED MANUSCRIPT S1 Serine protease (SP)1 family proteins participate in various physiological

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processes, such as digestion, development, and immune processes (Cerenius et al.,

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2008; Loof et al., 2011; Lu et al., 2014; Rawlings et al., 2004). SPs usually contain

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signal peptides and are produced as zymogens. Zymogens are converted to the active

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enzymes by proteolytic cleavage at specific site. Trypsin and chymotrypsin are major

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types of digestive proteases and usually highly expressed in midgut immediately after

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feeding (Brackney et al., 2010; Soares et al., 2011). As a typical member of SP family,

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bovine chymotrypsin features a catalytic triad consisting of Ser195, His57, and

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Asp102 amino acid residues in its catalytic center (Perona and Craik, 1995). A

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substrate binding cleft near the active site is the predominant factor in determining its

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substrate specificity. The RNA interfere experiment in a fruit fly, Bactrocera dorsalis,

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indicated that silence of a single trypsin induced the expressions of other trypsins, as a

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result counteracting the influence of knock down of a single trypsin on the digestion

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(Li et al., 2017). In immune activities, several SP zymogens can sequentially be

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activated to form a cascade pathway and finally act on effectors (Ross et al., 2003).

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The roles of SP cascades have been extensively studied in invertebrates (He et al.,

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2017; Rao et al., 2010; Wang et al., 2014; Zou et al., 2010). The proteolytic activation

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of prophenoloxidase (PPO) and Spätzle-induced synthesis of antimicrobial peptides

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(AMPs) are mediated by these enzyme cascades (Kanost and Jiang, 2015; Kanost et

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Abbreviations used are: SPs, serine proteases; SPHs, serine protease homologs; PPO, prophenoloxidase; AMPs, antimicrobial peptides; HP, hemolymph proteinase; JNK, c-Jun N-terminal kinase; PDVs, polydnaviruses; VLPs, virus-like particles; qPCR, quantitative real-time PCR; ANOVA, analysis of variance; VG, venom gland; DEPC, Diethy pyrocarbonate; cDNA, complementary DNA; cSPs, clip-domain serine proteases; cSPHs, clip-domain serine protease homologs; p. i, post infection; Pp, Pteromalus puparum; Am, Apis mellifera; Dm, Drosophila melanogaster; Ms, Mandu sexta; CCP, complement control protein; LDLA, low-density lipoprotein receptor class A; ANK, Ankyrin repeat region; FZ, Frizzled; CHIT, Chitin-binding; SR, scavenger receptor Cys-rich; CTL, C-type lectin; MSP, modular serine protease; GD, gastrulation defective.

ACCEPTED MANUSCRIPT al., 2004; Kellenberger et al., 2011; Povelones et al., 2013). In Manduca sexta,

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hemolymph proteases HP14, HP21 and PAP 1/2/3 are responsible for activating PPO

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pathway and HP8 stimulates AMP synthesis (An et al., 2009, 2013; Wang and Jiang,

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2008). In Drosophila melanogaster, genetic analyses reveal that a series of genes in

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the SP pathways are involved in mediating the Toll-Dorsal patterning of embryo for

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ventralization and activating AMP productions (Belvin and Anderson, 1996; Kambris

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et al., 2006). Researches also show that Drosophila serine proteases MP1, MP2 and

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Hayan are involved in melanization (Veillard et al., 2016) while Hayan also

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participates in c-Jun N-terminal kinase (JNK)-dependent cytoprotective program and

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is involved in systemic wound response (Nam et al., 2012).

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In addition to SPs, many serine protease homolog genes (SPHs) have been

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identified. SPHs lack amidase activity because of the mutation or absence of the

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catalytic residues. These SPHs promote the activation of PPOs (Felfoldi et al., 2011;

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Gupta et al., 2005; Wang and Jiang, 2017; Wang et al., 2014; Yu and Kanost, 2003).

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SPHs are also involved in somatic muscle attachment in Drosophila embryos, cell

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adhesion in the crayfish Pacifastacus leniusculus and regulation of TEP1 recruitment

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to microbial surfaces in Anopheles gambiae (Huang et al., 2000; Povelones et al.,

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2013; Zhang et al., 2013).

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Parasitic wasps, being invaluable in classical and augmentative biological control

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of various insect pests, are an abundant and diverse hymenopteran group on earth,

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which lay their eggs into internal body (endoparasitic wasps) or on the external

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surface (ectoparasitic wasps) of their hosts (Gehrer and Vorburger, 2012; Pennacchio

ACCEPTED MANUSCRIPT and Strand, 2006). To avoid host immune defense and allow their offspring to develop

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inside or outside host hemocoel for successful parasitism, several parasitic factors,

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including venom, polydnaviruses (PDVs), virus-like particles (VLPs), ovarian fluids

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and teratocytes, are produced by parasitoids and used alone or in combination,

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depending largely upon their life strategy (Asgari and Rivers, 2011; de Graaf et al.,

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2010; Pennacchio and Strand, 2006; Poirie et al., 2014; Teng et al., 2016). To date,

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previous studies are mainly focused on the parasitic factors produced by the wasps

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and the immune responses of their hosts (Gueguen et al., 2013; Thoetkiattikul et al.,

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2005; Wang et al., 2013; Zhu et al., 2009). Insight into the venom compositions of the

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parasitoids reveals SPs and SPHs are major components of venom and may

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participate in immune activities (Colinet et al., 2014; de Graaf et al., 2010; Vincent et

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al., 2010; Yan et al., 2016; Zhu, 2016, 2010). However, the reported physiological

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functions of SPs and SPHs in venom glands of parasitoids are limited to a few species.

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In Cotesia rubecula, a serine protease homolog named Vn50 is isolated from its

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venom gland, and defined to show the ability in inhibiting host humoral immune

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response by interfering with the PO cascades (Asgari et al., 2003; Zhang et al., 2004).

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In Nasonia vitripennis, serine proteases possess a possible cytotoxic function in cell

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death of Spodoptera frugiperda cell line (Formesyn et al., 2013). Recently, genome-wide analyses have helped to identify SP and SPH genes in

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several insect species, including D. melanogaster (Ross et al., 2003), Apis mellifera

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(Zou et al., 2006), Bombyx mori (Zhao et al., 2010), Nilaparvata lugens (Bao et al.,

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2013), M. sexta (Cao et al., 2015) and Plutella xylostella (Lin et al., 2015). SP

ACCEPTED MANUSCRIPT pathways, which have been revealed by genetic and biochemical analyses in M. sexta,

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Helicoverpa armigera, A. gambiae, D. melanogaster, Aedes aegypti and other insects,

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vary in food digestion, embryo development and immune responses (An et al., 2013;

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Jiang et al., 2003; Kuwar et al., 2015; Paskewitz et al., 2006; Zou et al., 2010).

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However, our current knowledge about SP and SPH pathways in parasitic wasps is

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still limited. Therefore, there is still a crucial need for extensive analyses using the

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combination of various techniques such as genomic and transcriptomic analyses to

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acquire a more comprehensive view on the composition and functional diversity of

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SPs and SPHs in hymenopteran parasitoids.

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Pteromalus puparum is a pupal endoparasitoid wasp of certain Papilionidae and Pieridae species, and is the dominant parasitic species in regulating the field

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population of the small white butterfly, Pieris rapae, which is an important

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agricultural pest of the crucifer and caper families such as cabbage, cauliflower,

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broccoli and collard, and spreads worldwide (Cai et al., 2004). P. puparum wasps

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complete their life cycles except adult stage within the hemocoels of their host, feed

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on host, and defend against host immune responses using the venom as the key

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parasitic factor. For this parasitoid, the composition and function of the venom, and its

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immune interrelationship with its host have been well investigated by our group (Fang

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et al., 2016, 2011a, 2011b; Wang et al., 2015, 2013; Yan et al., 2017, 2015; Zhu et al.,

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2015). To better uncover the interrelationship between P. puparum and its host P.

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rapae, the whole genome of P. puparum has recently been sequenced by our group,

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and the available gene annotation information enables us to identify SPs and SPHs in

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ACCEPTED MANUSCRIPT this parasitoid. In this current study, we characterized the SP and SPH genes and

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presented the expression patterns in different development stages and tissues based on

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transcriptomic and qPCR (quantitative real-time PCR) analyses. These data provide a

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foundation for discovering the potential function of these genes, which are crucial for

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the wasps to evade their host immune system and defend the microbial infections.

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2. Materials and Methods

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2.1 Insect rearing

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The laboratory P. rapae and P. puparum cultures were reared at 25 ± 1 °C with a

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photoperiod of 10 h: 14 h (light: darkness) as described previously (Fang et al., 2010;

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Zhang et al., 2005) and used in all experiments. Briefly, P. rapae larvae were fed with

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cabbage leaf until they pupated, and then exposed to 2-day old mated female wasps

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of P. puparum. Parasitized P. rapae pupae were maintained under the same

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environmental condition described above. Once emerged, the new wasp adults were

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collected and held in plastic finger-type vials and fed with 20% (v/v) honey solution.

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2.2 Identification of SP/SPH genes from P. puparum genome

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SP and SPH sequences of D. melanogaster, M. sexta, A. mellifera and other insects

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were downloaded from NCBI GenBank (https://www.ncbi.nlm.nih.gov/genome).

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These genes were used as queries to search P. puparum genome for matches using

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BLAST program (E-value 1e-5). Identified genes were validated manually by

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searching the non-redundant nucleotide database. The predicted sequences were

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categorized as SPs and SPHs based on the conserved His, Asp and Ser residues in

ACCEPTED MANUSCRIPT catalytic triad residues (Perona and Craik, 1995). Amino acid sequences containing all

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three residues in TAAHC, DIAL and GDSGGP motifs are considered as SPs. The

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sequences lack one or more of these residues were regarded as SPHs (Ross et al.,

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2003). The signal peptides and transmembrane (TM) regions were predicted based on

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SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and TMHMM Server v.2.0

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(http://www.cbs.dtu.dk/services/TMHMM/). The domain analyses of the retrieved

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protein sequences were carried out using Pfam (http://pfam.xfam.org/), SMART

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(http://smart.embl.de/) and PROSITE (http://prosite.expasy.org/). The function

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prediction was according to KEGG and UniprotKB/Swiss-Prot annotation.

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2.3 Sequence alignment and phylogenetic analysis

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P. puparum SP/SPH genes were aligned with those from D. melanogaster, M. sexta,

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B. mori, A. mellifera, N. vitripennis and other insect species. Multiple sequence

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alignments were performed with ClustalX 2.0 (Thompson et al., 1997), and

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Neighbor-joining method was used to construct phylogenetic trees by MEGA5.10

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(Tamura et al., 2011) with 1000 bootstrap replicates.

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2.4 Samples collection

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2.4.1

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Immunization by three microorganism species

Gram-positive

bacterium

Micrococcus

luteus,

Gram-negative

bacterium

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Escherichia coli and entomopathogenic fungus Beauveria bassiana were used for

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septic injury. The bacterium or fungus was freshly collected, washed three times with

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sterile PBS and resuspended at a density of 5 x 107 colony forming units in PBS,

ACCEPTED MANUSCRIPT respectively, following the protocol by Wang et al. (2017). The 2-day old mated

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female wasps of P. puparum were anesthetized with carbon dioxide for 30 s.

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Acupuncture needles that pre-dipped in a conidial suspension of microbe were used to

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penetrate the abdominal cuticle of the wasps. Control wasps were pricked with sterile

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PBS only. These adult females were collected at 6, 24 and 48 h post-prick in order to

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analyze the bacterium- or fungus-induced gene expressions.

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Sample collections from different tissue and development stages

For tissue extraction, the 2-day mated female wasps were anaesthetized in −70 °C

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refrigerator for 5 min, and dissected in DEPC (Diethy pyrocarbonate) water with 1

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unit/µl RNAase inhibitor (Toyobo, Osaka, Japan) on the ice plate under a stereoscope

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(Olympus, Japan). The tissues including fat body, gut, ovary, venom gland and the

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remaining carcass were isolated, washed, and then pooled into centrifuge tube with

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Trizol reagent (Invitrogen, USA), respectively.

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For different development stages, P. puparum embryos were dissected from P.

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rapae pupae less than 12 h after parasitism. Also, the parasitoid larvae were dissected

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from P. rapae pupae at 2, 4 and 6 d after parasitism, which were equal to the 1st, 2nd,

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3rd instar of the wasps. The yellow wasp pupae i.e. the early stage of pupae were

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acquired and divided into females and males. Two-day old mated female and male

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wasps were also obtained. P. puparum in different development stages were collected,

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washed and pooled into centrifuge tube with Trizol reagent (Invitrogen, USA),

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respectively.

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2.5 RNA extraction and qPCR Wasps from different development stages and immune challenged samples as well

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as different tissue samples of mature female wasps were used for RNA extraction. The

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total RNA was extracted using Trizol reagent according to the manufacture’s protocol.

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The concentration of total RNA was estimated by measuring the absorbance at 260

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nm. Single-stranded complementary DNAs (cDNAs) were synthesized using the

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PrimeScript™ One Step RT-PCR Kit (Takara, Japan). The larval single-stranded

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cDNA was made from equivalent mixtures of RNA samples extracted from 1st, 2nd,

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3rd instar of wasp larvae. All the primer sequences (Table S1) were designed using

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Primer 3 (Untergasser et al., 2012) and synthesized commercially (Sangon, China).

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QPCR was performed on a BIO-RAD CFX96™ Real-Time System (Bio-Rad, USA)

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using the iQ™ SYBR Green Supermix Kit (Takara, Japan), according to the

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manufacturers’ instructions. The cDNA (2 µl) was used as a template in each 25 µl

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reaction mixture. The cycling conditions for qPCR were as follows: enzyme

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activation at 95 °C for 30 secs, followed by 40 cycles with denaturation at 95 °C for 5

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sec, annealing at 60 °C for 34 sec. We have tested the expression stability of several

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candidate reference genes in different developmental stages, tissues and

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immune-challenged wasps. The expression level of P. puparum actin 1 was the most

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stable among different microbial infections. The transcript level of 18s rRNA

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presented good stability in different tissues and at differently developmental stages.

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Therefore, we chose actin 1 as an internal control standard for the relative expression

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levels of genes in different infected cases. Development- and tissues-specific relative

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ACCEPTED MANUSCRIPT expressions were normalized to reference gene by18s rRNA. Dissociation curves

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were checked at the end of PCR reactions. The mRNA expression levels were

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quantified using the 2∆∆Ct method (Livak and Schmittgen, 2001). Error bars represent

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the means ± standard deviations from three biological replicates. One-way analysis of

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variance (ANOVA) was performed in expression profiles of different development

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stages and tissues distribution and two-way ANOVA was used in expression profiles

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of immune response.

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2.6 Constructing, sequencing and analyzing RNA-seq libraries

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The construction and sequencing of cDNA libraries were performed by Nextomics Biosciences (Nextomics, Wuhan, China). Developmental stage samples and tissue

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samples were prepared for deep sequencing analysis. Various life stages including

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newly laid P. puparum embryos (n=200), 1st, 2nd, 3rd instar of wasp larvae with equal

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quantity (n=10), female and male wasps in pupal stage and adult stage (n=20). For

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tissue analysis, ovary, venom gland and carcass without venom gland were prepared

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from female adults. The isolated RNA was purified using Sera-mag Magnetic Oligo

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(dT) beads (Illumina, USA), and then transcribed using N6 primers followed by

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synthesis of second cDNA strand. After end pair processing and ligation of adaptor,

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RNA was amplified by PCR and purified using QIAquick Gel Extraction Kit (Qiagen,

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Germany). The larvae mRNA sequencing library was made from equivalent mixtures

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of RNA samples extracted from 1st, 2nd, 3rd instar of wasp larvae. Then nine mRNA

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libraries were sequenced using an Illumina HiSeq 2100 instrument (Illumina, USA)

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and raw reads were sorted out by barcodes.

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2.7 Analysis of RNA-seq Data Raw reads from each library were filtered to remove low quality reads and the resulting reads were termed clean reads. The expression levels were estimated

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by software TopHat and Cufflinks (Trapnell et al., 2012, 2010). The FPKM values of

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P. puparum SP and SPH genes were listed (Table S2). The expression profiles of

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SP/SPH genes in different development stages were visualized using the R statistical

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program version 3.1.3. Identification of differentially expressed genes between

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venom gland and carcass without venom gland were performed using the R

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package DEGSeq v1.2.2 (Wang et al., 2010). The p-values were adjusted using

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(Benjamini and Hochberg, 1995). Corrected p-value < 0.001, log2

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(FPKM_VG/FPKM_Carcass) >1 and FPKM_VG (Venom gland) >10 were set as

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the threshold. P. puparum SPs and SPHs which were differentially expressed in

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venom gland were listed in Table S3. Based on the previous venom proteomic

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information (Yan et al., 2016), part of these SPs and SPHs genes were marked and

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defined as putative venom proteins.

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3. Results

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3.1 Identification and characterization of SP and SPH genes in P. puparum

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One hundred and eighty-three predicted SP and SPH genes were identified from the

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genome of P. puparum and most of them were similar to chymotrypsin (S1) family

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(Table S4). The sequences of these 183 SP/SPH genes were listed in Table S5 and

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NCBI descriptions were provided in Table S6. The total number of SP/SPH genes in P.

ACCEPTED MANUSCRIPT puparum is 183, less than 204 in D. melangaster and 441 in A. aegypti, but far more

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than 57 in A. mellifera and 90 in N. lugens (Table S7). We also identified these

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SP/SPH genes in the genomes of N. vitripennis (Werren et al., 2010) and

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Microplitis demolitor in silico (Burke et al., 2014). The number of SP/SPH genes in P.

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puparum genome is much similar to the number (143 genes) in N. vitripennis and far

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more than that (74 genes) in M. demoliter (Table S7). Based on the presence or

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absence of the conserved His, Asp and Ser residues in the catalytic triad, the whole P.

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puparum SP/SPH genes were classified into 145 SP genes and 38 SPH genes (Table

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S4).

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In P. puparum, 183 SP and SPH genes are spread across 47 different scaffolds

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(Table S4). Apart from most of the scaffolds with less than 5 SP/SPH genes, 140

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SP/SPH genes are located in 14 scaffolds. We drew the location of genes on scaffolds

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with five or more SP/SPH genes (Fig. 1 & Fig. S1). Results indicated that large

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clusters of SP/SPH genes are common events in the genome of P. puparum.

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Twenty-four SP/SPH genes which form two clusters are located in scaffolds 17 with

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cluster 17-1 genes placed adjacently. Striking phenomenon also exists in scaffolds 5

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with 37 SP/SPH genes clustered into three clusters. Most of the SP/SPH genes in

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scaffolds 5 were considered as chymotrypsin or trypsin (Table S4). Genes in cluster

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5-2 share extremely similar structures at genome and protein levels and 16 of total 18

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genes were predicted to be SPs. These SPs expect PpSP55 were predicted to be

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activated by proteolytic cleavage between Arg and Ile, and consisted of signal

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peptides and single serine protease domains formed by two or three exons gene

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structures (Table S4). In following sections, we roughly described P. puparum SPs and SPHs based on

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their structures and functions.

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3.2 Structure features of the SPs and SPHs

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Signal domain SPs

Nearly half of P. puparum SP genes (84/183) are shorter than 300 residues, and

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only contain signal peptide and one serine protease domain (Table S4). Previous

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studies indicated that these enzymes were most likely to be trypsin or chymotrypsin

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and expressed in gut which is related to digestion. We analyzed the expression

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patterns of several SPs in the tissues of female wasps (Fig. 2) and SPs with signal

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domain were marked with black frame. PpSP17, PpSP72 and PpSP90 displayed

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similar expression patterns with strikingly high expressions in gut and very low levels

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in other tissues. Results also showed that PpSP14 was expressed specifically in fat

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body while PpSP21, PpSP56, PpSP83, PpSP95 and PpSP99 were exclusively

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expressed in venom gland. These results indicated that these proteases are widely

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distributed in different tissues to exert diverse functions.

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3.2.2

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Clip-domain SPs/SPHs (cSPs/cSPHs)

Clip-domain SPs (cSPs) and clip-domain SPHs (cSPHs) are involved in

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signal-amplifying reaction and play a significant role in mediating innate immunity

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through cleaving and activating downstream SPs/SPHs. There were 16 cSPs and 9

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cSPHs predicted in P. puparum genome. Clip domains are 37-55 amino acid

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sequences knitted by three disulfide bonds to form quite compact structures. The

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length of clip domains in P. puparum PpcSPs/cSPHs vary from 30 to 55 amino acids

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(Fig. S2). The domain of cSPs/cSPHs from P. puparum, A. mellifera, D. melanogaster and M.

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sexta were aligned (Fig. S3) and the phylogenetic analysis revealed that P. puparum

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cSPs/cSPHs were clustered together with other three insect cSP/cSPH genes (Fig. 3).

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The phylogenetic tree showed that cSPs and cSPHs could be roughly divided into

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three clades. A close ortholog relationship was observed between PpcSPH9,

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MsSPH53 and masquerade proteins from D. melanogaster. All of them contain 5 clip

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domains followed by a serine protease domain. PpcSP9 clustered with AmHP8,

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MsHP8, MsPAP1 and PpcSP8. We examined the microbe (E. coil, M. luteus and B.

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bassiana) induced expressions of PpcSP8 and PpcSP9. PpcSP8 was barely increased

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by bacteria or fungus (data not shown) whereas the expression of PpcSP9 was

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enhanced by the treatment of M. luteus and B. bassiana 24 h and 48 h post infection

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(p. i). The expression of PpcSP9 reached the highest in the fungus-infected samples

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24 h p. i. PpcSP6 shares a high amino sequence identity with AmHP21 and MsHP6.

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QPCR results showed the induction of PpcSP6 24 h and 48 h post fungus infection

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(Fig. 4). The expressions of PpcSPH1 and PpcSPH8 were also increased after immune

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challenge. The expression of PpcSPH1 was dramatically activated by each of three

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microbes 6 h p. i., and then gradually decreased in the E. coli and M. luteus-infected

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samples while it reached a peak 24 h p. i. in the B. bassiana-infected samples.

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PpcSPH8 was expressed highly in the M. luteus and B. bassiana-treated samples only

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at 24 h and 48 h p. i (Fig. 4).

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ACCEPTED MANUSCRIPT 321

3.2.3

Other complex domain SPs/SPHs

Ten of one hundred and eighty-three SP/SPH genes are far larger than the size of a

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typical serine protease gene, and these SP/SPH genes consist of other domains and

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modules (Fig. 5). These modules include complement control protein (CCP) domain,

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low-density lipoprotein receptor class A (LDLA) domain, CUB domain, Ankyrin

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repeat region (ANK), Frizzled (FZ) domain, SEA domain, Kringle domain, PAN

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domain, Chitin-binding (CHIT) domain, scavenger receptor (SR) domain and C-type

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lectin (CTL) domain. PpSP24 contains TM region, FZ domain, LDLA repeats, SR

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domain and serine protease domain. The domain structure is similar with Drosophila

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Corin, and may act as a pro-atrial natriuretic peptide-converting enzyme (Yan et al.,

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2000). PpSP74 contains 4 LDLA repeats, 2 CCP domains followed by serine protease

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domain whereas PpSP81 possess 2 more LDLAs than the domains in PpSP74. Both

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of PpSP74 and PpSP81 have domain architectures quite similar to M. sexta HP14b

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and modular serine protease (MSP) from D. melanogaster (DmMSP) (Fig. 5).

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PpSP81 was hardly induced by immune challenge (data not shown). Analysis of the

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expression response of PpSP74 gene to each of three microbes showed a relatively

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low expression in the B. bassiana-challenged sample 6 h and 48 h p. i, but displayed a

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striking induction at 24 h p. i (Fig. 4).

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3.3 Possible function of SPs/SPHs in development

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Extracellular serine protease processing events are essential during embryonic

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development. Drosophila embryonic dorsal-ventral polarity relies on the serine

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proteinase cascade in the egg perivitelline space (Belvin and Anderson, 1996). Several

ACCEPTED MANUSCRIPT identified SPs such as Nudel, gastrulation defective (GD), Snake and Easter are the

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key components associated with DV axis establishment. Binding to Pipe-sulfated

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glycoprotein, Nudel can be autoactivated firstly and then activate GD zymogen. GD

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interacts with Snake, which in turn activates terminal proteinase of the signal cascade,

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Easter. There were 3 GD genes, 1 Nudel-like gene, 2 Snake genes and 7 Easter genes

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predicted in P. puparum genome. We only identified one Nudel-like gene PpSP62 in P.

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puparum genome and this gene is a large mosaic protein consisting of a TM region

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with serine protease domain embedded between the second and third LDLA domains.

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The number of LDLA domains is 4, far less than LDLA domains in Nudel genes in A.

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mellifera, D. melanogaster, Culex quinquefasciatu, but the same with the number of

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domains in N. vitripennis and Trichogramma pretiosum Nudel-like genes (Fig. S4).

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The presence of the transmembrane region suggested that PpSP62 located at vitelline

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membrane served as a signal transduction member. The sequence alignment was made

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between PpSP62, 8 Nudel genes and 2 Nudel-like genes (Fig. S5). The phylogenetic

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tree revealed that genes from hymenoptera were divided into two clades. PpSP62 was

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closely clustered with Nudel-like genes from N. vitripennis and Trichogramma

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pretiosum. The Nudel genes of A. mellifera, Megachile rotundata and M. demoliter

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formed the other clade (Fig. 6a). PpSP67, PpSP121 and PpSP129 are similar to GD

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gene, which usually contains a putative signal peptide and a serine protease domain.

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The phylogenetic tree revealed that these three GD genes in P. puparum were

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clustered with N. vitripennis GD gene and PpSP121 was more closely related with N.

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vitripennis GD gene (Fig. S6 & Fig. 6b). Based on the transcriptome data, only

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ACCEPTED MANUSCRIPT PpSP129 had relatively high expression in the embryo stage. The FPKM values of

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embryos in PpSP67 and PpSP121 were less than 1.0 (Table. S2). The qPCR analyses

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also verified these results, indicating that only PpSP129 may function in the

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embryonic development (Fig. 7). PpcSP3 and PpcSP6 found in the genome of P.

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puparum were predicted as Snake genes (Table S4). These two genes possess signal

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peptides, clip domains and serine protease domains as most Snake genes characterized

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thus far. We also identified 7 Easter genes. Easter processes Spätzle into activating

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ligand for Toll receptor and finally triggers the intracellular Toll-dorsal pathway. Four

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Easter genes are closely linked with the same orientation and are located in scaffolds

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16 (Fig. 1). These 4 Easter-like genes are distributed in a cluster within only 14.5 kb.

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Another two Easter genes located in scaffolds 7 (Fig. S1) were also closely linked,

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indicating Easter genes in P. puparum undergo gene duplication. A search of the P.

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puparum genome showed 12 P. puparum Stubble genes. Stubble has been

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characterized as transmembrane protein in intracellular signaling during leg and wing

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imaginal disc morphogenesis (Bayer et al., 2003). Eleven of P. puparum Stubble

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genes lacked the transmembrane domains which may be due to the incomplete of

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N-terminal sequence. The number of Stubble genes in P. puparum is more than 8 in P.

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xylostella, 5 in N. lugens and 1 in D. melanogaster. The length of P.puparum Stubble

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genes varies from 262 to 946 amino acids, consisting of 5-12 exons (Table S4). These

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implied the abundance of Stubble-like genes in P. puparum may also be conductive to

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other physiological processes.

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3.4 Expression profile of SPs/SPHs

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ACCEPTED MANUSCRIPT 387

Several transcriptome databases from different development stages and tissues of P. puparum have been acquired. We obtained RNA-seq from different development

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stages of P. puparum, including embryos, larvae, female and male pupae, female and

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male adult wasps. The samples of venom, ovary and carcass from adult female wasps

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were also collected for transcriptome sequencing. Expression profiles of serine

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proteinase genes were determined by these databases.

The transcriptome (Table S2) revealed that some P. puparum SPs and SPHs owned

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very low FPKM values in these already existing databases. Some of these SP/SPH

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genes were possible to be pseudogenes. We retained all of the SPs and SPHs in

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consideration of that some genes were only highly expressed in a specific tissue, but

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not or barely expressed in other tissues. PpSPs and PpSPHs genes (133 in total with

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the FPKM values greater than 1) were profiled (Fig. 8). The hierarchical clusters were

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used to represent relative values of these genes and could be roughly divided into four

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groups.

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A few numbers of SPs/SPHs displaying highest expressions in embryos were listed

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in group I. Interestingly, more than half of these genes displayed similar expression

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patterns and were detected at high levels in both embryo and female adult stages. The

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SP and SPH genes highly expressed in adult female, the carcass and the ovary were

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also categorized into group I. Group II is the largest group of genes highly expressed

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in larval period. This group contains 53 SPs/SPHs, accounting for nearly 40% genes

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in this super family. In group III, genes can be divided into three parts. In the first two

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parts, eight and eleven SPs presented distinct expression patterns in mature female

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ACCEPTED MANUSCRIPT and male stages, respectively. Eleven SPs and SPHs with high transcript levels in both

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female and male adult stages were located in the third part. In group IV, eight

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SPs/SPHs expressed mainly in female or male pupal stage in the upper part and

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eighteen SPs/SPHs showed most abundance in the venom gland of the lower part.

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Screening the transcriptome of P. puparum adult females yielded several genes with

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notably high FPKM values in venom gland. To control false positive rate, the

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expression level cut-off was set as FPKM_VG (Venom gland) > 10, and a venom

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gland to carcass expression ratio log2 (FPKM_VG/FPKM_Carcass) > 1 and

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corrected p-value < 0.001 to define differentially expressed genes in the venom

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gland. Sixteen of eighteen PpSPs/PpSPHs profiled in group IV (Table S3) were

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differentially expressed in the venom gland relative to the carcass. Among them, six

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PpSPs and two PpSPHs contain signal peptides and identified in proteomic database,

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which were considered as venom proteins. In our previous work, eight PpSPs and a

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PpSPH were identified as venom proteins with the combined transcriptomic and

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proteomic analysis (Yan et al., 2016). It is reasonable to have these two distinct results

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since they mapped to different assembly sequences. We also used qPCR to validate

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RNA-seq databases (Fig. 2). PpSP56, PpSP99, PpSP115, PpSPH1, PpSPH23 and

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PpSPH26 had extremely higher transcript profiles in venom gland than those in other

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tissues. The expression of PpSPH1 in venom gland was 1,000 times of that in fat body,

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the second highest expression tissue in adult female. PpSP56, PpSP99, PpSP115,

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PpSPH23 and PpSPH26 also showed similar expression patterns, with transcript

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levels in venom gland dozens of times higher than that in other tissues. Compared to

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ACCEPTED MANUSCRIPT the number of SP and SPH genes specifically expressed in the venom gland, a few

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showed most abundance in the ovary. PpSP62 and PpSPH12 showing relatively high

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expression in the ovary were categorized into the lower part of group I.

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4. Discussion

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SPs and SPHs belong to a large family, which function in biological processes as

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food digestion, development and immunity. In the present article, P. puparum SP and

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SPH genes have been identified by comparative analysis of genes in other reported

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insects and confirmed by catalytic triad. There were 145 SPs and 38 SPHs identified

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in the genome of P. puparum. The ratio of SPs and SPHs is close to that in D.

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melanogaster or A. mellifera.

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SP/SPH genes displaying a lineage of tandem repeats distribution were observed in

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scaffolds of the P. puparum genome. These P. puparum SPs/SPHs presented large

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clusters in several scaffolds with similar structures or functions. This phenomenon is

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also widely identified in several species, such as D. melanogaster, B. mori and N.

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lugens (Bao et al., 2013; Ross et al., 2003; Zhao et al., 2010). Previous studies showed

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that gene expansion does not occur in SPs/SPHs of honey bee, A. mellifera, and this

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may be due to their sociality, which shapes the diversity and evolution of the genes

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(Gadau et al., 2012; Simola et al., 2013; Viljakainen et al., 2009). Therefore, the

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amazing expansion of this superfamily in P. puparum with a similar structure or

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function is the results of gene duplication and unequal crossing over.

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SPs with signal peptides and single serine protease domains are likely to be

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gut-related serine proteinases. SPs in P. puparum presented distinct expression

ACCEPTED MANUSCRIPT patterns. SPs most abundantly expressed in gut tissue may relate to digestion. For

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another part of SPs, the SPs highly expressed in fat body may indicate a role in wasp

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immune system, whereas the SPs mainly expressed in venom gland may indicate a

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function in regulating the host immune system. Signal serine proteinase domain SPs,

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which occupying half of the SP genes family in P. puparum, may also participate in

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various physiological processes. Further researches are needed to identify the function

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of these candidate SP genes.

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The size of P. puparum SP and SPH genes ranges from 175 to 2242 residues, with

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the average size of 360. Our results showed that 35 of the 183 P. puparum SP and

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SPH genes contain other modules. Clip domains constitute the largest group of

463

regulatory domains in P. puparum. These cSPs/cSPHs play a vital role in immune

464

reaction and embryo development. PpcSPH9 contains five clip domains and shares a

465

similar structure with masquerade. Drosophila masquerade gene participates in

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somatic muscle attachment in Drosophila embryo (Murugasuoei et al., 1995).

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Mutations in this Drosophila SPH affect axonal guidance and taste behavior

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(MurugasuOei et al., 1996). PpcSPH9 with penta clip domains may allow the attached

469

SPH domain to interact with multiple partners. We examined the microbe induced (E.

470

coil, M. luteus and B. bassiana) expressions of the clip-domain SP/SPH genes. QPCR

471

results indicated PpcSP9 can be activated by Gram positive bacterium and fungus. We

472

implied PpcSP9 may also be involved in immune response. PpcSPH1 responded

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quickly when infected by any of three microbes and may behave as a vital cofactor in

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upstream of the proteinase cascade. PpcSPH8 expressed highly in M. luteus and B.

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ACCEPTED MANUSCRIPT bassiana samples 24 h and 48 h p. i., indicating it may be regulated by the Toll

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pathway activated by gram-positive bacteria and fungus. Some SPs contain other

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types of domain additions. PpSP74 share structure similarity with M. sexta HP14b and

478

DmMSP. Previous studies showed that M. sexta HP14 and DmMSP could detect

479

bacteria and fungi by peptidoglycan-recognition molecules and then autoactivated and

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triggered the serine proteinase cascade in activating prophenoloxidase system or Toll

481

pathway (Buchon et al., 2009; Kim et al., 2008; Wang and Jiang, 2010). The

482

expression of PpSP74 have been strikingly induced 24 h post B. bassiana infection,

483

indicating that PpSP74 may also be a modular serine protease that could be recruited

484

into the PG recognition complex.

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Previous studies presented several kinds of SP and SPH genes that determine the

486

dorsal-ventral polarity of embryos in Drosophila (Cho et al., 2010). Among them,

487

Drosophila Nudel is an initiator of these SP cascades. In the structure of Nudel genes

488

in most insects, the SP and SPH domains separated by two or three LDLA domains

489

(Fig. S4). However, we only found Nudel-like genes in the parasitoids of P. puparum,

490

N. vitripennis and T. pretiosum. The sequence of PpSP62 was confirmed by gene

491

cloning. These nude-like genes are much shorter than Nudel genes. The phylogenetic

492

trees also showed that Nudel and Nudel-like genes in hymenoptera formed two clades.

493

These may indicate that some Nudel-like genes in hymenoptera parasitoids such as

494

PpSP62 may lose part of C-terminal sequences in evolutionary events.

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RNA-seq databases provided us with gene temporospatial expression patterns.

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Nearly 2/5 of the P. puparum SPs/SPHs expressed exclusively at the larval stage. P.

ACCEPTED MANUSCRIPT puparum wasps grow up inside the hemocoel of their host, may encounter defense

498

factors like proteinase inhibitors secreted by their host to block the serine protease

499

activity, which is similar to the interaction mechanism between herbivorous insects

500

and their host (Azzouz et al., 2005; Chougule et al., 2005). Meanwhile, larvae are

501

considered as a rapid growth stage. It is necessary to secrete massive proteinase for

502

digesting sufficient food. Therefore, the wasps need to generate abundant SPs/SPHs to

503

compete with the defense stress and grow up quickly in a host-parasitoids

504

co-evolution system (Saadat et al., 2014). The number of SPs/SPHs mainly expressed

505

in female or male pupal stages is 8, much less than that (53) in larval stage. In pupal

506

stage, the host provides these immature wasps with natural barriers for eclosion and

507

protects them from the attack of enemies. Besides, these pupae stop the ingestion of

508

food. It is expected to see the reduced expression levels of SPs/SPHs involved in

509

immunity and digestion. Insight into the distribution of adult female tissues shows

510

that several P. puparum SPs/SPHs are highly expressed in the venom gland. In most

511

host-parasitoid interaction mechanism, the venom secreted by the venom gland is an

512

essential requirement for successful parasitism (Asgari and Rivers, 2011; Fang et al.,

513

2011b; Martinson et al., 2014). Wasps introduce parasitoid factors to immobilize their

514

host. These include protein secretions from venom glands, PDVs and so on. Abundant

515

SPs/SPHs as venom proteins are discovered in parasitoids (Colinet et al., 2014; de

516

Graaf et al., 2010) and other venomous animals (Mukherjee et al., 2016). The serine

517

proteases in N. vitripennis venom induced cell apoptosis of Sf21 cell line (Formesyn

518

et al., 2013). A clip-SPH called Vn50 isolated from the venom of C. rubecula blocked

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ACCEPTED MANUSCRIPT the melanization of its host through significantly reduced proteolysis of proPO

520

(Asgari et al., 2003; Zhang et al., 2004). Previous studies also revealed that a clip-SP

521

from Bombus ignitu venom SP (Bi-VSP) is a multifunctional enzyme. In the

522

immunity of B. ignite, BI-VSP acts as a proPO-activating factor, thereby triggering

523

the PO cascade. Bi-VSP also exhibits fibrin(ogen)olytic activity when interacts with

524

mammals and could induces a lethal melanization response in target insects (Choo et

525

al., 2010). Since N. vitripennis and P. puparum are close relatives that belong to the

526

same family, P. puparum venom SP and SPH genes may also act as cytotoxic

527

compounds in cell death related processes. Studies also showed that serine protease

528

and their homologs with clip domain generated striking melanization in the host or

529

enemies. We made an assumption that clip-domain SPs/SPHs specifically expressed

530

in the venom gland of P. puparum can also participate in the serine protease cascade

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of the host (P. rapae) and interfere in the host signal transduction of immune system.

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All in all, P. puparum serine proteases and their homologs have been identified in

533

different development stages, tissues and microbe-challenged samples, which provide

534

a better understanding of the roles of these enzymes in digestion, development and

535

immunity in this parasitoid. Further research is needed to gain more insights in their

536

physiological processes and to provide feasible target genes used in biological control

537

of insect pests.

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ACCEPTED MANUSCRIPT Funding:

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The study is supported by grants from National Natural Science Foundation of China

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(Grant no. 31472038, 31272098, http://www.nsfc.gov.cn/), Major International

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(Regional) Joint Research Project of National Natural Science Foundation (Grant

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no.31620103915, http://www.nsfc.gov.cn/), and China National Science Fund for

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Distinguished Young Scholars (Grant no. 31025021, http://www.nsfc.gov.cn/) as well

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as the Foundation for Innovative Research Group from Ministry of Agriculture,

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China.

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Figure Legends

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Fig. 1. Scaffold location of Pteromalus puparum SP and SPH genes. Gene names

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and predicted functions are shown on the right of the bar. The distance of two adjacent

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genes (kilobases, kb) is presented on the left, while the distance cannot match the

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length of bar is marked in red. Scaffold number is shown at the top of each bar.

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Fig. 2. Tissue-specific expressions of SP and SPH genes. Total RNA was extracted

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from the gut (G), fat body (FB), ovary (O), venom (V) and carcass (C, the remaining

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body) of the adult female P. puparum, and used to analyze the expression patterns of

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these SPs and SPHs using qPCR. P. puparum 18s rRNA was used as a housekeeping

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gene. Error bars represent the means ± standard deviations from three biological

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replicates. A one-way ANOVA was used to determine the significant difference with

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different lowercase letter (a-c) (p <0.05).

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Fig. 3. Phylogenetic analysis of clip domain SP/SPH genes. The domain of amino

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acid sequences of Pteromalus puparum (Pp), Apis mellifera (Am), Manduca sexta

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(Ms) and Drosophila melanogaster (Dm) were aligned. Phylogenetic tree was

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constructed by Neighbor-joining method, using the program Mega 5.10. Red spots at

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the nodes denote bootstrap values greater than 500 from 1000 trials.

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Fig. 4. Expression levels of SP/SPH genes following different immune challenge.

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Expression levels of SP/SPH genes following the infection of Gram-negative

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(Escherichia coli) or Gram-positive (Micrococcus luteus) bacterium or

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entomopathogenic fungus (Beauveria bassiana) were analyzed using qPCR. Time

ACCEPTED MANUSCRIPT points along the x-axis represent the hours post-infection. Error bars represent the

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means ± standard deviations from three biological replicates. P. puparum actin 1 was

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used as a housekeeping gene. A two-way ANOVA was used to determine the

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combined effects of infection and time. The different lowercase letters (a-c) represent

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the significant difference at the different time points after infection with the same

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pathogen (p <0.05), and the capital letters (A~C) indicate the significant difference at

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the same time points after different pathogenic infection (p <0.05).

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Fig. 5. Domain organizations of SPs/SPHs with complex domains in Pteromalus

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puparum.

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Fig. 6. Phylogenetic analysis of GD (a) and Nudel (b) genes. Phylogenetic tree was

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constructed by Neighbor-joining method, using the program Mega 5.10. Red spots at

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the nodes denote bootstrap values greater than 500 from 1000 trials. The GenBank

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accession number for sequences used in phylogenetic analysis in Fig. 6a: PpSP62

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(PPU07067-RA, Pteromalus puparum); Nv_Nudel-like (XP_003424379.2, Nasonia

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vitripennis); Tp_Nudel-like (XP_014224565.1, Trichogramma pretiosum); Md_Nudel

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(XP_008548271.1, Microplitis demolitor); Am_Nudel (XP_006559739.1, Apis

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mellifera); Mr_Nudel (XP_012141152.1, Megachile rotundata); Tc_Nudel

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(XP_015840900.1, Tribolium castaneum); Ap_Nudel (XP_001949959.4,

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Acyrthosiphon pisum); Dm_Nudel (NP_523947.2, Drosophila melanogaster);

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Cq_Nudel (XP_001843380.1, Culex quinquefasciatus) and Cb_Nudel

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(XP_019889456.1, Cerapachys biroi). Amino acid sequences used for phylogenetic

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tree in Fig. 6b: PpSP67 (PPU07692-RA, Pteromalus puparum); PpSP121

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(PPU13886-RA, Pteromalus puparum); PpSP129 (PPU16927-RA , Pteromalus

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puparum); NvGD (XP_003427708.1, Nasonia vitripennis); Am_GD

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(XP_006563318.1, Apis mellifera); MrGD (XP_012143735.1, Megachile rotundata);

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Dm_GD (NP_001259478.1, Drosophila melanogaster); Bm_GD (XP_012548092.1,

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Bombyx mori) and Tc_GD (KYB27136.1, Tribolium castaneum).

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Fig. 7. Confirmation of developmental stage expressions of SP and SPH genes:

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Total RNA was extracted from the embryo (E), larvae (L), female pupae (FP), male

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pupae (MP), female adult (FA) and male adult (MA), and used to analyze the

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expression pattern of these SPs and SPHs using qPCR. P. puparum 18s rRNA was

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used as a housekeeping gene. Error bars represent the means ± standard deviations

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from three biological replicates. A one-way ANOVA was used to determine the

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significant difference with different lowercase letter (a-d) (p <0.05).

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Fig. 8. Expression profiles of Pteromalus puparum SP and SPH genes across

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different developmental stages and tissue distributions. Log2 FPKM values for the

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SPs/SPHs are presented by bar colors where the darker red represent higher

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expression values, the darker green represent lower expression values.

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Supplementary Material

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Fig. S1. Scaffold location of Pteromalus puparum SP and SPH genes. Gene names

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and predicted functions are shown on the right of the bar. The distance of two adjacent

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genes (kilobases, kb) is presented on the left, while the distance cannot match the

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length of bar is marked in red. Scaffold number is shown at the top of each bar.

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Fig. S2. Alignment of 25 Pteromalus puparum clip domain sequences by Clustal

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X2. PpcSPH9 has five clip domains represented by PpcSPH9-1, -2, -3, -4 and -5. Six

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conserved Cys residues are marked with black.

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Fig. S3. Alignment of serine proteinase domains from clip SPs/SPHs of

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Pteromalus puparum (Pp), Apis mellifera (Am), Manduca sexta (Ms) and

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Drosophila melanogaster (Dm) by Clustal X2.

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Fig. S4. Domain organizations of Nudel and Nudel-likegenes in 11 insect species.

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PpSP62 (PPU07067-RA, Pteromalus puparum); Nv_Nudel-like (XP_003424379.2,

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Nasonia vitripennis); Tp_Nudel-like (XP_014224565.1, Trichogramma pretiosum);

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Cb_Nudel (XP_019889456.1, Cerapachys biroi); Am_Nudel (XP_006559739.1, Apis

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mellifera); Dm_Nudel (NP_523947.2, Drosophila melanogaster); Cq_Nudel

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(XP_001843380.1, Culex quinquefasciatus); Tc_Nudel (XP_015840900.1, Tribolium

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castaneum); Mr_Nudel (XP_012141152.1, Megachile rotundata); Md_Nudel

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(XP_008548271.1, Microplitis demolitor); Ap_Nudel (XP_001949959.4,

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Acyrthosiphon pisum).

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Fig. S5. Alignment of amino acid sequences from Nudel or Nudel-like genes of

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Pteromalus puparum and other 10 insects by Clustal X2.

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Fig. S6. Alignment of serine protease domains of GD homolog genes from

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Pteromalus puparum and other 6 insects by Clustal X2.

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Table S1. Primers used for qPCR analysis of gene expressions.

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Table S2. FPKM values of the Pteromalus puparum serine proteases and their

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homologs at different development stages and tissues obtained from the RNA-seq

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data.

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Table S3. Differentially expressed Pteromalus puparum serine proteases and their

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homologs in the venom gland.

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Table S4. Prediction of serine proteases and their homologs in Pteromalus

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puparum.

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Table S5. The predicted amino acid sequences of 183 Pteromalus puparum serine

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proteases and their homologs.

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Table S6. NCBI blast descriptions of serine proteases and their homologs in

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Pteromalus puparum.

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Table S7. Gene counts for clip-domain and non-clip-domain serine proteases and

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their homologs in ten insect species.

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