Identification of a small pacifastin protease inhibitor from Nasonia vitripennis venom that inhibits humoral immunity of host (Musca domestica)

Accepted Manuscript Identification of a small pacifastin protease inhibitor from Nasonia vitripennis venom that inhibits humoral immunity of host (Musca domestica) Cen Qian, Dan Liang, Ya Liu, Pei Wang, Saima Kausar, Guoqing Wei, Baojian Zhu, Lei Wang, Chaoliang Liu PII:

S0041-0101(17)30092-2

DOI:

10.1016/j.toxicon.2017.03.005

Reference:

TOXCON 5592

To appear in:

Toxicon

Received Date: 4 January 2017 Revised Date:

2 March 2017

Accepted Date: 6 March 2017

Please cite this article as: Qian, C., Liang, D., Liu, Y., Wang, P., Kausar, S., Wei, G., Zhu, B., Wang, L., Liu, C., Identification of a small pacifastin protease inhibitor from Nasonia vitripennis venom that inhibits humoral immunity of host (Musca domestica), Toxicon (2017), doi: 10.1016/j.toxicon.2017.03.005. 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.

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ACCEPTED MANUSCRIPT Identification of a Small Pacifastin Protease Inhibitor from Nasonia vitripennis Venom that

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Inhibits Humoral Immunity of Host (Musca domestica)

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Cen Qian1, Dan liang1, Ya Liu, Pei Wang, Saima Kausar, Guoqing Wei, Baojian Zhu, Lei Wang,

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Chaoliang Liu*

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College of Life Sciences, Anhui Agricultural University, 130 Changjiang West Road, Hefei

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230036, PR China

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*Corresponding author: College of Life Sciences, Anhui Agricultural University, 130 Changjiang

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West Road, Hefei 230036, China.Tel:+86 551 65786360; Fax:+86 551 65786360; E-mail:

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[email protected].

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These authors contributed to this work equally.

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Abstract Nasonia vitripennis is an important natural enemy of many flies. Pacifastin protease inhibitors

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(PPIs) play an important role in development and innate immunity of insects. In this study, cDNA

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sequences of a small pacifastin protease inhibitor in N. vitripennis (NvSPPI) was characterized and

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its open reading frame (ORF) contains 243bp. Real-time quantitative PCR (RT-qPCR) results

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revealed that NvSPPI mRNA were detected specifically in the venom apparatus, while they were

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expressed at low levels in other tissues tested. In the venom apparatus, NvSPPI transcript was

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highly expressed on the fourth day post eclosion and then declined gradually. The NvSPPI gene

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was recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused

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with glutathione S-transferase were purified. Inhibition of recombinant GST-NvSPPI to three

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serine protease inhibitors (trypsin, chymotrypsin, and protease K) were tested and results showed

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that recombinant NvSPPI could inhibit the activity of trypsin. Meanwhile, we evaluated the

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influence of the recombinant GST-NvSPPI on the phenoloxidase (PO) activity and

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prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results

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showed PPO activation in host hemolymph was inhibited by recombinant NvSPPI; however, there

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was no significant inhibition on the PO activity. Our results suggested that NvSPPI could inhibit

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PPO activation in host hemolymph and trypsin activity in vitro.

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Key words: Nasonia vitripennis; Serine protease inhibitors; Prophenoloxidase activation;

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Humoral immunity; Biological control

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ACCEPTED MANUSCRIPT 1. Introduction

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The pacifastin protease inhibitors (PPIs), characterized by multiple pacifastin light chain domains

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(PLDs), comprise a large family of protease inhibitors. The current knowledge on the function of

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PPIs is mainly on their inhibition of proteolytic enzymes, especially serine protease (Breugelmans

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et al., 2009; Gai et al., 2008; Simonet et al., 2003). The first member of the pacifastin family was

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found in the crustacean species, Pacifastacus leniusculus (Crustacea: Decapoda) (Hergenhahn et

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al., 1987). Subsequently, PPIs were found in Eriocheir sinensis (Crustacea: Decapoda), Penaeus

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monodon (Crustacea: Decapoda) Schistocerca gregaria (Orthoptera: Acrididae), Bombyx mori

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(Lepidoptera, Bombycidae) and other arthropods (Breugelmans et al., 2009; Gai et al., 2008;

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Liang et al., 1997; Liu et al., 2015; Sangsuriya et al., 2016; Simonet et al., 2002; Simonet et al.,

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2004). All PLDs of PPIs are characterized by a conserved pattern of six cysteine residues (Cys1 –

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Xaa9-12 – Cys2 – Asn –Xaa – Cys3 – Xaa – Cys4 – Xaa2-3 – Gly – Xaa3-6 – Cys5 – Thr –Xaa3

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– Cys6) (Breugelmans et al., 2009). The 3-D structure analysis shows that these six residues form

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three disulfide bridges through Cys1–Cys4, Cys2–Cys6 and Cys3–Cys5 models, which give PPIs

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a typical fold and remarkable stability (Breugelmans et al., 2009; Gáspári et al., 2002;

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Kellenberger et al., 2003; Mer et al., 1996; Roussel et al., 2001).

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Although many PPIs were discovered in arthropods, the functional analyses were carried out

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mainly on the crustacean species and the locust species. In crayfish, the transcript levels of PPI in

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hemocytes could be up-regulated through immune challenge. RNA interference (RNAi) of

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crayfish PPI could not only induce a higher phenoloxidase activity after immune challenge but

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give rise to the enhancement of nodules and phagocytosis. These results indicated that PPIs in the

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crayfish were involved in both humoral and cellular immune responses (Hergenhahn et al., 1987;

ACCEPTED MANUSCRIPT Liu et al., 2007). In swimming crab, Portunus trituberculatus (Crustacea: Decapoda), PPIs were

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involved in antibacterial defense and prophenoloxidase cascade (Sangsuriya et al., 2016;

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Sivakamavalli et al., 2016 ). In S. gregaria, the transcript levels of PPIs were more abundant in the

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fat body of immune challenged individuals, which suggested PPIs In S. gregaria might participate

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in the immune response (Franssens et al., 2008). In B. mori, the PPI could inhibit a fungal

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trypsin-like cuticle degrading enzyme, suggesting its protective function of defensing against

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entomopathogenic fungi (Breugelmans et al., 2009). In other insects, no direct evidence is

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available on the possible functions of PPIs.

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Nasonia Vitripennis (Hymenoptera: Pteromalidae) is a model parasitic wasps (Shuker et al., 2003; Werren et al., 2010). Parasitic wasps are natural enemy insects that play an important role in

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biological control. They lay eggs into hosts or on the surface of hosts along with maternal and

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embryonic factors such as venom, polydnavirus (PDV), virus-like particles (VLP), ovarian

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proteins, teratocytes, and proteins secreted from larvae to interfere with the host immune

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responses for successful parasitization (Dong et al., 2007; Dong et al., 2009; Zhu et al., 2008 ).

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Unlike ichneumonid and braconid parasitoids, N. vitripennis only injects venom into its host (flies)

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after oviposition. So it must interfere with the host immune response through venom proteins.

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Using proteomics methods, De Graaf et al (2010) previously identified 79 venom proteins from N.

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vitripennis, one of which is small serine protease inhibitor-like venom protein (NCBI accession

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number NP_001155083). Our group also determined the transcriptome and proteome of venom

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gland and other residual tissues in N. vitripennis by RNA-seq (RNA sequencing) and LC-MS/MS

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(liquid chromatography–tandem mass spectrometry) methods (Qian 2013), and we also detected a

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small protease inhibitor protein with a pacifastin domain in venom (NvSPPI), as described by de

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Graaf et al. The first venom PPI, cysteine-rich venom protein 4 (CVP4), was found in the venom of an endoparasitoid wasp (Pimpla hypochondriaca), that is known to influence the immune system of

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the host organism (Parkinson et al., 2004). But no direct evidence is available on the possible

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immune functions of the venom PPIs. Here, we molecularly characterized a small pacifastin

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protease inhibitor in N. vitripennis venom (NvSPPI) and determined its tissue and developmental

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expression patterns. We also tested the role of NvSPPI in inhibition of host (Musca domestica,

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Diptera: Muscidae) immune response, such as the inhibition of recombinant NvSPPI on three

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serine protease inhibitors’ in vitro and PO activity and PPO activation of host hemocytes. These

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results will provide further insight into the role of PPIs in insects’ immune responses and offer a

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new ideal for the biological control of insect pests.

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2.1. Insect rearing

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Cultures of Musca domestica and N. vitripennis were collected from the experimental field of

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Zhejiang University, Hangzhou, China. In brief, the host larvae were fed on an artificial diet

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composed of 15% milk powder, 35% wheat bran, and 50% water, in 500 mL glass canning jars (at

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25 ± 1 °C, light:dark = 10h:14h, relative humidity (R. H.) = 75%) until eclosion. M. domestica

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adults were maintained on a mixture of sugar and milk powder (10:1) and water, within a stainless

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steel-mesh cage (55 cm × 55 cm × 55 cm) under the same conditions just described. Freshly

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pupated hosts were exposed to mated female wasps (pupae:wasps = 1:10) in a 500 mL glass

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canning jar for 24 h. The parasitized pupae were maintained under the conditions just described.

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After emerging, the female wasps were collected and held in glass containers (also under the

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conditions just described) and fed ad lib on 20% (v/v) honey solution to lengthen the life span for

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3-4 days until dissection of the venom reservoir and gland.

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Female N. vitripennis from 0 to 7 days post eclosion were collected and paralyzed for 5 min at

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−70 °C. The head, thorax, gut, ovary, remaining abdomen carcass (the rest of the abdomen after

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dissection), and venom apparatus (containing venom reservoir and gland) were then collected on

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ice under a stereoscope (Lecia, Frankfurt, Germany). Samples were stored at −70 °C.

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2.3. RNA extraction and cDNA cloning

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The collected tissues were homogenized in liquid nitrogen, and total RNA were extracted using

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the TRizol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s

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instructions.RNA integrity was confirmed by ethidium bromide gel staining and RNA quantity

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was determined spectrophotometrically at A260/280. Single-stranded cDNAs were synthesized by

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using PrimeScript™ One Step RT-PCR Kit (Takara, Dalian, China). Oligonucleotide primers

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(Table 1) were designed based on cDNA sequences of N. vitripennis SPPIs (GenBank accession

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numbers is NM_001161611). PCR conditions were as follows: an initial delay at 95 °C for 5 min;

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34 cycles of denaturation at 95 °C for 30 min; annealing at 55 °C for 30s; and extending at 72 °C

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for 1 min. PCR products were fractionated in a 1% agarose gel by electrophoresis and purified

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with the DNA gel extraction kit (Aidlab, Beijing, China), and then cloned into the pGEM®-T easy

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cloning vector (Promega, Madison, Wisconsin, USA). Positive clones were selected by PCR and

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confirmed by sequencing at Invitrogen, Shanghai, China.

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The cDNAs and deduced amino acid sequences were analyzed by DNAStar software (version 5.02,

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DNAStar, Madison, Wisconsin, USA) and online Blast. The signal peptides were analyzed by

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Signal P. Multiple sequence alignments were carried out with DNAman software (Lynnon Biosoft,

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Quebec, QC, Canada) and Clustal W2. The phylogenetic tree was constructed by using MEGA 5.1

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(Tokyo Metropolitan University, Tokyo, Japan) with the neighbor-joining (NJ) method. The

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predicted tertiary structure of the protein was predicted by phyre2 online

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(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) and Raswin software.

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2.5. Protein expression, purification, and antibody preparation

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The primers (Table 1) containing BamH I and Xho I restriction enzyme sites were designed to

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amplify the ORF cDNA sequences of NvSPPI by PCR. PCR products and the pGEX-4T-2 vector

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were ligated after they were double digested by BamH I and Xho I (Takara, Dalian, China). The

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recombinant plasmids were confirmed by sequencing, transformed into Escherichia coli BL21

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(DE3) cells (AxyGen, Shanghai, China), and induced by 1 mM isopropyl thiogalactoside (IPTG)

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for protein expression. The recombinant proteins were analyzed by 12% SDS-PAGE and then

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purified using GST·BindTM Resin Kit (Novagen, Hilden, Germany) according to the protocols.

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Protein concentrations were determined by using the BCA Protein Assay Kit (Novagen, Hilden,

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Germany).

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ACCEPTED MANUSCRIPT 2.6. Real-time quantitative PCR (RT-qPCR)

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RT-qPCR was used to determine the transcription profiles of NvSPPI in different tissues and ages

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of N. vitripennis females. The 18S rRNA gene (accession number GQ410677) was used as an

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internal control. Ten microliters of each first-strand cDNA product were diluted with 90 µL of

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sterilized water before use. The RT-qPCR system was performed with each 20 µL reaction

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containing 10 µL SsoFast EvaGreen SuperMix (Bio-Rad, Hercules, California, USA), 1 µL

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forward primer (200 nM), 1 µL reverse primer (200 nM), 1 µL diluted cDNA, and 7 µL sterile

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water. The thermal cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s

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and 60 °C for 34 s. Amplification was monitored on the iCycleriQ TM Real-Time PCR Detection

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System (Bio-Rad, Hercules, California, USA). The specificity of the SYBR-Green PCR signal

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was further confirmed by melting curve analysis. The experiments were repeated three times as

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independent biological replicates. The mRNA expression was quantified using the 2−∆∆Ct method

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(Livak and Schmittgen, 2001).

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2.7. Serine protease inhibition assays

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Protease inhibition assays were performed with purified recombinant NvSPPI protein using the

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method described by Qian et al. (2015). Three typical serine proteases (bovine pancreatic trypsin,

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bovine pancreatic chymotrypsin, and protease K, 200 ng/mL) (Sigma, Taufkirchen, Germany) and

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their corresponding substrates (N-benzoyl-Val-Gly-Arg-p-nitroanilide,

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N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilid, and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilid) (Sigma,

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Taufkirchen, Germany) were selected for determination of the spectrum of enzyme inhibition by

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recombinant NvSPPI as follows. The recombinant NvSPPI (1 µg) was pre-incubated with a

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ACCEPTED MANUSCRIPT reaction buffer (100 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5) containing 200 ng/mL

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serine protease for 30 min at room temperature, and then 200 µL substrate was added (0.1 mM,

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100 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5) before measuring the absorbance at 405

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nm every min for 30 min. Substrates alone were used as blanks. Buffer and buffer with bovine

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serum albumin (BSA) were used as controls. One unit of enzyme activity was defined as an

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increase of absorbance by 0.001 per min. The experiments were repeated three times as

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independent biological replicates.

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2.8. Prophenoloxidase (PPO) activation and phenoloxidase (PO) activity assays

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PPO activation and PO activity assays were performed with purified recombinant NvSPPI

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according to the method described by Qian et al. (2015). M. domestica in the white pupal stage

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were sterilized by 75% alcohol, rinsed with sterilized water and finally air-dried at room

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temperature before use. Hemolymph was collected from pupae by puncturing the pupal cuticle

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with a sterilized dissecting pin and rapidly transferred into a sterilized 1.5 mL Eppendorf tube. The

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collected hemolymph was then centrifuged at 3300 g for 5 min at 4 °C, and the supernatant was

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transferred into another 1.5mL Eppendorf tube. Two microliters of each hemolymph sample were

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incubated with or without Micrococcus luteus (0.5 µg) in 10 µL of Tris buffered saline (TBS) at

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pH 7.4 in wells of a 96-well plate for 60 min at room temperature. Then 200 µL of L-dopamine (2

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mM in TBS, pH 6.5) was added and absorbance at 470 nm was monitored. One unit of PO activity

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was defined as an increase of absorbance at470 nm by 0.001 per min. Plasma samples with low

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PO activity when incubated alone but high PO activity after incubation with M. luteus were

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selected for subsequent PPO and PO activation assays.

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ACCEPTED MANUSCRIPT For PPO activation assays, each 2 µL of prepared hemolymph was incubated with 10 µL TBS,

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10 µL TBS/M. luteus (0.5 µg)/ NvSPPI (2 µg) mixture, 10 µL TBS/M. luteus (0.5 µg)/BSA (2 µg)

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mixture, 10 µL TBS/M. luteus (0.5 µg)/GST(2 µg) mixture, and 10 µL TBS/PTU (phenyl thiourea,

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saturated), respectively, for 20 min at 25 °C. Finally, 200 µL of L-dopamine substrate (2 mM) was

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added to each sample, and the PO activity was measured at 470 nm in a plate reader for 30 min.

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One unit of PO activity was defined as an increase of absorbance at 470 nm by 0.001 per minute.

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All of the above experiments were repeated three times as independent biological replicates.

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For PO activity assays, each 2 µL of prepared hemolymph was incubated with 10 µL TBS/M.

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luteus(0.5 µg) mixture for 10 min at 25 °C. Then 2 µL TBS, 2 µL TBS, 2 µL NvSPPI (2 µg), 2 µL

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BSA (2 µg), 2 µL GST (2 µg), and 2 µL PTU was added, respectively, and incubated for 10 min at

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25 °C. Finally, 200 µL of L-dopamine substrate (2 mM) were added to each sample, and the PO

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activity was measured at 470 nm in a plate reader for 30 min. One unit of PO activity was defined

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as an increase of absorbance at 470 nm by 0.001 per min. All of the above experiments were

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repeated three times as independent biological replicates.

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2.9. Statistical analysis

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All data were calculated as the mean ± standard deviation. Differences between samples were

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analyzed by one-way analysis of variance (ANOVA). Means were compared by least significant

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difference (LSD) tests. All statistical calculations were run using DPS software (version 8.01)

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(Tang and Zhang, 2013) and statistical significance was set at p < 0.05.

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3. Results

ACCEPTED MANUSCRIPT 3.1. Molecular Characterization of NvSPPI

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Fragment of NvSPPI containing 243 nucleotides were amplified and sequenced. It encods 80

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amino acids with one predicted secretory N-terminal signal peptides (Figure 1A). BLASTn results

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showed that this cDNA sequence is completely consistent with N. vitripennis SPPI in the NCBI

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database (NP_001155083). The predicted molecular weight and isoelectric point (pI) of the

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NvSPPI protein were 8.61 kDa and 7.56, respectively. It had a typical pacifastin domain and

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mainly consisted of β-pleated sheets (Figure 1B). The six residues form three disulfide bridges

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through Cys58–Cys77, Cys61–Cys72 and Cys48–63, which is consistent with the ‘Cys1–Cys4,

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Cys2–Cys6 and Cys3–Cys5’ model (Figure 1B). A multiple sequence alignment of PLDs from

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NvSPPI and other PPIs showed that although the numbers of PLDs in these species varied, typical

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PLD motifs were highly similar, including the six cysteine residues that formed disulfide bonds

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between cysteine numbers 1–5, 2–4, and 3–6 (Figure 2). Phylogenetic analyses of NvSPPI with

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homologs in other species using their mature peptide region sequences indicated that NvSPPI

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showed the closest genetic distances to Ceratosolen solmsi marchali (Hymenoptera: Agaonidae)

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(Figure 3).

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3.2. Expression and purification of recombinant NvSPPI

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The recombinant protein fused with glutathione S-transferase at its N-terminuses, GST-NvSPPI,

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was successfully detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE) with molecular weights of about 32 kDa. The recombinant protein was mainly

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detected in the supernatant. As soluble fusion protein, GST-NvSPPI was purified with the

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GST•Bind™ Resin Kit (Novagen, Hilden, Germany) and the purified proteins were analyzed by

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SDS-PAGE (Figure 4). The protein concentration of purified GST-NvSPPI was 1.7 mg/mL, and

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the concentration of the GST fusion proteins was 2.1 mg/mL.

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NvSPPI expressions were detected by RT-qPCR using 18S rRNA as the reference gene (Figures 5).

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NvSPPI mRNAs were expressed specifically in the venom apparatus, while they were detected at

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low levels in other tissues tested. Transcript levels of NvSPPI were monitored at different

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developmental stages from 0 to 7 days post eclosion (dpe) in the venom apparatus of female adults,

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and results showed that NvSPPI expression was hardly detected at 0 and 1 dpe, reached to the

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highest level at 4 dpe, and declined gradually afterward.

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3.4. Serine protease inhibition qctivity of NvSPPI

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To define whether NvSPPI has an impact on serine protease activity, we determined its enzyme

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activity inhibition spectrum to trypsin, chymotrypsin, and proteinase K with recombinant

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GST-NvSPPI. Results showed that recombinant GST-NvSPPI significantly inhibited the activity

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of trypsin (F = 3.067, p = 0.04), but it had no significant inhibition on chymotrypsin (F= 0.371, p

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= 0.776) and proteinase K (F = 0.174, p = 0.911) (Figure 6).

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3.5. Effects of NvSPPI on prophenoloxidase (PPO) activation and phenoloxidase (PO) activity

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To test whether NvSPPI played a role in PPO activation and PO activity of hemolymph of host

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pupae, inhibition of PPO activation and PO activity were tested with recombinant GST-NvSPPI

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and compared with positive and negative controls. The PO activity between samples incubated

ACCEPTED MANUSCRIPT with NvSPPI /M. luteus was significantly different compared with the BSA and GST controls (F =

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48.421, p < 0.001) (Figure 7A). The results indicated that recombinant GST-NvSPPI could inhibit

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PPO activation in the hemolymph of the host. When PPO in the hemolymph was pre-activated

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with M. luteus, recombinant GST-NvSPPI had no effect on PO activity (F =44.726, p < 0.001)

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(Figure 7B). These results suggested that NvSPPI could inhibit PPO activation in the host

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hemolymph but could not inhibit PO activity once PPO was activated.

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4. Discussion

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As reported, arthropod PPIs usually possess one to several conserved PLDs. Such as in B. mori, it has thirteen PLDs of [9]. However, in Drosophila, no PPIs gene is predicated in Flybase

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(http://flybase.org/). This may be due to a ‘lost’ in the genome of fruit flies after the divergence of

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mosquitoes and flies 250 million years ago (Bolshakov et al., 2002). It can be speculated that the

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evolution of PPIs family might have coincided with adaptations to specific biological conditions

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(Simonet et al., 2003).

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All PLDs of PPIs are characterized by a conserved pattern of six cysteine residues and form

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three disulfide bridges through Cys1–Cys4, Cys2–Cys6 and Cys3–Cys5 models. Detailed analysis

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of the 3-D structure of PPIs shows that the exposed canonical peptidase-binding loop (P3-P3’)

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contains the P1-P1’reactive bond that interacts directly with the catalytic site of the enzyme

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(Kellenberger et al., 1995; Malik et al., 1999; Patthy et al., 2002). Howevre, because of different

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intramolecular interactions in the inner core region of PPIs, they can be divided into two separate

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groups (group I and group II) (Kellenberger et al., 2005). The core of group I is usually stabilized

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by an interaction of a Lys10 with a Try25(Lys10/ Try25), while the core of Group II is always

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ACCEPTED MANUSCRIPT stabilized by an interaction of a Phe10 with an Ala26(Phe10/Ala26) (Breugelmans et al., 2009).

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Here, we characterized the NvSPPI in N. vitripennis had a typical PLD consisting of six cysteine

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residues, and the core of NvSSPI showed the model of Phe10/Ala26. So NvSSPI belongs to the

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Group II PPIs.

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Studies on P.leniusculus and S. gregaria revealed that the specificity and selectivity of PPIs

depend on the nature of their P1 residue (Breugelmans et al., 2009; Gáspári et al., 2002; Malik et

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al., 1999; Roussel et al., 2001; Simonet et al., 2005). In general, the domain with the P1 site of Arg

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or Lys inhibits trypsin; and the P1 site of Tyr, Leu, Phe, or Met inhibits chymotrypsin

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(Breugelmans et al., 2009). Here, the P1 site of NvSPPI is Arg, so it was supposed to inhibit the

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activity of trypsin but not chymotrypsin. Results of our serine protease inhibition assays with

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recombinant NvSPPI were consistent with this principle.

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In B.mori, the expression profiles of both PPI-1s (BMPP-1a and BMPP-1b) transcripts showed

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significant differences between the different tissues. The BMPP-1a showed the high transcript

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levels in ventral nerve cord, while the BMPP-1b showed the high transcript levels in head

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(Breugelmans et al., 2009). PPI in S. gregaria (sgpp-2) showed the high transcript levels in foregut

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and fat body (Simonet et al., 2002). Here, NvSPPI was expressed mainly in N.vitripennis venom

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apparatus, in which their transcribed levels were hundreds of times higher than that in other tissues

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tested. N. vitripennis is an ectoparasitoid of flies and deposits its eggs along with the venom to

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ensure the development of the offspring by inhibiting the immune response or growth and

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development of the host (Abt and Rivers, 2007; Asgari, 2006; Beckage and Gelman, 2004). The

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high expression of NvSPPI in venom suggested that NvSPPI was probably involved in inhibition

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ACCEPTED MANUSCRIPT of the host immune response. For the expression profiles of NvSPPI in venom apparatus, the

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expression levels of NvSPPI reached the maximum at 4 days post eclosion, then declined

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gradually. This is consistent with the parasitic time and biological rhythm of female wasps. The

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first three days post eclosion were the main mating and oviposition periods; after oviposition peak,

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female wasps would re-express NvSPPI for the subsequent parasitism.

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In order to further verify whether NvSPPI protein is involved in inhibition of the host immune response, PPO activation and PPO activity assays were carried out. PPO and PO

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activation assays of hemolymph from M. domestica (host) pupae showed that NvSPPI inhibited

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PPO activation. Unlike vertebrates, invertebrates don’t have complex and memory property

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acquired immune system, but only rely on the innate immune system to resist the invasion of

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pathogens and infection (Jiravanichpaisal et al., 2006; Kimbrell and Beutler et al., 2001). Insects,

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as the largest groups in invertebrates, possess their effectively innate immune system composed by

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humoral and cellular immunity. Cellular immunity is mediated by insect hemocytes and it involves

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agglutination, phagocytosis, encapsulation and nodule formation (Sun et al., 2015; Williams,

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2007;). Humoral immunity includes antibacterial peptide synthesis and the PPO activation cascade

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(Kanost et al., 2004). For PPO activation cascade, activation of zymogen PPO to active PO

325

involves a serine protease cascade (Cerenius and Soderhall, 2004; Cerenius et al., 2008) and is

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tightly regulated by serine proteases and serpins (Li et al., 2016; Liu et al., 2015;Wang et al., 2016;

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Zhang et al., 2016). So NvSPPI protein plays a role in repressing host melaninization through

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inhibition of trypsin activity and PPO activation. This will provide a new ideal for the biological

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control of insect pests.

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ACCEPTED MANUSCRIPT 5. Conclusions

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Although PPIs were predicated to exist in many insects by bioinformatics analysis of available

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genome data, few direct evidences are available on the possible functions of PPIs in insects,

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particularly in parasitic wasps. Here, our results indicate that recombinant NvSPPI inhibits the

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humoral immunity of host fly through inhibition of trypsin activity and PPO activation. Our

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results enrich the knowledge of immune function of PPIs in insects. However, more detailed

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studies are needed to further investigate the mechanism of PPIs inhibition of host humoral

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immunity.

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339 Acknowledgements

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N. vitripennis was provided by Institute of Insect Sciences, Zhejiang University. Dan liang, Ya Liu,

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Pei Wang, Saima Kausar and Baojian Zhu performed the experiments, Chaoliang Liu, Cen Qian

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and Guoqing Wei designed the study, Cen Qian and Lei Wang wrote the paper. This work was

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supported by the earmarked fund for Modern Agro-industry Technology Research System [grant

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number CARS-22-SYZ10]; Sericulture Biotechnology Innovation Team [grant number

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2013xkdt-05]; PhD programs in Biochemistry and Molecular Biology [grant number xk2013042];

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and the National Natural Science Foundation of China [grant number 31402018].

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Conflict of Interest

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The authors declare no conflict of interest. The founding sponsors had no role in the design of the

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study; in the collection, analyzes, or interpretation of data; in the writing of the manuscript and in

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the decision to publish the results.

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characterization of Ostrinia furnacalis serpin1, a regulator of the prophenoloxidase activation

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Zhu, J.Y., Ye, G.Y., Fang, Q., Wu, M.L., Hu, C., 2008. A pathogenic picorna-like virus from the endoparasitoid wasp, Pteromalus puparum: initial discovery and partial genomic

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ACCEPTED MANUSCRIPT Figure Legends

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Fig.1 Sequence analysis of NvSPPI.

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(A) Nucleotide and deduced amino sequences of NvSPPI cDNAs from N. vitripennis. Signal

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peptides are underlined; the initiator codon “ATG” and terminator codon “TAA” are shaded. (B)

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Predicted structural domain and tertiary structure of NvSPPI. The pacifastin domain was predicted

512

by Protein Blast of NCBI online. The tertiary structure was predicted by phyre2 on line. Three red

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square showed six residues form three disulfide bridges through Cys58–Cys77, Cys61–Cys72 and

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Cys48–63 (consistent with the ‘Cys1–Cys4, Cys2–Cys6 and Cys3–Cys5’ model).

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Fig.2 Multiple sequences alignment and amino acid identity analysis of PLDs between

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NvSPPI and other insect PPIs.

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Six conserved cysteine residues in PLDs are highlighted by black shadows. Disulfide bond

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between cysteines residues are marked by black lines. The key active sites are indicated by P3, P1,

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P1' and P3', respectively. The corresponding species of abbreviations and their GenBank accession

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numbers are as follows: CsmPD-1: Ceratosolen solmsi marchali (XP_011499775); TpPD-1,2,3,4:

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Trichogramma pretiosum (XP_014223671); PxPD-1,2: Papilio xuthus (KPJ04192); NlPD-1,2:

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Neodiprion lecontei (XP_015517821); DsPD-1: Diachasma alloeum (XP_015122112); PmPD-1,2:

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Papilio machaon (KPJ07734); DpPD-1,2,3,4,5: Danaus plexippus (EHJ66762); FaPD-1: Fopius

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arisanus (JAG78941); ObPD-1,2,3: Operophtera brumata (KOB67284); WaPD-1: Wasmannia

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auropunctata (XP_011698661); SgPD-1,2: Schistocerca gregaria (CAF18560); BmPD-1:

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Bombyx mori (XP_004927292).

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ACCEPTED MANUSCRIPT Fig.3 Phylogenetic analysis of NvSPPI and other insect PPIs’ amino acid sequences based on

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the neighbor-joining method.

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The GenBank accession numbers of amino acid sequences are as follows: Ceratosolen solmsi

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marchali (XP_011499775); Danaus plexippus (EHJ66762); Operophtera brumata (KOB67284);

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Diachasma alloeum (XP_015122112); Fopius arisanus (JAG78941); Eriocheir sinensis

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(ACF35640); Pacifastacus leniusculus (AAC64661); Schistocerca gregaria PP-5,4t,4a

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(CAF18560, CAC82510,CAI36149); Trichogramma pretiosum (XP_014223671); Neodiprion

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lecontei (XP_015517821); Wasmannia auropunctata (XP_011698661); Bombyx mori

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(XP_004927292).

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Fig.4 SDS-PAGE analysis of NvSPPI recombinant protein.

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M: Protein molecular weight marker; 1: Not induced by IPTG; 2: Supernatant after IPTG

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induction; 3: Purified proteins. Arrows indicate the positions of recombinant proteins on

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SDS–PAGE gels.

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Fig.5 Transcriptional profiles of NvSPPI in different tissues and developmental stages of N.

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vitripennis.

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(A) Tissue distribution of NvSPPI in N. vitripennis. Abdomen carcass represents the rest of

547

abdomen post dissection. Venom apparatus contains venom reservoir and gland, and venom

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released. (B) Transcript levels of NvSPPI in venom apparatus of N. vitripennis at different

549

developmental stages. Female adults were sampled on days 0 to 7 post eclosion. All values in the

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figure are represented as mean ± standard deviation. Bars labeled with different letters are

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significantly different (one-way ANOVA followed by LSD test, p < 0.05).

552 Fig.6 Effects of recombinant NvSPPI on the activity of three serine proteases.

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GST: GST-tag protein expressed by pGEX-4T-2; BSA and Buffer: treated by BSA and reaction

555

buffer respectively; NvSPPI: treated by recombination proteins respectively. All values in the

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figure are represented as mean ± standard deviation. Bars labeled with different letters are

557

significantly different (one-way ANOVA followed by LSD test, p < 0.05).

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Fig.7 Effects of recombinant NvSPPI on the PPO activation (A) and PO activity (B) of

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hemolymph of M. domestica pupae.

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(A) Screened hemolymph was incubated with TBS alone, or TBS/M. luteus, NvSPPI /M. luteus,

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BSA/M. luteus, GST/M. luteus, PTU/M. luteus in TBS for 20 min at 25 °C. PPO activation was

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assayed using L-dopamine as a substrate, as described in Materials and Methods. (B) Screened

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hemolymph was incubated with M. luteus in TBS for 10 min at 25 °C, and then incubated with

565

TBS, NvSPPI, BSA, GST, or PTU for 10 min at 25 °C. PO activity was assayed using L-dopamine

566

as a substrate, as described in Materials and Methods. All values in the figure are represented as

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mean ± standard deviation. Bars labeled with different letters are significantly different (one-way

568

ANOVA followed by LSD test, p < 0.05).

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ACCEPTED MANUSCRIPT Table 1 Primers used in this study

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Primer name

Primer sequence（ （5'-3'） ）

Gene cloning

NvSPPI-F

ATGTCAAAAGTTTTGAAAGTCGGTTTGC

NvSPPI -R

TTAATGTTGCGGACAAGCCATTC

RT- NvSPPI -F

GAAGAAAATGGAGCGCCAAAAG

RT- NvSPPI R

TTAATGTTGCGGACAAGCCATTC

RT-18S-F

TGGGCCGGTACGTTTACTTT

RT-18S-R

CACCTCTAACGTCGCAATAC

NvNvSPPI -F-B

CGCGGATCCGAAGAAAATGGAGCGCCAAAAG

NvNvSPPI -R-E

CCGGAATTCTTAATGTTGCGGACAAGCCATTC

Recombinant expression

Note: “____” present Restriction Enzyme cutting site: BamHI

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” present Restriction Enzyme cutting site: EcoRI

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RT-qPCR

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Primers function

ACCEPTED MANUSCRIPT We identified a small pacifastin protease inhibitor from Nasonia vitripennis (NvSPPI). 1. NvSPPI is detected specifically in the venom apparatus of N. vitripennis. 2. NvSPPI can inhibit trypsin activity in vitro.

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3. NvSPPI can inhibit PPO activation in host (Musca domestica) hemolymph.

ACCEPTED MANUSCRIPT Ethical Statement

Identification of a Small Pacifastin Protease Inhibitor from Nasonia vitripennis

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Venom that Inhibits Humoral Immunity of Host (Musca domestica) Cen Qian1, Dan liang1, Ya Liu, Pei Wang, Saima Kausar, Guoqing Wei, Baojian Zhu,

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Lei Wang, Chaoliang Liu*

•

1) All authors agree to its submission and the Corresponding author has been authorized by co-authors;

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With the submission of this manuscript I would like to undertake that:

2) This Article has not been published before and is not concurrently being

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considered for publication elsewhere; 3) This Article does not violate any copyright or other personal proprietary

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right of any person or entity and it contains no abusive, defamatory, obscene or fraudulent statements, nor any other statements that are unlawful in any

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way.

•

My Institute’s College of Life Sciences, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, China, representative is fully aware of this submission.