A Transcriptome Analysis Suggests Apoptosis-Related Signaling Pathways in Hemocytes of Spodoptera litura After Parasitization by Microplitis bicoloratus

Microplitis bicoloratus parasitism induction of apoptotic DNA fragmentation of host Spodoptera litura hemocytes has been reported. However, how M. bicoloratus parasitism regulates the host signaling pathways to induce DNA fragmentation during apoptosis remains unclear. To address this question, we performed a new RNAseq-based comparative analysis of the hemocytes transcriptomes of non-parasitized and parasitized S. litura. We were able to assemble a total of more than 11.63 Gbp sequence, to yield 20,571 unigenes. At least six main protein families encoded by M. bicoloratus bracovirus are expressed in the parasitized host hemocytes: Ankyrin-repeat, Ben domain, C-type lectin, Egf-like and Mucin-like, protein tyrosine phosphatase. The analysis indicated that during DNA fragmentation and cell death, 299 genes were up-regulated and 2,441 genes were down-regulated. Data on five signaling pathways related with cell death, the gap junctions, Ca2+, PI3K/Akt, NF-κB, ATM/p53 revealed that CypD, which is involved in forming a Permeability Transition Pore Complex (PTPC) to alter mitochondrial membrane permeabilization (MMP), was dramatically up-regulated. The qRT-PCR also provided that the key genes for cell survival were down-regulated under M. bicoloratus parasitism, including those encoding Inx1, Inx2 and Inx3 of the gap junction signaling pathway, p110 subunit of the PI3K/Akt signaling pathway, and the p50 and p65 subunit of the NF-κB signaling pathway. These findings suggest that M. bicoloratus parasitism may regulate host mitochondria to trigger internucleosomal DNA fragmentation. This study will facilitate the identification of immunosuppression-related genes and also improves our understanding of molecular mechanisms underlying polydnavirus-parasitoid-host interaction.


Introduction
Polydnaviruses (PDVs) have a very special life cycle. Unlike many viruses, they are not always obligate intracellular parasites, replicating inside living host cells to produce virions that can transfer genes to other cells [1][2][3][4]. Rather, PDVs are obligate symbionts of many endoparasitic wasps in the families Braconidae (carrying bracovirus) and Ichneumonidae (carrying ichnovirus). Both viruses have similar life cycles, wherein viral DNAs are integrated into a wasp's genome via Wasp Integration/Excision Motif (WIM) [5] and transmitted vertically to the wasp's offspring in a proviral form. Viruses replicate in the nucleus of the calyx cell in wasp ovaries. Mature virions are stored in the lumen of the calyx and oviduct, and the suspension of virus and protein is called calyx fluid. When a female wasp finds a host, she injects calyx fluid, venom produced by the venom gland and one or more eggs into the hemocoel of the host caterpillar. Virions infect host cells and discharge their circular dsDNA into the host nuclei, which then rapidly integrates into the host genome via the Host Integration Motif (HIM) [6]. Virulence genes are then transcribed in host cell nuclei and the cytoplasm, resulting in expression of virulence proteins. During the development of the wasp's offspring, the host hemocoel contains innate suppressive proteins from virus gene expression. In addition, specifically among the bracoviruses, the induction of host hemocyte apoptosis causes host immunosuppression [1,3].
Apoptosis or programmed cell death (PCD), is common to all metazoan phyla, including insects. Braconidae-induced apoptosis, however, is specifically characterized by internucleosomal DNA fragmentation. Apoptotic DNA fragmentation involves a variety of elements, including AIF, EndoG and DFF40. Every element is regulated by different signaling pathways, defined as extrinsic and intrinsic pathways. Extrinsic apoptosis pathway is triggered by the ligand-induced oligomerization of specific cell surface receptors, and this process induces the intracellular assembly of the deathinducing signaling complex for the activation of a caspase cascade initiated from caspase 8 that results in activation of caspase 3 and further cascade activation of DFF (cleavage of DFF45 releases DFF40 into the nucleus). DFF, a heterodimeric protein comprising 45 kDa and 40 kDa subunits termed ICAD/DFF45 and CAD/ DFF40 [7]. The DFF complex is localized in the cellular cytoplasm, resulting in the triggering of extrinsic apoptotic stress, and activated caspase 3 cleaves DFF45 and dissociates DFF40. Caspase 7 and Granzyme B also can cleave DFF45 but with a lower efficiency than caspase 3 [8]. Activated DFF40 translocates into the nucleus. In the nucleus, the activation of DFF40 is enhanced by interaction with the chromosomal protein Histone H1 and it cleaves chromosomal DNA at internucleosomal sites into fragments of ,200 bp. [9][10][11]. In contrast, the intrinsic pathway is also controlled by mitochondria, which collects and integrates pro-and anti-apoptotic signal stimuli from other organelles as well as from the extracellular microenvironment, such as DNA damage produced by Ataxia-Telangiectasia Mutated (ATM), endoplasmic reticulum (ER) stress and calcium overload. The intrinsic pathway can mediate caspase-independent and caspase-dependent apoptosis. Following intrinsic apoptotic stress triggering, EndoG is released from the mitochondrial intermembrane space and moves to the nucleus to produce nucleosomal DNA fragmentation, giving rise to 200,5,000 bp sized fragments in a caspase-independent manner. AIF is another endonuclease released from the mitochondrial intermembrane space. It is a flavoprotein that produces DNA fragments up to 5,000 bp in size, and it also does not require caspase activation [12]. Releasing cytochrome c can also mediate cell death via activation of caspase 8, which triggers a caspase-dependent apoptosis.
Numerous viruses are well known to modulate the mitochondrial apoptosis of infected host cells by altering Mitochondrial Membrane Permeabilization (MMP) in a direct and indirect manner with viral proteins. MMP regulation is performed via the Voltage-Dependent Anion Channel (VDAC) of the outer membrane (OM), the Adenine Nucleotide Translocase (ANT) of the inner membrane (IM), and cyclophilin D (CypD) of matrix proteins. Viral proapoptotic proteins are direct inducers of MMP. They include viral protein R (Vpr), which directly interacts with ANT and VDAC, thereby triggering MMP associated with mitochondrial membrane potential (DY m ) loss, mitochondrial intermembrane space (IMS) protein release, and caspase cascade activation. Viral proapoptotic proteins are also indirect MMP facilitators and promote apoptosis via both p53-dependent andindependent mechanisms [13]. The alteration of membrane permeability may release apoptotic-promoting factors from the mitochondria, such as AIF, EndoG, and Cyt c in the IMS, ultimately resulting in nuclear translocation. All of these signaling pathways involved in apoptotic DNA fragmentation are stimulated by intrinsic stress through the mitochondria via EndoG and AIF, in a process that is also called caspase-independent cell death, involving release of Cyt c, and extrinsic stress through caspase cascades via DFF40, which is also called caspase-dependent cell death [14].
After apoptotic stimulation, DFF40, EndoG and AIF migrate to the nucleus under the control of critical apoptosis-involved signaling pathways, including the gap junction signaling pathway, Ca 2+ signaling pathway, PI3K/Akt signaling pathway, NF-kB signaling pathway, and ATM/p53 signaling pathway. The gap junction signaling pathway induces apoptosis via regulation of the permeability of the plasma membrane resulting in alteration of intracellular and extracellular communication via transmission of small molecules, such as apoptotic signaling ATP. Gap junction proteins are the target proteins of activated caspase 3 [15] and also Ca 2+ . The Ca 2+ signaling pathway is involved in apoptosis via altering the permeability of the mitochondrial membrane to release apoptosis-inducing factors to trigger apoptotic caspasedependent and -independent pathways [13]. Apoptotic caspasedependent signaling pathways include the PI3K/Akt signaling pathway and NF-kB signaling pathway via regulation of caspase 3, and the apoptotic caspase-independent signaling pathways include regulation of the ATM/p53 signaling pathway by AIF expression [16]. The PI3K/Akt signaling pathway is crucial to many aspects of cell growth and survival, and its inhibition increases DNA fragmentation by the help of caspase 3 [17]. Baculoviruses inhibit cell apoptosis through activating the PI3K/Akt signaling pathway [18]. Nuclear Factor-kB (NF-kB) transcription factors regulate the expression of antimicrobial peptides (AMPs) and many genes involved in cell survival, such as c-IAP1/2, XIAP, and Bcl-XL. All NF-kBs are homo-or heterodimers of Rel proteins, such as p50/ p65 subunits. p53 plays an important role in suppressing tumorigenesis through inducing genomic stability via DNA repair, cell cycle arrest and apoptosis. p53 promotes AIF activity and caspase-independent cell death by binding to a p53-responsive element (p53RE) in the AIF promoter, which ultimately results in efficient induction of large-scale DNA fragmentation (5 kb) [16].
In this paper, we aimed to clarify the mechanism of parasitism induction of host hemocyte apoptosis. To test the hypothesis that parasitism regulates host apoptotic signaling pathways to produce apoptotic DNA fragmentation involved in nuclear elements to the nucleus, resulting in internucleosomal DNA fragmentation from 5 kb to 200 bp, we sequenced the RNA from hemocytes of the Oriental Leafworm Moth Spodoptera litura parasitized by the wasp Microplitis bicoloratus and compared the transcriptome of hemocytes from non-parasitized controls. Using this transcription data, we obtained an overview on how M. bicoloratus parasitism regulates apoptosis signaling pathways during the immunosuppression and induced killing of host S. litura hemocytes. Furthermore, we proposed M. bicoloratus bracovirus products to regulate mitochondria permeability to trigger internucleosomal DNA fragmentation and block a set of key genes in the cell survival signaling pathway.

Gap junction signaling pathway regulation by M. bicoloratus parasitism
Gap junction proteins form gap junction channels connecting cells for cell-cell communication and form hemichannels facilitating extracellular and intracellular communication including between ER and mitochondria to exchange small molecular, such as ATP and Ca 2+ , to trigger apoptosis [24]. In the insect circulating hemocytes, gap junction proteins form hemichannels to allow communication between the cell and environment. Under lipopolysaccharide (LPS) immunochallenge, hemichannel dye uptake decreases [25]. Typically, the decrease of the transcription level of hemichannel components and the decrease in opening of hemichannels on the cell surface result in the decrease of dye uptake. Gap junction proteins, Spli-Inx2 and Inx3, have been characterized and functioned [26] and in this study, Spli-inx1 and inx4 also were detected from hemocytes ( Fig. S1 and Table S4). Comparisons with S and M transcriptome data indicated that all 26 elements of the gap junction signaling pathway existed in the hemocytes. During immune challenge by M. bicoloratus parasitization, 2 genes (Spli-GNAS, ADCY5) were not expressed in the parasitized host hemocytes. To determine the differential expression of genes, all transcriptome were assembled into a combined pool, and S1, S2, M1, and M2 were mapped using this pool to obtain reads and the RPM values of S and M. Furthermore, the analysis obtained the fold change and p-value between parasitized and non-parasitized. These analyses indicated that 12 genes (ADCY8, CPKC, GNAS, INX1, INX2, INX3, INX4, ITPR1, PKA, PLCB, PRKG, and TUBA) were down-regulated ( Table 2). The qRT-PCR results indicate that the parasitization downregulated 3 key molecules, Inx1, Inx2, Inx3, on the cell membrane, not Inx4 (Fig. 1). These molecules are involved in forming hemichannels and gap junctions, suggesting that there might be disruptions of intracellular between ER, mitochondria and extracellular molecular exchanges.
Ca 2+ signaling pathway regulation by M. bicoloratus parasitism with respect to apoptosis Calcium ions (Ca 2+ ) control every aspect of cells as cellular messengers. Ca 2+ ions also can become death signals when delivered at physiologically aberrant conditions. Mitochondria eventually decide whether Ca 2+ signals are life or death signals via regulation of the mitochondrial membrane proteins Bcl-2 and Bax/Bak [27]. Comparisons of the transcription data from the S and M pools indicate that all 31 elements of the Ca 2+ signaling pathway existed in the examined hemocytes. Under M. bicoloratus parasitism, 3 genes (Spli-ANT, CypD, PLCG2) increased in Table 2. Cont.  Table 3). The qRT-PCR results indicate that the parasitism upregulated a key molecule, CypD, in the mitochondria (Fig. 1). This molecule is involved in forming a permeability transition pore complex (PTPC), suggesting that the M. bicoloratus alters Ca 2+ signaling pathway to promote apoptosis.

NF-kB signaling pathway regulation by M. bicoloratus parasitism
The NF-kB signaling pathway regulates gene expression via regulation of nuclear transcription factor. Comparison of the transcription data from the S and M pools indicates that all 18 elements of NF-kB signaling pathway existed in the hemocytes.

ATM/p53 signaling pathway regulation by M. bicoloratus parasitism
The ATM/p53 signaling pathway plays an important role in cell cycle control and apoptosis. In normal cells, the p53 protein level is low. DNA damage and stress signaling may trigger an increase of p53 protein levels, which has three major functions: cell cycle arrest, DNA repair and apoptosis. The cell cycle arrest prevents replication of proteins involved in DNA repair. Apoptosis avoids proliferation of cells containing abnormal DNA. p53 is a transcriptional activator that regulates the expression of MDM2. A comparison of the transcription data from the S and M pools indicate that all 21 elements of the ATM/p53 signaling pathway existed in the hemocytes. Under M. bicoloratus parasitism, 1 gene (Spli-SESN), was expressed in the parasitized host hemocytes, and 1 gene (CYC) was not expressed in the parasitized host hemocytes. Another 3 genes (Spli-PPM1D, PTEN, and TSC2) were downregulated ( Table 6). The qRT-PCR results indicate that the parasitism increased expression of a key molecule, p53, in the ATM/p53 signaling pathway (Fig. 1).

Discussion
M. bicoloratus parasitism regulated host hemocyte apoptosis, resulting in DNA fragmentation. In this study, we examined the impacts of both the apoptotic caspase-dependent and -independent signaling pathways on the host hemocytes based on transcriptome data. Our results demonstrated that bracovirus proteins are expressed in the host hemocytes, suggesting their roles in DNA fragmentation by regulating key signaling pathways,     resulting in the triggering of caspase-dependent and -independent pathways. First, we found that M. bicoloratus parasitism regulated genes involved in forming the PTPC, which control mitochondrial apoptosis. Following M. bicoloratus parasitization, Spli-CypD was dramatically up-regulated (Table 3, Fig. 1). PTPC, which is a large multiprotein structure assembled at the contact sites between outer membrane (OM) and inner membrane (IM) of mitochondria, regulates MMP. PTPC activation provokes a sudden increase in the IM permeability to solutes of low molecular weight, causing the unregulated entry of water and osmotic swelling of the mitochondrial matrix. Numerous studies suggest that the PTPC is assembled by ANT (in the IM), VDAC (in the OM) and mitochondrial matrix protein cyclophilin D (Cyp D) [13]. According to our data, under M. bicoloratus parasitism, PTPC can form in the mitochondria of host hemocytes. Some DNA viral proteins may be direct inducers of MMP, and some may be indirect MMP facilitators, resulting in the activation of the mitochondrial apoptosis pathway [13]. This suggests an inducing condition of PTPC. M. bicoloratus parasitism may promote cell death via regulation of PTPC formation to release factors involved in DNA fragmentation from mitochondria into nuclei to cleave DNA.
PTPC formed suddenly during immunochallenge, AIF, EndoG, and Cyt c in the mitochondria were released from the intermitochondrial space into the cytoplasm. EndoG and AIF directly move into the nucleus to digest DNA [28,29]. In mammals, the endonuclease DFF40 initiates DNA fragmentation. A recent report found that in Caenorhabditis elegans, there is an unexpected connection between Dicer and DNA degradation during cell death [30]. The Dicer-family RNase III enzymes include helicase, PAZ, RNaseIII, and dsRNA-binding domains [30]. CED-3 cleaves DCR-1, the C. elegans Dicer orthologue, as a candidate, at a specific position to yield a short isoform termed tDCR-1, which lacks the helicase and PAZ domain, and gains the capacity to cleave DNA into fragments [31]. Once DNA suffers double-strand breaks, the ATM signaling pathway activates and interacts with many different proteins to induce cell cycle arrest, increase DNA repair, and inhibit apoptosis, which involves the p53 signaling pathway, NF-kB signaling pathway and PI3K/Akt signaling pathway via the activation of IKKb and p53 [32]. Typically, the activated ATM signaling pathway should inhibit host cell apoptosis for cell survival [33,34].
At this point, we wish to examine how the parasitism inhibited the ATM-triggered DNA repair and cell survival signaling pathways. During DNA damage in the host hemocytes, ATM is expressed ( Table 6). The ATM signaling pathway is responsible for DNA repair via activation of the related cell survival signaling pathway [35]. DNA damage may activate protein kinases, such as ATM, to phosphorylate p53 at one of these three residues, which thereby increases the p53 level. After the DNA damage is repaired, the ATM kinase is no longer active. p53 will be quickly dephosphorylated and destroyed by the accumulated MDM2 [36]. p53 is conserved across eukaryotic organisms, and the decrease of transcriptional levels of genes regulated by p53 leads to a subdued resistance to pathogens infections. In C. elegans, p53/ CEP-1 are inhibited by the nucleolar protein NOL-6, a nucleolar RNA-associated protein, causing innate immune suppression [37].
It is well known that PI3K/Akt signaling pathway regulates cell survival and apoptosis. PI3K is composed of heterodimers of inhibitory adaptor/regulatory (p85) and a catalytic (p110) subunits. p85 binds and integrates signals from various cellular proteins, including transmembrane tyrosine kinase-linked receptors and intracellular proteins, providing an integration point for activation of p110. Akt, which contains a PH domain in the Nterminal region, is the primary downstream mediator of the effects of PI3K. The PH domain of Akt interacts with 39-phosphoinositides, contributing to recruitment of Akt to the plasma membrane. Recruitment to the membrane results in a conformation change, contributing to exposure of two crucial phosphorylation sites, serine 473 and threonine 308, for activation. An unexpected finding is that p85 was not expressed under M. bicoloratus parasitism (Table 4). HSV-1, herpes simplex virus, induces the phosphorylation of Akt during infection of oral epithelial cells, leading to anti-apoptosis, and inhibition of HSV-1induced PI3K activity increases DNA fragmentation [17]. Insect baculovirus AcMNPV activates PI3K/Akt signaling pathway antiapoptosis to replicate itself in the host cell via enhancing phosphorylation of Ser 473 of Akt [18]. In our laboratory, overexpression of the gap junction proteins Inx2 and Inx3 caused dramatic apoptosis in Sf9 and Spli221 cells but no phosphorylation of Akt in Hi5 cell lines, which reveals an anti-apoptosis function [26].
NF-kB signaling pathway regulates cell survival and apoptosis. In innate immunosuppression in invertebrates, it is well known that PDV protein vankyrins, which lack the phosphorylation and ubiquitination domains, function as IkB mimics via completion for the NF-kB site with IkB [38]. This results in retention of NF-kB in the cytoplasm, which inhibits immune gene expression for products such as antimicrobial peptides (AMPs) [39]. Three vankyrin genes were expressed in the host hemocytes (Table 1). NF-kB is constituted of p50 and p65 subunits. Normally, the p50/ p65 complex is released from IkB and translocated to the nucleus to activate the transcription of genes involved in cell survival. During the immunochallenge, p50 and p65 were down-regulated by M. bicoloratus parasitism ( Table 5, Fig. 1) suggesting that M. bicoloratus blocked the critical signaling pathway to promote cell apoptosis.
Ca 2+ overload from the ER to mitochondria is required for initiation of programmed cell death. An unexpected result concerns Ca 2+ loading between the endoplasmic reticulum and mitochondria. Previously, we proposed that innexin hemichannels on the ER can be Ca 2+ channels, providing a pannexin 3-like function in the mammal to deliver Ca 2+ [24,31]. In such a case, inx genes should be up-regulated to produce more hemichannels, but 3 inx genes were been down-regulated, only inx4 was up-regulated ( Table 2, Fig. 1). This suggests a disruption in hemichannel activation under M. bicoloratus parasitism.
In Table 1 and Fig. 1, we show six types of gene transcriptions in the parasitized host hemocytes related to the Ankyrin-repeat, PTP, C-type lectin, Ben domain, Mucin-like and EGF-like families. Recent research indicates that C-type lectin (SIGN-R1) enhances uptake and the processing of circulating apoptotic cells in the spleen [40]. CpBV-lectin encoded by C. plutellae bracovirus is secreted into plasma and binds to the surface of parasitoid eggs to induce host immunosuppression via inhibition of host hemocyte non-self recognition [41]. In our research system, considering the interaction between M. bicoloratus bracovirus proteins and apoptosis, whether MbBV-lectin provides a relative contribution to apoptotic cell clearance, similar to SIGN-R1, requires further examination. However, it is reasonable to indicate that most important genes displayed less transcription in the host hemocytes during apoptosis. The Ben domain-containing proteins are well known to be involved in the transcriptional repression through its interaction with histone deacetylase, and overexpression causes cell cycle arrest [42]. The ankyrin-repeat protein family acts as inhibitors of nuclear transcription factors via binding of NF-kB homodimers [39]. Protein tyrosine phosphatases are the largest family encoded by bracovirus, and PTPs are well known as a regulator of apoptosis in human [43], such as PTP-1B regulation of the PI3K/Akt cascade to influence the nuclear localization of FOXO1, a transcription factor that regulates the expression of several pro-apoptotic genes [44], and SHP-1 that disrupts antiapoptotic pathways through the regulation of the p85 subunit of PI3K [45], and TC-PTP also regulates p53 expression during apoptosis [46]. PTP-H2 from MdBV is a functional tyrosine phosphatase [47] and induces apoptosis of Sf21 cells [48]. MbCrp (egf-like) disrupts the cytoskeleton of host hemocytes [49].
In conclusion, our findings demonstrated that M. bicoloratus parasitism could regulate critical signaling pathways of host hemocytes to promote apoptosis to suppress host cellular immunity. Bracovirus may regulate proteins to form a PTPC structure that altered mitochondrial permeability, resulting in the release of DNA fragmentation elements, causing DNA damage and keeping ATM expression. This might have implications for better understanding of the mechanism of innate immunosuppression via the apoptosis pathway. However, analysis of the bracovirus proteins regulation of the critical signaling pathway may involve three levels in the cell, as a ligand binding to receptor on the cell surface, as a mini-protein to compete with scaffold proteins, as a nuclear factor to promote gene expression, as a host translation inhibitory factor to inhibit host protein translation or utilization of an RNAi mechanism [50] to inhibit gene expression on the mRNA level. The proteins responsible for specific signaling molecules in host hemocytes remain to elucidated.

Insect rearing and experimental animals
The S. litura colony was reared on an artificial diet (formulated according to [51]) at 2761uC, RH 60-80%, and under a 12:12 h photoperiod regimen. The parasitoid M. bicoloratus colony was maintained on S. litura larvae reared in the laboratory according to established methods [52]. Adults were also provided with honey as a dietary supplement.

Isolation of hemocytes from larvae of S. litura
Hemocytes were collected 5 days post-parasitization from parasitized S. litura larvae (more than 1,000) (when immature parasitoids in the host developed to the second larvae [52], approximately 21% hemocytes underwent apoptosis [1]) and named 'M' (parasitized by M. bicoloratus) in this paper. The fourth instar S. litura larvae were used to collect hemocytes to serve as the control group, named 'S' (non-parasitized S. litura hemocytes) in this paper.

Total RNA extraction
Total RNA was isolated from hemocytes using an RNeasy Plus Universal Mini Kit (QIAGEN, Maryland, USA), which is specific for genome DNA elimination, according to the manufacturer's instructions. The concentration of each RNA sample was determined by measuring OD at A260/A280 using the NanoDrop 2000 and running 1 x TBE agarose gel. High quality samples (with an A260/A280 ratio .2.0, A260/A230.2.0, concentration. 500 ng/ul) showing 28S and 18S RNA bands clearly were stored at 280uC until use. RNA was prepared from at least two biological replicates and used for independent library preparations.
Transcription mRNA sequencing, assemble, gene predicted Sequencing libraries were prepared using a RNA-Seq sample preparation kit from Illumina following the manufacturer's instructions. The transcription sequences were sequenced using an Illumina Hiseq2000, and the total base number was more than 26.3 Gb per sample. There were two replications for the M1, M2, S1, and S2 pools. RNA-seq de novo assembly was performed using Trinity [53]. GetORF in EBOSS were used to find protein from contigs [54].
Gene Ontology (GO) and KEGG data GO Slim test were assigned to the NR-annotated transcripts using a local Blast2GO pipeline b2g4pipe [55] with access to a local GO MySQL database (version of April 2013). The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for analysis of molecular networks [56].

Definition of up-or down-regulated genes based on fold change
Clean reads were mapping to assembled contigs, to get RPM value based on reads number [57]. Statistical analysis of data was performed using DESeq [58]. Transcript abundances for each gene were expressed as a weighted mean of counts from each replicate normalized to the overall library size (known as 'base mean'). p-values (adjusted for false discovery rate) were generated for each gene in pair-wise comparisons between different conditions. In our analyses, we used an adjusted p-value of 0.001 as a criteria for identifying significant differences in gene expression.
Total RNA isolation, cDNA synthesis and qRT-PCR Total RNA was isolated from hemocytes of parasitized S. litura larvae 5 days post-parasitization using RNAiso Plus (TaKaRa, Dalian, China), according to manufacturer's instructions, including DNase treatment. The concentration and purity of each RNA sample was determined by measuring OD at A260/A280 using NanoDrop 2000. Samples with an A260/A280 ratio .2.0) were used to synthesize cDNA using Oligo d (T) 18 primers following manufacturer's instruction (TaKaRa, Dalian, China). All cDNA samples were stored at 280uC for preservation. qRT-PCR was performed using SYBR PCR Kit (Takara, Dalin, China) with the ABI 7500 system following the cycling parameters: 50uC, 2 min; 95uC, 10 mim; 95uC, 5 sec, 60uC, 34 sec, 40 cycles; 95uC, 15 sec; 60uC, 1 min; 95uC, 30 sec; 60uC, 15 sec. The 2-DDCT method was used to get the relative mRNA levels [59]. 18S rDNA gene was used as the housekeeping genes for normalization. Three replications have been carried out for per sample.

GenBank accession numbers
The whole RNA-Seq project was deposited into DDBJ/DRA/ GenBank under the accession DRA001149. Figure S1 Completed ORF and short qRT-PCR products.