Expression pattern of immunoglobulin superfamily members in the silkworm, Bombyx mori

Gene 548 (2014) 198–209

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Gene journal homepage: www.elsevier.com/locate/gene

Expression pattern of immunoglobulin superfamily members in the silkworm, Bombyx mori Lei He a,1, Guangli Cao a,b,1, Moli Huang a, Renyu Xue a,b, Xiaolong Hu a,b, Chengliang Gong a,b,⁎ a b

School of Biology & Basic Medical Science, Soochow University, Suzhou 215123, China National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, China

a r t i c l e

i n f o

Article history: Received 12 February 2014 Received in revised form 9 July 2014 Accepted 11 July 2014 Available online 11 July 2014 Keywords: Bombyx mori IgSF Tissue expression Cypovirus

a b s t r a c t Immunoglobulin superfamily (IgSF) proteins are involved in cell adhesion, cell communication and immune functions. In this study, 152 IgSF genes containing at least one immunoglobulin (Ig) domain were predicted in the Bombyx mori silkworm genome. Of these, 145 were distributed on 25 chromosomes with no genes on chromosomes 16, 18 and 26. Multiple sequence alignments and phylogenetic evolution analysis indicated that IgSFs evolved rapidly. Gene ontology (GO) annotation indicated that IgSF members functioned as cellular components and in molecular functions and biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that IgSF proteins were involved in signal transduction, signaling molecules and interaction, and cell communication. Microarray-based expression data showed tissue expression for 136 genes in anterior silkgland, middle silkgland, posterior silkgland, testis, ovary, fat body, midgut, integument, hemocyte, malpighian tubule and head. Expression pattern of IgSF genes in the silkworm ovary and midgut was analyzed by RNA-Seq. Expression of 105 genes was detected in the ovary in strain Dazao. Expression in the midgut was detected for 74 genes in strain Lan5 and 75 genes in strain Ou17. Expression of 34 IgSF genes in the midgut relative to the actin A3 gene was significantly different between strains Lan5 and Ou17. Furthermore, 1 IgSF gene was upregulated and 1 IgSF gene was downregulated in strain Lan5, and 4 IgSF genes were upregulated and 2 IgSF genes were downregulated in strain Ou17 after silkworms were challenged with B. mori cypovirus (BmCPV), indicating potential involvement in the response to BmCPV-infection. These results provide an overview of IgSF family members in silkworms, and lay the foundation for further functional studies. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The immunoglobulin superfamily (IgSF) is composed of genes encoding at least one immunoglobulin (Ig) domain. The Ig domain is highly conserved and has a representative sandwich structure of two opposing antiparallel β-pleated sheets, stabilized by a disulfide bridge (Bork et al., 1994; Halaby et al., 1999; Harpaz and Chothia, 1994). Searches of the InterPro and Pfam databases show several types of Ig domains such as I-subtype, I-subtype 2, C2-set_2, V-set, V-type, Ig, I-set and Ig-like (Huang et al., 2009). These domains are found in cell Abbreviations: IgSF, immunoglobulin superfamily; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RPKM, reads per kb per million reads; DSCAM, Down syndrome cell adhesion molecule; EGFR, epidermal growth factor receptor; FPKM, fragments per kb of exon model per million reads; VEGFR1, vascular endothelial growth factor receptor 1; DPR6, defective proboscis extension response 6; OPCML, opioid-binding protein/cell adhesion molecule-like protein; Ig, immunoglobulin; EST, expressed sequence tag; BmCPV, Bombyx mori cypovirus; MYLK, myosin light-chain kinase; BmMDB, B. mori Microarray Database; Sns, sticks and stones; Nrg, neuregulin; HIF-1, hypoxia-inducible factor 1. ⁎ Corresponding author at: School of Biology & Basic Medical Science, Soochow University, Suzhou 215123, China. E-mail address: [email protected] (C. Gong). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.gene.2014.07.030 0378-1119/© 2014 Elsevier B.V. All rights reserved.

adhesion molecules, cell receptors, antigens, cell surface glycoproteins, and other proteins (Ossiboff and Parker, 2007; Soroka et al., 2010; Wang and Springer, 1998). Ig domains give IgSF proteins specific adhesion and recognition capability, including cell–cell adhesion, cell-surface recognition and pathogen recognition (Hutter et al., 2000; Hynes and Zhao, 2000). Thus, IgSF proteins are important in cell adhesion, cell communication and immune functions. IgSF proteins are divided into six classes: cell surface I, cell surface II, cell surface III, secreted proteins, extracellular matrix proteins, and muscle proteins, based on broad functional similarities (Vogel et al., 2003). Most IgSF members are integral membrane proteins on the surface of lymphocytes and participate in diverse activities in the mammalian immune system. IgSF proteins are crucial for transducing the signals of cytoplasmic and nuclear effectors and delivering antigens to compartments for degradation (Litman et al., 1999). Recently, 142 IgSF proteins in Drosophila melanogaster and 80 in Caenorhabditis elegans were identified using hidden Markov models (Vogel et al., 2003). In Anopheles gambiae, 138 proteins with at least one type of Ig domain were identified, of these, 85 IgSF genes show significant induction by treatment with Plasmodium, gram-negative or gram-positive bacteria. These results suggest essential roles for IgSF proteins in response to infection (Garver et al., 2008). Recent reports showed that IgSF members are

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essential for the invertebrate immune response. For example, Down syndrome cell adhesion molecule (DSCAM), an IgSF member, is distributed on the cell surface of hemocytes and is a hypervariable pattern recognition receptor in the innate immune system of Eriocheir sinensis (Jin et al., 2013). In A. gambiae, DSCAM is involved in defense against bacterial and plasmodial parasites (Dong et al., 2006). Hemolin, an IgSF protein, is expressed in the silk glands of Galleria mellonella in response to bacterial challenge and might mark apoptotic cells for elimination by hemocytes (Shaik and Sehnal, 2009). In Manduca sexta, hemolin binds to molecules on the surface of bacteria, triggering a protective response involving humoral and cellular reactions and inhibiting hemocyte aggregation (Eleftherianos et al., 2007; Ladendorff and Kanost, 1991). Hemolin is also involved in antiviral defense in Lepidopteran insects (Terenius, 2008). These findings demonstrate the importance of IgSF proteins in immunity and response to infection (Garver et al., 2008; Litman et al., 1999). The silkworm Bombyx mori is the most well-studied model organism of the Lepidoptera because of its long history of domestication and its economic value. The genome sequence of silkworm is well established (Mita et al., 2004; Xia et al., 2004). IgSF genes in the silkworm genome were predicted using EBI InterPro (http://www.ebi.ac.uk/interpro/) and the Sanger database Pfam browser (http://pfam.sanger.ac.uk/), yielding 133 IgSF silkworm genes (Huang et al., 2009). Challenge with the gramnegative bacterium Escherichia coli or the gram-positive bacterium Bacillus thuringiensis results in upregulation or downregulation of 37 IgSF silkworm genes, indicating that these genes might participate in the immune response (Huang et al., 2009). Studies of IgSFs in mammals, D. melanogaster, C. elegans and mosquitoes show that IgSF members execute diverse functions in multiple physiological systems (Dong et al., 2006; Garver et al., 2008; Jin et al., 2013; Litman et al., 1999). Understanding silkworm IgSF genes more comprehensively will benefit additional studies on IgSF immune functions. In this study, we identified silkworm IgSF proteins with at least one type of Ig domain, predicted their functions based on GO annotation and KEGG analysis and analyzed the expression of the IgSF genes using microarrays and RNA-Seq. Until now, few studies have investigated the involvement of IgSF members in the invertebrate response to viral infection, so BmCPV-induced expression of IgSF genes in the silkworm midgut was also analyzed in this study.

Silkworm gene expression data were stored at the B. mori Microarray Database (BmMDB) (http://www.silkdb.org/microarray/). Using microarrays, IgSF gene expression in the anterior silkgland, middle silkgland, posterior silkgland, testis, ovary, fat body, midgut, integument, hemocyte, malpighian tubule and head was determined in silkworms from day 3 of the fifth instar stage, surveyed as described previously (Xia et al., 2007). Signal values higher than 300 were assumed to be expressed genes. To obtain gene expression levels in different tissues, we applied a linear normalization method to normalize individual channel data (Xia et al., 2007). Normalized signal intensity values of repeated experiments were merged and average values were calculated to make heat maps of the expression of IgSF genes. Hierarchical clusters were determined using the average linkage method with MeV software (Eisen et al., 1998).

2. Materials and methods

2.7. Expression profile of IgSF genes based on RNA-Seq

2.1. In silico cloning of silkworm IgSF genes

Domesticated silkworm strains Dazao, Lan5 and Ou17 were used in this study and reared in a 12 hour (h) light/12 h dark photoperiod, 25 °C and 70–85% relative humidity. To analyze the expression profiles of IgSF genes in ovaries, RNA was isolated from 20 ovaries (Strain Dazao) at the third day and sixth day of the fifth instar stage, and the second day of the pupal stage using RNAout kits (Tiandz, Mianyan, China), followed by treatment with 10 U DNaseI at 37 °C for 1 h. The mRNAs were purified using Micropoly (A) Purist™ mRNA purification kits (Ambion, Woodward Austin, USA) and 10 μg of the resulting mRNA was used for cDNA synthesis with PrimeScript Reverse Transcriptase (TaKaRa, Dalian, China) following previously described methods (Ng et al., 2005). Equal amounts of cDNAs from different periods were mixed and fragmented into 300–500 bp sections. A cDNA library was constructed using TruSeq DNA Sample Prep Kit-Set A (Illumina, San Diego, USA) and amplified using a TruSeq PE Cluster kit (Illumina, San Diego, USA) after purification with Ampure beads (Agencourt, Beverly, USA). Sequencing was performed with an Illumina Hiseq 2000 (Illumina, San Diego, USA). To determine IgSF gene expression in the response to BmCPVinfection, silkworms in strains Lan5 and Ou17 at the first day of the third instar stage were fed mulberry leaves coated with BmCPV (107 polyhedron/ml) for 8 h, then fed normal leaves. Midguts were dissected from silkworms at 36, 72, 108, 144 and 180 h postinfection for total RNA isolation, and five midguts were sampled at each time point. Normal silkworm midguts were isolated at same time. RNA-Seq for midgut samples was performed as above.

Silkworm genomic data were obtained from the silkworm database (http://silkworm.swu.edu.cn/silkdb/) (Xia et al., 2004). Hypothetical IgSF gene sequences were obtained using in silico cloning based on IgSF sequences for D. melanogaster and Tribolium castaneum using tBLASTn (http://www.ncbi.nlm.nih.gov/blast) to align silkworm genomic and expressed sequence tag (EST) databases. IgSF members were also predicted by homology searches using BLASTp (http://www.ncbi. nlm.nih.gov/blast). Proteins containing at least one Ig domain were regarded as IgSF proteins. Predicted IgSF gene sequences were compared with B. mori genomic sequences using BLASTn (http://www. ncbi.nlm.nih.gov/blast) to characterize exons and introns. 2.2. Sequence analysis Signal peptides were predicted by the SignalP 3.0 Server (http:// www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al., 2004). Transmembrane domains were analyzed using TMPRED (http://www.ch.embnet. org/software/TMPRED_form.html) (Hofmann and Stoffel, 1993). Conserved motifs and protein domains were predicted using Conserved Domain Search Service (http://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi) (Marchler-Bauer et al., 2011), SMART (http://smart.emblheidelberg.de/) (Letunic et al., 2012) and Pfam (http://pfam.sanger.ac. uk/) (Punta et al., 2014).

2.3. GO and KEGG analyses GO assignment used the HMMPfam method, and GO classification of predicted IgSFs was by BGI-WEGO web services (http://wego.genomics. org.cn) (Ye et al., 2006). KEGG pathway analysis for IgSFs was carried out by the KEGG Automatic Annotation Server (http://www.genome.jp/ tools/kaas/) (Moriya et al., 2007). 2.4. Construction of phylogenetic trees Phylogenetic trees with IgSF proteins were generated by neighborjoining method using MEGA 5.0 (http://www.megasoftware.net/) based on amino acid sequences according to a previous study (Hall, 2013). The bootstrap value was set to 1000. 2.5. IgSF gene mapping Silkworm IgSF gene mapping was by SilkMap (http://www.silkdb. org/silksoft/silkmap.html), a tool to map protein or nucleotide sequences onto silkworm chromosomes. 2.6. Expression patterns of IgSF genes based on microarray data

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To analyze gene expression abundance, clean reads were mapped directly to reference gene sequences of B. mori after eliminating low-quality sequences using TopHat software (Trapnell et al., 2010). To analyze the mRNA abundance of different genes, all read numbers were converted into reads per kb per million reads (RPKM) or fragments per kb of exon model per million reads (FPKM) (Mortazavi et al., 2008). RPKM and FPKM were calculated using MA-plot-based method with random sampling model of the DEGseq software package (Wang et al., 2010). Genes differentially expressed between the two samples were identified using a q-value false discovery rate-adjusted p-value of 0.05 (Wang et al., 2010). 2.8. Real-time PCR To validate the RNA-Seq data, real-time PCR was carried out with a real-time reverse transcription-PCR system (Cycler 1000; Bio-Rad, Hercules, CA, USA) with SYBR green. Primers used for determining the relative expression of genes were designed based on cDNA sequences (Supplemental Table 1). The B. mori housekeeping gene actin A3 was used as an internal control for the normalization. A 20 μl real-time PCR reaction solution contained 0.2 μg cDNA, 5 pmol of each primer, and 10 μl of SYBR Green Real-time PCR Master Mix (ABI, Vernon, CA, USA). All

samples were run in quintuplicate. Real-time PCR was: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min, with a final cycle at 95 °C for 15 s, 60 °C for 30 s and 95 °C for 15 s. Relative gene expression was estimated according to the 2−ΔΔCt method (Livak and Schmittgen, 2001). 3. Results 3.1. IgSF members in the silkworm To confirm the identity of IgSF members in the silkworm genome, the Pfam and SMART databases were searched using BLASTp, validating 152 IgSF proteins containing at least one Ig domain type (Supplemental Table 2). Kettin (BGIBMGA000622), a large IgSF protein, had 35 Ig domains. In addition, 9 IgSF genes had alternative splicing forms, and 7 genes had paralogs. They were distributed at different genomic locations (Supplemental Table 2, Fig. 1). Several large silkworm IgSF protein subfamilies such as titin, beat, hemolin, and kekkon1 were identified. Multiple sequence alignment showed that members of the same family shared high homology, and gene mapping showed that members of the same family were adjacently located on the same chromosome (Fig. 1).

Fig. 1. Distribution of IgSF members on silkworm chromosomes.

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3.2. Ig domains of silkworm IgSF proteins

3.4. Phylogenetic relationships of IgSFs

Three types of Ig domains, immunoglobulin, immunoglobulin C-2 type, and immunoglobulin-like were found using SMART analysis and 36 types of other associated domains were also found (Fig. 2, Table 1B). Putative signal peptide sequences were found on 67 proteins that might be secreted to the extracellular matrix to participate in interactive processes as intercellular messengers; 66 proteins had transmembrane domains that might result in their location at the cell surface (Table 1A) (Litman et al., 1999). In addition, of the 36 types of associated domains, the most abundant was fibronectin III, found in 43 silkworm IgSF proteins. We also found 11 IgSF proteins with leucine-rich repeat domains that are involved in biological processes including signal transduction, cell adhesion, disease resistance, apoptosis and the immune response, and are important in the formation of protein–protein and protein–ligand interactions (Kobe and Kajava, 2001; Rothberg et al., 1990; Wei et al., 2008).

To determine the evolutionary relationships of silkworm IgSFs, we constructed phylogenetic trees based on IgSF amino acid sequences using the neighbor-joining method (Fig. 3). The IgSFs were divided into three large groups. Groups A and B had more members than group C, which had five IgSF members. The three groups had distant phylogenetic relationships, and appeared to have evolved independently. Groups A and B were divided into two subgroups. Generally, members with analogous functional domains grouped into a cluster. The fasciclin-2 precursor (BGIBMGA006873) had three homologues, but they grouped into different clusters, indicating that their functions diverged during evolution.

3.3. Mapping silkworm IgSF genes The distribution of IgSF members on silkworm chromosomes is in Fig. 1. The silkworm IgSF genes were not randomly distributed on chromosomes. No IgSF genes were found on chromosomes 16, 18 and 26; Chromosomes 2, 3, 4, 15 and 20 each had 1 gene; and chromosome 12 had the most IgSF genes with 15. Mapping IgSF genes in the silkworm revealed that certain chromosomes had more genes. This chromosome distribution of IgSF genes was distinct from the distribution in the C. elegans genome, in which 22 genes are on the X chromosome (Teichmann and Chothia, 2000). This difference suggested divergence in the evolutionary pattern of the silkworm compared to C. elegans.

3.5. GO analysis GO classification was assigned to the silkworm IgSF genes using the BGI-WEGO. Functional annotations indicated functions in cellular components as well as molecular functions and biological processes. IgSF members were mainly enriched in extracellular region, protein binding, anatomical structure formation, cell adhesion, development, immune system process, reproduction, and response to stimulus (Fig. 4). Most functions were associated with cell adhesion and the immune system, suggesting probable roles in adhesion, pathogen recognition, and immune responses. 3.6. KEGG analysis KEGG analysis showed relationships between IgSF proteins and 20 pathways (Table 2), with enrichment mainly in signal transduction, signaling molecules and interaction, and cell communication. Several

Fig. 2. Typical domain architectures of silkworm IgSF members. Horizontal line, sequence length; symbols, domains.

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genes were involved in multiple pathways simultaneously, such as the fibroblast growth factor receptor 2, FMS-like tyrosine kinase 1, and myosin light-chain kinase (MYLK). 3.7. Microarray-based IgSF gene expression profiles in different tissues Transcriptional levels of IgSF genes were determined in 10 tissues on day 3 of the fifth instar stage using genome-wide microarray analysis. Silkworm IgSF genes showed expression in multiple tissues (Fig. 5). A few IgSF members showed constitutive expression in all tissues but slight differences existed in different tissues. For example, DSCAM (BGIBMGA005745, probe sequence number: sw21135) and lachesin (BGIBMGA011387, probe sequence number: sw20431) were expressed at high levels in 10 different tissues. Interference hedgehog-like protein (BGIBMGA008552, probe sequence number: sw01501) and peroxidasin (BGIBMGA000553, probe sequence number: sw08623) were expressed to different degrees in different tissues. Some IgSF members exhibited tissue-specific expression, whereas others exhibited differential expression in different tissues. For example, hemicentin (BGIBMGA009442, probe sequence number: sw08717), which is involved in organization in epithelial cells and cell attachment (Vogel and Hedgecock, 2001; Vogel et al., 2006), was expressed at high level only in the gonads, especially in the ovary. Opioid-binding protein/ cell adhesion molecule-like protein (OPCML) (BGIBMGA010275, probe sequence number: sw01127), a putative tumor suppressor gene involved in promoter methylation status in women with ovarian cancer (Czekierdowski et al., 2006; Duarte-Pereira et al., 2011; Zhou et al., 2011), was mainly expressed in the midgut. Members of the titin family regulate the elasticity of myofibrils and thick filaments in the skeletal muscle of various organisms. Titin mutations are a common cause of myofibrillar myopathy with early respiratory failure (Fabian et al., 2007; Pfeffer et al., 2014; Tsuji et al., 2002). In the silkworm, titin1 (BGIBMGA000623, probe sequence number: sw17684) and titin2 (BGIBMGA000624, probe sequence number: sw03048) showed high expression in the head and integument. In some cases, members of the same family had different expression patterns. For example, in the DSCAM family, BGIBMGA005745 (probe sequence number: sw21135) was expressed at high levels in 10 different tissues, while BGIBMGA013795 (probe sequence number: sw03517) was expressed at a low level; another member of this family, BGIBMGA013735 (probe sequence number: sw16175), was expressed more strongly in the testis, head, integument and midgut than in other tissues. The differences in tissue expression patterns among gene family members might indicate a divergence in the functions of the family members (Huang et al., 2009). 3.8. Expression pattern of IgSF genes in ovary and midgut To obtain a global view of the expression pattern of IgSF genes in different silkworm tissues, high-throughput RNA-Seq was performed using ovary and midgut samples. After removing low-quality reads, 6,234,820 paired-end reads with an average length of 100 bp were obtained from ovary samples. The ratio of valid data to raw data was 98.87%. For midgut samples, 123,208,938 paired-end reads with an average length of 92 bp were obtained; the ratio of valid data to raw data was 81.93% (Table 3). More than 80% of reads could be uniquely aligned to the genome. To validate the RNA-Seq data, expression of randomly selected genes relative to the housekeeping gene actin A3 was determined by real-time PCR. The results showed that transcriptional levels of the selected genes, as determined by real-time PCR, were similar to the RNA-Seq results (Supplemental Fig. 1). This suggested that the RNA-Seq data was credible. Generally, gene expression could be divided into low (RPKM 0.1– 3.75), moderate (RPKM 3.75–15) and high expression (RPKM N 15). Genes with RPKM 0–0.1 were considered not expressed (Hall, 2013). IgSF gene expression levels are in Table 4.

The silkworm ovary had more moderately expressed and highly expressed genes than the midgut and fewer non-expressed genes. Clear differential expression between IgSF genes in the midgut was seen when strains Lan5 and Ou17 were compared (Fig. 6). However, in both strains Lan5 and Ou17, a single IgSF gene (BGIIBMGA010275, OPCML) was highly expressed, 7 genes were moderately expressed, 51 genes were expressed at low levels, and 68 IgSF genes were not expressed. The midgut expression levels of IgSF genes relative to the constitutively expressed actin A3 gene were compared in the silkworm strains Lan5 and Ou17. Genes with a fold-change more than 2.0 are in Table 5. The relative expression of 31 IgSF members was higher in strain Lan5 than in strain Ou17. In Ou17, the relative expression of 3 IgSF members, lachesin, turtle-like protein A and beat, was higher than in Lan5. Of note, the relative expression level of lachesin (BGIBMGA006354, BGIBMGA006355) was more than 335 times higher in strain Ou17 than in strain Lan5. 3.9. Expression of IgSF genes in response to BmCPV infection To analyze the expression of IgSF genes in the midgut after treatment with BmCPV, high-throughput RNA-Seq was performed using midgut samples from silkworms from strains Lan5 and Ou17 infected Table 1 Representation of the silkworm IgSF proteins. Percentages are proteins containing one or more of the indicated domains out of the total IgSF proteins. Representation of the silkworm IgSF (A) Signal peptide and transmembrane regions Signal peptide Transmembrane Both Neither (B) Functional domains Immunoglobulin Immunoglobulin C-2 type Immunoglobulin_like Fibronectin III Leucine-rich repeats Leucine rich repeat C-terminal domain Leucine-rich repeats_TYP Leucine rich repeat N-terminal domain Thrombospondin type 1 repeats Tyrosine kinase Epidermal growth factor-like domain (EGF) Src homology 3 domain Protein kinase Pleckstrin homology domain RhoGEF Domain present in ZO-1 and Unc5-like netrin receptors DEATH domain Complement control protein (CCP) Domain present in hormone receptors PHD zinc finger Olfactomedin-like domains CLECT domain Serine/threonine protein kinases CUB domain Calcium-binding EGF-like domain EGF_like domain Laminin-type epidermal growth factor-like domain von Willebrand factor (vWF) type A domain (VWA) Low-density lipoprotein receptor domain class A Laminin B domain Laminin G domain BPTI/Kunitz family of serine protease inhibitors (KU) Four-disulfide core domain (WAP) Semaphorin domain Kringle domain SERine Proteinase Inhibitors Protein tyrosine phosphatase SEC14 domain Spectrin repeats

Genes (%) 67(44.1) 66(43.4) 34(22.4) 53(34.9)

111(73.0) 106(69.7) 43(28.3) 43(28.3) 11(7.2) 11(7.2) 7(4.6) 5(3.3) 5(3.3) 4(2.6) 4(2.6) 3(2.0) 2(1.3) 2(1.3) 2(1.3) 2(1.3) 2(1.3) 2(1.3) 2(1.3) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7) 1(0.7)

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Fig. 3. Neighbor-joining tree of silkworm IgSF members based on amino acid sequences. Bootstrap value was set to 1000.

Fig. 4. GO categories of IgSF genes and all silkworm genes. Percentages show the proportion of all silkworm IgSF genes with a particular function.

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Table 2 IgSF genes involved in pathway distribution. Primary metabolic

Primary metabolic

Three metabolic

Reference number

KO number

Gene name

Environmental information processing

Signal transduction

MAPK signaling pathway Hippo signaling pathway—fly

BGIBMGA010090 BGIBMGA010335 BGIBMGA0103351 BGIBMGA012742 BGIBMGA001183 BGIBMGA002036 BGIBMGA010090 BGIBMGA001183 BGIBMGA001183

K05093 K16680 K16680

FGFR2; fibroblast growth factor receptor 2 ED; echinoid ED; echinoid

K16690 K05096 K00907 K05093 K05096 K05096

VN; protein vein FLT1, VEGFR1; FMS-like tyrosine kinase 1 MYLK; myosin light-chain kinase FGFR2; fibroblast growth factor receptor 2 FLT1, VEGFR1; FMS-like tyrosine kinase 1 FLT1, VEGFR1; FMS-like tyrosine kinase 1

HIF-1 signaling pathway Calcium signaling pathway PI3K-Akt signaling pathway Signaling molecules and interaction

Cellular processes

BGIBMGA006873- K06491 2 BGIBMGA010594 K07523 BGIBMGA012049 K05695

NCAM; neural cell adhesion molecule

Adherens junction

BGIBMGA001183 BGIBMGA010090 BGIBMGA010090 BGIBMGA002036 BGIBMGA001183 BGIBMGA002036 BGIBMGA012049

K05096 K05093 K05093 K00907 K05096 K00907 K05695

Endocrine system

Insulin signaling pathway

BGIBMGA012049

K05695

Circulatory system

Vascular smooth muscle contraction Gastric acid secretion Axon guidance

BGIBMGA002036

K00907

LRRC4C, NGL1; netrin-G1 ligand PTPRF, LAR; receptor-type tyrosine–protein phosphatase F FLT1, VEGFR1; FMS-like tyrosine kinase 1 FGFR2; fibroblast growth factor receptor 2 FGFR2; fibroblast growth factor receptor 2 MYLK; myosin light-chain kinase FLT1, VEGFR1; FMS-like tyrosine kinase 1 MYLK; myosin light-chain kinase PTPRF, LAR; receptor-type tyrosine–protein phosphatase F PTPRF, LAR; receptor-type tyrosine–protein phosphatase F MYLK; myosin light-chain kinase

BGIBMGA002036 BGIBMGA010901 BGIBMGA012667 BGIBMGA010594

K00907 K07521 K07521 K07523

MYLK; myosin light-chain kinase UNC5; netrin receptor unc-5 UNC5; netrin receptor unc-5 LRRC4C, NGL1; netrin-G1 ligand

Transport and catabolism

Endocytosis

Cell motility

Regulation of actin cytoskeleton Focal adhesion

Cell communication

Organismal systems

Cytokine–cytokine receptor interaction Cell adhesion molecules (CAMs)

Digestive system Development

with BmCPV. After removing low-quality reads, 113,182,370 paired-end reads with an average length of 94 bp were obtained. The ratio of valid data to raw data was 83.99% (Table 3). Genes differentially expressed by RNA-seq after infection with BmCPV were selected for real-time PCR analysis to validate the RNA-Seq data. The results showed that the selected genes had a concordant direction of change for both RNA-Seq and real-time PCR (Supplemental Fig. 2), demonstrating that the RNASeq data was credible. The IgSF gene expression response to BmCPV infection was clearly different between strains Lan5 and Ou17 (Fig. 6). Based on gene expression after infection with BmCPV, sticks and stones (Sns) (BGIBMGA000162) expression was upregulated and hemicentin 1 (BGIBMGA001050) expression was downregulated in strain Lan5. BmCPV infection caused upregulation of Sns (BGIBMGA000162, BGIBMGA000163), turtle-like protein (BGIBMGA005030) and defective proboscis extension response 6 (DPR6) (BGIBMGA013547) upregulation; and downregulation of nephrin-like (BGIBMGA003796) and turtle-like protein A (BGIBMGA008685) in strain Ou17.

Table 3 Summary of transcriptome sequence data. Lan5-BmCPV and Ou17-BmCPV, silkworm strains infected with BmCPV. Sample

Strain

Raw data

Valid data

Ratio of valid data

Average length (bp) of reads

Ovary Midgut Midgut Midgut Midgut

Dazao Lan5 Ou17 Lan5-BmCPV Ou17-BmCPV

6,306,078 75,393,654 75,000,000 74,388,572 60,592,364

6,234,820 61,771,838 61,437,100 61,499,610 51,682,760

98.87% 81.93% 81.92% 82.67% 85.30%

100 92.69 91.99 93.11 95.11

4. Discussion The silkworm genome is reported to encode 133 IgSF proteins (Huang et al., 2009). In this study, we predicted 152 IgSF proteins in the silkworm, with several proteins newly identified (Supplemental Table 2). In comparison with D. melanogaster, A. gambiae, and A. mellifera, the silkworm contains more Ig domain types. Moreover, the C2-set_2 type domain is found in B. mori, Antheraea mylitta and Heliconius erato, but not in other insects such as D. melanogaster and A. mellifera by BLAST search of insect databases for the C2-set_2 type domain. Therefore, the C2-set_2 type domain might be unique to Lepidoptera. The IgSF family in D. melanogaster comprises 142 proteins (Litman et al., 1999). The silkworm IgSF also contains numerous large subfamilies. We found that members in the same subfamily had high homology but were distributed on different chromosomes. Nonetheless, some IgSF members with high sequence similarity were adjacent on the same chromosome. For example, the kekkon1 homologues BGIBMGA009485 and BGIBMGA009489, BGIBMGA009335 and BGIBMGA009336 were located on different regions of chromosome 14, but BGIBMGA009485 was adjacent to BGIBMGA009489, and BGIBMGA009335 was adjacent to BGIBMGA009336, respectively, suggesting that some closely related members were generated by a gene duplication event during evolution. Some members were duplicated once and some were duplicated several times (Ohtani et al., 2011). The phylogenetic relationship of the silkworm Ig, I-set, V-set and C2-set_2 domains showed high divergence among the four domains (Huang et al., 2009) and an evolutionary rate for immune-related IgSF genes that was higher than other IgSF genes (Gibbs et al., 2007; Kosiol et al., 2008; Nielsen et al., 2005; Yu et al., 2006). In this study, we found that silkworm IgSF members could be divided into three groups

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205

Fig. 5. Hierarchical cluster analysis of IgSF gene expression in multiple tissue types.

with distant phylogenetic relationships. Different homologues of some IgSF members were located on different branches of the phylogenetic tree, suggesting divergence in the functions of the homologue during evolution and rapid evolution of IgSF genes in the silkworm. RNA-Seq results showed that vascular endothelial growth factor receptor 1 (VEGFR1) was highly expressed in silkworm ovaries at the fifth instar and early pupal stages. KEGG analysis indicated that VEGFR1 is involved in PI3K-Akt signaling pathway, hypoxia-inducible factor 1 (HIF-1) signaling pathway, cytokine–cytokine receptor interaction, focal adhesion and endocytosis. Signaling mediated by the epidermal growth factor receptor (EGFR) guides dorsal migration of border cells

Table 4 Expression levels of IgSF genes. Lan5-BmCPV and Ou17-BmCPV, silkworm strains infected with BmCPV. Samples

RPKM 0.1–3.75 3.75–15 N15 0–0.1*

IgSF genes in the ovary (strain Dazao) IgSF genes in the midgut (strain Lan5) IgSF genes in the midgut (strain Ou17) IgSF genes in the midgut (strain Lan5-BmCPV) IgSF genes in the midgut (strain Ou17-BmCPV) *

47 64 62 66 57

Genes with RPKM 0–0.1 were considered not expressed.

29 9 11 12 10

29 1 2 1 1

47 78 77 73 84

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Fig. 6. Venn diagram for expression level of IgSF members in the silkworm midgut of strains Lan5 and Ou17. A, High expression; B, moderate expression; C, low expression; D, no expression; Lan5 and Ou17, midgut expression for strains Lan5 and Ou17; Lan5-BmCPV and Ou17-BmCPV, expression in the midgut in strains Lan5 and Ou17 after BmCPV infection.

and modulates specific eggshell structural gene expression in centripetal follicle cells (Bernardi et al., 2007; Duchek and Rorth, 2001). Also, EGFR cooperates with hold up in establishing oocyte positioning, follicle cell fate and egg polarity during Drosophila oogenesis (Rotoli et al., 1998). The HIF-1-mediated transcriptional cascade is a major regulator of border cell locomotion (Djagaeva and Doronkin, 2010) and endocytosis is involved in yolk protein uptake by developing oocytes in Drosophila (Khokhar et al., 2008). The PI3K-Akt signaling pathway is important in apoptosis. In many cancers, this pathway is hyperactive, reducing apoptosis and allowing proliferation (Song et al., 2014; Yap et al., 2008). Therefore, we suggest that high expression of VEGFR1 might result in proliferation and survival of oocytes, oocyte positioning, follicle cell fate and migration and yolk protein uptake. Neuregulin (Nrg), in the EGF protein family, activates EGFR and has multiple essential functions in vertebrate embryogenesis (Burden and Yarden, 1997; Schnepp et al., 1998). Fasciclin 2 is a specific inhibitor of EGFR signaling in Drosophila development and a morphogenetic switch that organizes epithelial cell cluster polarity and motility (Mao and Freeman, 2009; Szafranski and Goode, 2004). Nrg and fasciclin 2 were moderately expressed in silkworm ovaries at the fifth instar and early pupal stages, so we propose that Nrg might be important in oogenesis, and fasciclin 2 might be important for silkworm egg morphogenesis. MYLK is a serine/threonine-specific protein kinase that phosphorylates the regulatory light chain of myosin II, and participates in a calcium signaling pathway in Xenopus oocyte maturation (Machaca, 2007). In this study, we found that MYLK was moderately expressed in silkworm ovaries at the fifth instar and early pupal stages. Therefore, MYLK might also be associated with oocyte maturation in silkworms. Unc-89 is essential for maintaining muscle cell architecture in C. elegans (Spooner et al., 2012), and mutations in the C. elegans Unc89 gene result in disorganized muscle structure in nematodes with no

M-lines and with thick filaments that are not organized into A-bands (Benian et al., 1996). Members of the giant titin/twitchin-like myosin-associated protein family might have dynamic functions during contraction–relaxation cycles in muscle in addition to their functions as cytoskeletal proteins (Heierhorst et al., 1994). Twitchin regulates the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibers (Fabian et al., 2007). Titin is responsible for much of the elasticity of myofibrils in various organisms and most of the elastic force that returns stretched muscles to their resting length (Tskhovrebova and Trinick, 2003). In this study, we found that the muscle protein Unc-89 (BGIBMGA013531), twitchin-like (BGIBMGA004546, BGIBMGA004547) and titin2 (BGIBMGA000624) were moderately expressed in the midgut, suggesting that Unc-89 and twitchin/titin were associated with midgut peristalsis. Peroxidasins are in subfamily 2 of the peroxidase– cyclooxygenase superfamily and might function in antimicrobial defense (Soudi et al., 2012). In silkworms, expressed peroxidasin (BGIBMGA000553) might be involved in resistance of silkworms to pathogenic microorganism. In a previous report, 37 silkworm IgSF genes responded to bacterial infection by the gram-negative bacterium E. coli and the gram-positive bacterium B. thuringiensis. In general, more IgSF genes were downregulated than upregulated during E. coli and B. thuringiensis infections (Huang et al., 2009). Bacterial infections elicit more prominent responses than Plasmodium infections in mosquitoes, suggesting that some IgSF members might be important in the immune response (Garver et al., 2008). BmCPV is a pathogenic microorganism of silkworms that multiplies in the cytoplasm of midgut cells. In this study, we found that only 7 IgSF genes responded to BmCPV infection. Differential expression of IgSF genes decreased after BmCPV infection,

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207

Table 5 Differentially expressed genes in the midgut in silkworm strains Lan5 and Ou17. Gene name

Protein name

Differences in gene expression between strains Lan5 and Ou17

BGIBMGA006354 BGIBMGA006355 BGIBMGA008684 BGIBMGA008685 BGIBMGA008686 BGIBMGA010627 BGIBMGA005029 BGIBMGA005030 BGIBMGA013547 BGIBMGA014206 BGIBMGA000881 BGIBMGA010535 BGIBMGA005031 BGIBMGA005032 BGIBMGA005033 BGIBMGA005642 BGIBMGA008174 BGIBMGA010925 BGIBMGA006348 BGIBMGA000160 BGIBMGA000162 BGIBMGA000163 BGIBMGA000164 BGIBMGA000981 BGIBMGA008511 BGIBMGA008512 BGIBMGA011386 BGIBMGA011387 BGIBMGA005271 BGIBMGA012593 BGIBMGA010347 BGIBMGA012742 BGIBMGA003286 BGIBMGA003287 BGIBMGA001703 BGIBMGA013795 BGIBMGA004513 BGIBMGA004514 BGIBMGA009489 BGIBMGA003448 BGIBMGA006342 BGIBMGA000999 BGIBMGA000139 BGIBMGA008552 BGIBMGA007764 BGIBMGA004750 BGIBMGA004751 BGIBMGA000888 BGIBMGA012240

Lachesin Turtle-like protein A Beat Turtle-like protein Defective proboscis extension response (Dpr 6) ATP-dependent RNA and DNA helicase Neurotrimin-like protein Turtle-like protein Protein tyrosine phosphatase Insulin-related peptide binding protein Leucine-rich transmembrane protein Roundabout Sticks and stones (Sns) Jerky protein homologue-like Semaphorin-2A Lachesin Beat Basigin Beat-IIIa Pro-neuregulin-2 Frazzled protein Klingon Down syndrome cell adhesion molecule (DSCAM) Tyrosine phosphatase 69D, drome Kekkon1 (kek1) Lachesin precursor Beat Tyrosine-protein kinase-like 7 Interference hedgehog-like protein Interleukin-1 receptor accessory protein-like 2 Sidestep Roundabout Basement membrane-specific heparin sulfate proteoglycan core protein-like protein Nephrin

−335.60 −2.92 −2.17 15.28 14.69 13.81 7.97 6.26 5.42 5.20 5.08 4.61 4.52 4.35 4.28 3.97 3.84 3.68 3.60 3.52 3.41 3.08 2.73 2.69 2.57 2.55 2.51 2.46 2.41 2.21 2.12 2.08 2.03

BGIBMGA004887

indicating that BmCPV infections elicited less prominent responses than bacterial infection in silkworm. The resistance of silkworm to BmCPV depended on the strain. The resistance of strain Ou17 was 270 times higher than the resistance of strain Lan5. RNA-Seq indicated that the expression level of IgSF genes in the midgut was clearly different between strains Lan5 and Ou17. Sns, a cell-adhesion molecule, is required for adhesion of migrating myoblasts to myotubes and for initiation of myoblast–myotube fusion (Bour et al., 2000; Gildor et al., 2012; Kocherlakota et al., 2008). In this study, we found that Sns was upregulated in both strains Lan5 and Ou17 after BmCPV infection. Nonetheless, the function of Sns in BmCPV infection is unknown. Hemicentin is involved in maintaining the architectural integrity of vertebrate tissues and organs and is abundant on the surface of many cell types. Recently hemicentin 1 was found to have a unique function in mitotic cytokinesis (Jordan et al., 2011; Xu et al., 2013). The silkworm midgut is composed of intestinal stem, columnar, and goblet cells. Intestinal stem cells differentiate into columnar and goblet cells. Hemicentin 1 was downregulated after BmCPV infection in silkworm strain Lan5, suggesting that mitotic cytokinesis of silkworm intestinal stem cells might be repressed in strain Lan5. Nephrins belong to the IgSF of cell-adhesion receptors and function in cell adhesion and signaling and regulate the structure and function of podocytes and maintain normal glomerular ultrafiltration in the vertebrate kidney (Ristola and Lehtonen, 2014; Tryggvason, 1999). Insect nephrocytes have anatomical, molecular and functional similarity to glomerular podocytes, which are vertebrate kidney cells (Weavers et al., 2009). In the midgut of the silkworm strain Ou17, nephrin-like protein was downregulated after BmCPV infection, suggesting that the silkworm excretory system might be involved in the response to BmCPV infection. Defective proboscis extension response (DPR), a

2.03

member of the IgSF, is required for the gustatory response to salt and functions in sensory physiology (Nakamura et al., 2002). Silkworm DPR6 was upregulated after BmCPV infection in strain Ou17, suggesting that the behavioral response of silkworm to salt might be changed by BmCPV infection. The turtle proteins are required for development and adult viability and are important for bilateral coordinated movement such as the coordinated righting behavior and tactile escape response (Bodily et al., 2001). Recent reports suggest that turtle proteins also function in the development of class-specific dendritic arborization neurons, dendritic morphologies, and dendritic and axonal self-avoidance in Drosophila (Ferguson et al., 2009; Long et al., 2009; Sulkowski et al., 2011). In our work, the turtle isoform BGIBMGA005030 was upregulated and turtle isoform BGIBMGA008685 was downregulated after BmCPV infection in silkworm strain Ou17. Nonetheless, the interaction of turtle proteins and BmCPV infection remains unknown. From the analysis of the results given above, we speculate that the differential expression of IgSF genes in the midgut between strains Lan5 and Ou17 might have resulted in the different resistance to BmCPV observed in the two silkworm strains. The mechanisms of significantly differentially expressed IgSF genes in BmCPV infection should be further studied. 5. Conclusions In this study, 152 IgSF genes containing at least one Ig domain were predicted in the B. mori silkworm genome. Mapping the IgSF genes onto silkworm chromosomes showed that the IgSF genes were not randomly distributed on chromosomes. Phylogenetic evolution analysis indicated that IgSFs evolved rapidly. Enrichment analysis suggested that the IgSF proteins were involved in cell adhesion, cell communication and

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immune responses. Microarray expression data showed that the 136 genes had different expression patterns in different tissues. RNA-Seq analysis indicated that silkworm ovaries had more moderately expressed and highly expressed genes than the midgut and fewer non-expressed genes. Expression levels of 34 IgSF genes in the midgut were significantly different between strains Lan5 and Ou17. 7 IgSF genes were differentially expressed in the midgut after BmCPV infection. These results provide an overview of IgSF family members in silkworms and give valuable clues to understanding the functions of the silkworm IgSFs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.07.030. Acknowledgments We are grateful to peer reviewers for their constructive comments. We gratefully acknowledge the financial support of the National Natural Science Foundation of China (31272500), State Key Laboratory of Silkworm Genome Biology (SKLSGB201200011), the National Basic Research Program of China (973 Program, 2012CB114600), the Specialized Research Fund for the Doctoral Program of Higher Education (20113201130002) and a project funded by the Priority Academic Program of Development of Jiangsu Higher Education Institutions. References Bendtsen, J.D1., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340 (4), 783–795. Benian, G.M., Tinley, T.L., Tang, X.X., Borodovsky, M., 1996. The Caenorhabditis elegans gene unc-89, required for muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains. J. Cell Biol. 132 (5), 835–848. Bernardi, F., Duchi, S., Cavaliere, V., Donati, A., Andrenacci, D., Gargiulo, G., 2007. Egfr signaling modulates VM32E gene expression during Drosophila oogenesis. Dev. Genes Evol. 217 (7), 529–540. Bodily, K.D., Morrison, C.M., Renden, R.B., Broadie, K., 2001. A novel member of the Ig superfamily, turtle, is a CNS-specific protein required for coordinated motor control. J. Neurosci. 21 (9), 3113–3125. Bork, P., Holm, L., Sander, C., 1994. The immunoglobulin fold-structural classification, sequence patterns and common core. J. Mol. Biol. 242 (4), 309–320. Bour, B.A., Chakravarti, M., West, J.M., Abmayr, S.M., 2000. Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Gene Dev. 14 (12), 1498–1511. Burden, S., Yarden, Y., 1997. Neuregulins and their receptors: a versatile signaling module in organogenesis and oncogenesis. Neuron 18 (6), 847–855. Czekierdowski, A., Czekierdowska, S., Szymanski, M., Wielgos, M., Kaminski, P., Kotarski, J., 2006. Opioid-binding protein/cell adhesion molecule-like (OPCML) gene and promoter methylation status in women with ovarian cancer. Neuroendocrinol. Lett. 27 (5), 609–613. Djagaeva, I., Doronkin, S., 2010. Hypoxia response pathway in border cell migration. Cell Adhes. Migr. 4 (3), 391–395. Dong, Y., Taylor, H.E., Dimopoulos, G., 2006. AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system. PLoS Biol. 4 (7), 1137–1146. Duarte-Pereira, S., Paiva, F., Costa, V.L., Ramalho-Carvalho, J., Savva-Bordalo, J., Rodrigues, A., Ribeiro, F.R., Silva, V.M., Oliveira, J., Henrique, R., et al., 2011. Prognostic value of opioid binding protein/cell adhesion molecule-like promoter methylation in bladder carcinoma. Eur. J. Cancer 47 (7), 1106–1114. Duchek, P., Rorth, P., 2001. Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis. Science 291 (5501), 131–133. Eisen, M.B., Spellman, P.T., Brown, P.O., et al., 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. U. S. A. 95 (25), 14863–14868. Eleftherianos, I., Gokcen, F., Felfoldi, G., Millichap, P.J., Trenczek, T.E., ffrench-Constant, R.H., Reynolds, S.E., 2007. The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta. Cell. Microbiol. 9 (5), 1137–1147. Fabian, L., Xia, X.Q., Venkitaramani, D.V., Johansen, K.M., Johansen, J., Andrew, D.J., Forer, A., 2007. Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator. J. Cell Sci. 120 (13), 2190–2204. Ferguson, K., Long, H., Cameron, S., Chang, W.T., Rao, Y., 2009. The conserved Ig superfamily member turtle mediates axonal tiling in Drosophila. J. Neurosci. 29 (45), 14151–14159. Garver, L.S., Xi, Z., Dimopoulos, G., 2008. Immunoglobulin superfamily members play an important role in the mosquito immune system. Dev. Comp. Immunol. 32 (5), 519–531. Gibbs, R.A., Rogers, J., Katze, M.G., Bumgarner, R., Weinstock, G.M., Mardis, E.R., Remington, K.A., Strausberg, R.L., Venter, J.C., Wilson, R.K., et al., 2007. Evolutionary and biomedical insights from the rhesus macaque genome. Science 316 (5822), 222–234.

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