ISJ 12: 214-224, 2015 ISJ 12: 214-224, 2015 ISSN 1824-307X RESEARCH REPORT Paralogous gene conversion, allelic divergence of attacin genes and its expression profile in response to BmNPV infection in silkworm Bombyx mori G Lekha, T Gupta, K Trivedy, K Ponnuvel Genomics Division, Seri Biotech Research Laboratory, Carmelaram Post, Kodathi, Bangalore 560 035, India Accepted July 27, 2015 Abstract The genomic organization, structure and polymorphism of attacin gene within the mulberry silkworm Bombyx mori strains have been analyzed. Genomic contig (AADK01007556) of B. mori attacin gene contains locus with two transcribed basic attacin genes, which were designated as attacin I and attacin II. Survey of the naturally occurring genetic variation in different strains of silkworm B. mori at the promoter and coding regions of two attacin genes revealed high levels of silent nucleotide variations (1- 4 % per nucleotide heterozygosity) without polymorphism at the amino acid level (non- Synonymous substitution). We also investigated variations in gene expression of attacin I and attacin II in silkworm B. mori infected with nucleopolyhedrovirus (BmNPV). Two B. mori strains, Sarupat, CSR-2 which were resistant and susceptible to BmNPV infection respectively were used in this study. Expression profiles of B. mori genes were analyzed using microarray technique and results revealed that the immune response genes including attacin were selectively up regulated in virus invaded midguts of both races. Microarray data and real-time qPCR results revealed that attacin I gene was significantly up-regulated in the midgut of Sarupat following BmNPV infection, indicating its specific role in the anti-viral response. Our results imply that these up-regulated attacin genes were not only involved in anti-bacterial mechanism, but are also involved in B. mori immune response against BmNPV infection. Key Words: Bombyx mori; attacin; microarray; genomic organization; differential expression   Introduction Insects fight bacterial infection, in part, through the extra cellular circulation of a variety of short, general antibacterial peptides (Iwanaga and Lee, 2005). Although over 400 different innate immune peptides have been identified in eukaryotes (Hoffmann et al., 1999), most insects produce relatively small number, fewer than 10 peptide classes and these have to effectively combat a wide range of potential pathogens. Among the different antibacterial proteins produced in insects, attacin, a high molecular weight protein has a major role in insect innate immunity. The amino acid sequence deduced from cloned attacin cDNA of B. mori revealed that the cDNA encodes an attacin precursor protein (Sugiyama et al., 1995). The putative mature protein of Bombyx mori attacin ___________________________________________________________________________ Corresponding author: Kangayam M Ponnuvel Genomics Division Seri Biotech Research Laboratory Carmelaram Post, Kodathi Bangalore 560 035, India E-mail: kmpvel@yahoo.com revealed varying levels of identity in amino acid sequences with those of Hyalophora cecropia acidic (70.4 %) and basic (68.3 %) attacins and Sarcophaga peregrina sarcotoxin IIA (18.8 %). Injection of Escherichia coli cells into B. mori larvae resulted in rapid induction of the expression of B. mori attacin gene that continued at least for 48 h mainly in fat bodies and hemocytes (Sugiyama et al., 1995). Taniai et al. (1996) isolated a genomic clone encoding attacin from genomic library of B. mori, and determined the nucleotide sequence of the 5'-upstream region. Mature attacin peptides are typically 190 - 214 amino acids in length (Sarcophaga peptides are longer) and adopt a “random coil” structure in solution (Gunne et al., 1990). This loose, flexible structure is devoid of disulfide bonds and does not take a rigid conformational shape which may allow relatively free amino acid substitution, explaining the low level of amino acid identity between attacin homologs in distant taxa. Dushay et al. (2000) reported cloning of two closely linked attacin genes from D. melanogaster. A comparison of their protein coding sequences 214   mailto:kmpvel@yahoo.com revealed that the amino acid sequences were more highly conserved than the nucleotide sequences, suggesting expression of both the genes (Wheelan et al., 2001). In this paper we present data on the quantity of polymorphism in the B. mori attacin genes and their expression profile in resistant and susceptible race. Further, the genomic structure of the attacin gene was analyzed and compared with attacin sequences of selected Indian silkworm strains. The structure of exon and intron as well as the phylogenetic relation of B. mori attacin gene to that in other insects were also compared and analyzed. It is also reported that attacin gene has two paralogous genes i.e., attacin I as well as attacin II, both the genes are found to be expressed after bacterial infection (Tanaka et al., 2008). The organization of both attacin genes and its position are explained in this report which are found to be located on the 6th chromosome. There are few antibacterial proteins such as gloverin, lebocin, serpin and these genes have been found to be involved in the immune response against the viral infection, especially against BmNPV infection (Cheng et al., 2014). There is not much information about the role of attacin gene against BmNPV infection. A microarray analysis was carried out to identify the genes associated with BmNPV resistance. There are many antibacterial proteins found to be upregulated after BmNPV infection. Among those antibacterial genes, the expression of attacin gene was significantly upregulated after BmNPV infection in the microarray analysis, indicating its prominent role in antiviral immunity. In the present study the differential expression of both attacin I and attacin II genes has also been analyzed after BmNPV infection to know the role of these genes in the antiviral immune response in silkworm B. mori. Materials and Methods Selection of silkworm races The silkworm Bombyx mori races viz., Sarupat and CSR-2 were selected for the study, as these are known to be most resistant and most susceptible to BmNPV. These two silkworm races were used for the microarray as well as for quantification and gene expression analysis using qPCR. Virus and inoculations B. mori multiple nucleopolyhedrovirus stock was maintained at this laboratory and used as viral inoculum. The viral inoculum was prepared by counting the number of viral polyhedra in a Neubauer chamber. The oral inoculation of BmNPV occlusion bodies was carried out in healthy newly moulted ‘0 day’ fifth instar larvae (first day after 4th moult) of Sarupat and CSR-2 races with viral dosage of 40,000 polyhedral inclusion bodies (PIB) per larva. Three replications containing twenty-five silkworms were maintained for each silkworm race. Similarly, the uninoculated control batches were reared separately under disease free environment. Silkworms feeding on BmNPV-free mulberry leaves were placed in labelled boxes until feeding was complete and then transferred to a controlled room where they remained until the end of the experiment. Collection of tissue BmNPV-infected fifth instar larvae (n = 6) were dissected and the midgut tissues was removed at different (6, 12, 18, 24, 30) h after post infection (hpi). They were quickly washed in diethylpyrocarbonate (DEPC)-treated solution and immediately frozen at -80 °C for further analysis.   RNA isolation and cDNA synthesis The RNA was extracted from different tissues like hemocytes, midgut, fat body and cuticle with TRIzol reagent (Invitrogen, USA), and then denatured in formaldehyde, formamide and electrophoresed in 2.0 % agarose gels. The first strand cDNA was synthesized using DNase treated RNA sample (2 μg) along with 1μl oligo (dT) (0.01mM) (Eurofin India Pvt Ltd, Bangalore) was added followed by incubation at 70 ºC for 3 min. Finally, 1X reverse transcriptase buffer (4μl), 10 mM dNTP (2 μl), 5 mM DTT (2μl) and M-MLV Superscript III reverse transcriptase (Invitrogen, USA) (0.5 μl) was added to obtain a final volume of 20 μl. The reaction mixture was incubated at 42 ºC for 60 min and terminated by heating at 75 ºC for 10 min according to the manufacturer’s protocol. Identification of attacin gene and genomic contig The cDNA of attacin gene was already identified and deposited. The attacin cDNA sequence was blast (BLAST) searched with B. mori genomic DNA database (Xia et al., 2004), for identification of corresponding contig homologous sequence for attacin gene. The genomic DNA sequence showing homologous sequence to B.mori attacin gene was identified and subsequently translated to determine putative amino acid sequence. The amino acid sequence was further analyzed through conserved domain search for the presence of the two functional domains in attacin (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb/cg i). EST expression in different tissues The specific expression of attacin I and attacin II in different tissues were identified by performing blastn followed by retrieval of the EST from the library. The different tissues selected are hemocytes, midgut, fat body and cuticle. Microarray experiment and data analysis A genome wide oligonucleotide microarray containing 24,924 probes were used to investigate the gene expression profiles of BmNPV infected as well as control midguts of Sarupat and CSR-2 silkworm B. mori at 12 h after post infection. The complete sets of raw and normalized data from this study have been deposited in the NCBI Gene Expression Omnibus (GEO) repository. Amplification of attacin gene in different silkworm races The genomic DNA isolated from silk moths using standard protocols (Nagaraja and Nagaraju, 215   http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb/cgi http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb/cgi 1995) was used as template in the PCR reaction. The up and down gene specific primers for attacin gene in the B. mori genomic contig were designed using the software program Primer3 (http://frodo.wi.mit.edu/cgibin/ primer3). The forward primer used was 5’-GGCTGGAAAGCTGGAACTAA- 3’ and the reverse was 5’- AGTCCATAGCCTGGGAACCT-3’. The reaction was done in an Eppendorf thermal cycler, PTC200, using 20 μl reaction mixture containing 50-100 ng of genomic DNA as template, 2.0 μl of 10X PCR buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1 µl each of forward and reverse primers and 0.3 U of Taq DNA polymerase (MBI fermentas). The PCR schedule was 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 2 min and a final extension of 7 min at 72 °C. The PCR amplified products were purified through Gel-spin column (Bangalore Genei) and M13 primer was used for the sequencing reaction. The amplified PCR product 699 bp length (Fig. 3) was cloned in TA cloning vector with M13 sequences flanking the 5’ and 3’ region and sequenced with gene specific primer as sequencing primer. Expression in Sarupat and CSR-2 midgut The BmNPV infected and control midgut samples were collected at different intervals of post infection from 0 to 30 h. The RNA was isolated from the midgut tissues and cDNA was synthesized. The cDNA was used as template to quantify attacin I and attacin II gene expression by qPCR. Results Tissue specific expression profile Tissues were collected from haemocytes, fat body, midgut and cuticle of fifth instar third day larvae for tissue specific expression analysis. The attacin I and attacin II expression was analyzed using forward primer 5’- GCAGGCAAGGTCAATTTGTT-3’ and reverse 5’- CGGTTGATGACGTCAGAGTG-3’ for attacin I. Forward primer-5’TCGAGGTCGTATTGCAGACA-3’ and 5’GGCTCCCACGAAGATCTGTA-3’ of reverse primer for attacin II. The reactions were conducted on a Stratagene MxPro-Mx3005P Real-Time PCR system (Agilent technologies) using the SYBR Premix Ex Taq Kit (TaKaRa), according to the manufacturer's protocol. Each amplification reaction was performed using a 20 μl reaction mixture, under the following conditions: denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and at 55 °C for 30 s. The experiment was performed in triplicate and results were standardized to the expression level of the constitutive β actin gene. A non-template control (NTC) sample was also run to detect contamination, if any. The microarray analysis was carried out to investigate the gene expression profiles in silkworm B. mori against BmNPV infection. The results indicated that some of the antibacterial proteins including attacin gene were upregulated after BmNPV infection and thus indicating their potential role in the antiviral immune response (Sagisaka et al., 2010). Therefore, an attempt has been made to study the differential expression of attacin gene in BmNPV resistance. In addition to that the organization of paralogous attacin genes, their tissue specific expression and variation in the promoter and coding regions were analyzed. The attacin cDNA sequence (accession no. S78369) was blast (BLAST) searched with B. mori genomic DNA database and a single contig (accession no AADK01007556) possessing the attacin gene sequence was identified. Two paralogous sequences similar to attacin gene sequence were present in the locus. Further, there were three exon with two intron regions of 91 bp and 79 bp length respectively. These two gene sequences were arranged in a direction opposite to the single contig with a gap of 3.4 kb length and the paralogous genes were designated as attacin I and attacin II (Fig.1). The conserved domain analysis showed the presence of two sub domains the in G domain of attacin, similar to that of other insect attacins. The data indicates that both attacin I and II genes are conserved across the taxa. Attacin I revealed full length cDNA of 831 bp length similar to the original cDNA sequence (accession no. S78369), while attacin II sequence matched partially with a length of 683 bp. Analysis of 5’ regulatory sequences of both the attacin genes indicated that attacin I and II possess Fig. 1 The B. mori attacin genomic organization. The position of the genes and their transcriptional directions ( ) are shown underneath. The overall structure of attacin I and II and the distance from the start codon (+1) to the functional parts shown in base pairs. 216   Fig. 2 Comparison of promoter regions of attacin I and II of silkworm B. mori. The G-box, TATA box and Cap site are boxed. G-box, TATA box and cap site followed by exon/intron region. The G-box and TATA box were found to be located at two different positions on the 5’ region in both the attacin genes. The G-box was located in 105 bp region of attacin I gene, whereas, it was in 138 bp region in attacin II gene. Similarly, the TATA box location was at 79 bp region in attacin I and at 84 bp region in attacin II of the upstream region from the starting codon. Interestingly, no difference was found in the cap site location (position at 52 bp) of both attacin I and attacin II upstream region (Fig. 2). The attacin gene sequences of Pure Mysore, Daizo, Nistari, NB4D2, CSR19 and cDNA sequence (Acc. No. S78369) were compared using multiple alignment program (Clustal W). The results indicate single nucleotide variations at 22 places (Fig. 3). The sequences were clustered through phylogenetic analysis and the dendogram obtained indicated that all the multivoltine races formed a separate cluster, while, the bivoltine races formed another (Fig. 4). Further, nucleotide variation was analyzed for possible changes in the amino acid level. These genes exhibit high levels of silent nucleotide variations (synonymous substitution), but within the silkworm races they are not excessively polymorphic at the amino acid level, as most of the nucleotide variation did not yield changes in the amino acid sequence. Among the 14-nucleotide variations observed within the amplified region, most of the changes caused no change in the amino acid sequence. The amino acid serine was coded by various degenerative codons due to which there were no changes at amino acid level. Similar findings were observed in the other amino acids such as valine, isoleucine, leucine and alanine. EST expression in different tissues The complete sequences of attacin I and II were blast searched in the silk base database and the EST sequences with maximum homology expressed in different tissues were retrieved. A total of 37 transcripts were retrieved from the EST library, of which 34 transcripts belonging to attacin I and 3 transcripts were that of attacin II. Out of 34 transcripts, attacin I expressed maximum (44 %) in fat body tissues (Fig. 5) followed by corpora allata (17 %). In case of attacin II, among the three transcripts, two were expressed in the fat body while one was from corpora allata. 217   Fig. 3 Multiple alignment of attacin gene sequences from different silkworm races of B. mori. The intron regions are boxed. Fig. 4 The evolutional tree was obtained by the neighbor-joining method based on the multiple alignments of attacin DNA sequences of different silkworm strains. The numbers on each branch indicate the percentage of the most parsimonious trees, which were found in 1000 bootstrap replications performed with MEGA5 programme. 218   Fig. 5 The pie chart indicating the number of attacin I transcripts in different tissues. Microarray experiment and data analysis The genes associated with BmNPV infection were identified in Sarupat (resistant) and CSR- 2(susceptible) B. mori silkworm races since these races reveal divergent responses with respect to BmNPV infection. Oligonucleotide microarray containing 24,924 probes were used to investigate the gene expression profiles in the midgut tissue of BmNPV infected and uninfected silkworms after 12 hours post infection (hpi). Results revealed that, 735 and 589 genes were up-regulated and downregulated, respectively, at 12 hpi, in Sarupat, whereas, 2183 genes were up-regulated and 2115 down-regulated in CSR-2 (data not shown). It was observed that immune related proteins showed higher expression in BmNPV infected tissues, of which attacin I and attacin II had a significantly up and down regulation in resistant and susceptible silkworm races, respectively (Fig. 6). Based on this data, it was concluded that attacin I was upregulated in the BmNPV infected Sarupat midgut tissue, however, higher expression of attacin II was found in BmNPV infected CSR-2 being a susceptible race. To validate the expression of these genes, the primers were designed for attacin I and attacin II for further qPCR analysis.  Tissue specific expression profile The qPCR analysis revealed that the attacin I expression was higher in the fat body followed by midgut, cuticle and hemocytes (Fig. 7). The decrease in the expression in the hemocytes possibly occurred because the viral infection damaged physical functions, resulting in the reduction of the gene expression (Cheng et al., 2014). None of the earlier report indicate expression of attacin in the mid gut tissues. In the present study the significant amount of transcripts were found to be expressed in the midgut tissues. It has already been reported that the attacin gene is expressed in the fat body and similar findings has been observed in the present study. Fig. 6 Heat map of hierarchical clustering of differentially expressed genes in BmNPV infected and uninfected midguts at 12 h of post infection in Sarupat and CSR-2 (clustering type: hierarchical clustering, Distance metric: Pearson correlation). The colors in the heat map display the relative values of all tiles; green indicates the lowest expression, yellow indicates the intermediate expression, and red indicates the highest expression. The numerical values give the actual values on a log 2 scale, which were associated with each color. 219   http://www.ncbi.nlm.nih.gov/pubmed/?term=Cheng%20Y%5Bauth%5D Fig. 7 Relative gene expression patterns of attacin I upregulated during BmNPV infection in Sarupat. RNA was isolated at 12 hours post infection. The relative expression levels of each gene was normalized using the Ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. In each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. All samples were tested in triplicate. The mean value ±SD was used for analysis of relative transcript levels for each time point using the ΔΔCt method. A Non-template control (NTC) sample was also run to detect contamination if any. ■- BmNPV infected □-uninfected Fig. 8 Relative gene expression patterns of gene expression in BmNPV infected and uninfected samples of Sarupat with attacin I. RNA was isolated at 6 hourly intervals from 0 to 30h. The relative expression levels of each gene at different time points were normalized using the Ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. In each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. All samples were tested in triplicate. The mean value ±SD was used for analysis of relative transcript levels for each time point using the ΔΔCt method. A Non-template control (NTC) sample was also run to detect contamination if any. ■- BmNPV infected □-uninfected Quantification of attacin I and II gene expression in BmNPV infected larvae In addition to microarray data, the expression of attacin I and attacin II in the midgut tissues was confirmed through qPCR analysis in the control as well as BmNPV infected silkworm races i.e., Sarupat and CSR-2 at different time intervals. In the BmNPV resistant race (Sarupat), the expression of attacin I has gradually increased from 6 hpi to 18 hpi and then the expression was maintained steadily up to 30 hpi (Fig. 8). The expression of attacin II was lesser in Sarupat, when compared with attacin I expression, which also showed a gradual decrease in the expression up to 18 hpi (Fig. 9). In CSR-2, which is a BmNPV susceptible race, the expression of attacin I in BmNPV infected samples were found to be lesser when compared to that of the control samples (Fig. 10). On the contrary to Sarupat, the expression of attacin II increased from 0 h to 30 h in BmNPV infected larvae of CSR-2. The highest level of expression of attacin II gene was observed in CSR-2 up to 18 hpi, thereafter the expression gradually decreased and steadily maintained up to 30 h of post infection (Fig. 11). 220   Fig. 9 Relative gene expression patterns of gene expression in BmNPV infected and uninfected samples of Sarupat with attacin II. RNA was isolated at 6 hourly intervals from 0 to 30h. The relative expression levels of each gene at different time points were normalized using the Ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. In each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. All samples were tested in triplicate. The mean value ±SD was used for analysis of relative transcript levels for each time point using the ΔΔCt method. A Non-template control (NTC) sample was also run to detect contamination if any. ■- BmNPV infected □-uninfected. Discussion It is already known that B. mori genome consists of two attacin genes viz. attacin I and attacin II which play an anti-bacterial role. However, the genome-wide microarray analysis followed by real time PCR revealed that attacin gene not only functions as an anti-bacterial gene but also has an anti-viral role in silkworm B. mori. In order to identify the specific role against BmNPV infection and to analyze differential expression of the attacin genes, an attempt has been made to analyze the expression profiles of these genes in BmNPV infected Sarupat (BmNPV resistant) and CSR-2 (BmNPV susceptible) silkworm races. The up regulation of attacin I gene in the BmNPV infected midgut samples of Sarupat indicated that attacin I was specifically being expressed in response to BmNPV infection in the resistant race whereas the attacin II gene expression was comparatively lesser in Sarupat. In case of CSR-2, the expression of attacin I in BmNPV infected samples were found to be lesser than that in the control samples. However, the expression of attacin II was comparatively higher in the BmNPV infected samples of silkworm race CSR-2. This observation indicates that among the two attacin genes, attacin I has association with BmNPV resistance. Families of attacin-like peptides (usually two to four functional genes per haploid genome) have been identified in the lepidopteran species B. mori (Sugiyama et al., 1995), H. cecropia (Hultmark et al., 1983), Hyphantria cunea (Shin et al., 1998), Trichoplusiani (Kang et al., 1996), and Heliothis virescens (Ourth et al., 1994), as well as in the dipteran species S. peregrina (Ando et al., 1987) and D. melanogaster (Asling et al., 1995; Dushay et al., 2000; Hedengren et al., 2000). The antibacterial peptides are often conserved across evolutionarily distance taxa but, little is known about the level and structure of the polymorphism within different species (Choe et al., 2002). Sugiyama et al. (1995) cloned the attacin gene and its 5’ upstream regulatory region was characterized. In the present study it was observed that the two attacin genes of B.mori were arranged in opposite directions in a single contig with a gap of 4.2 kb length and these two genes were designated as attacin I and attacin II. These genes are transcribed in opposite directions and interrupted at homologous position by two introns. The major difference between these two attacin genes is the size of exon III. In attacin I it is 351 nt while in attacin II it is only 203 nt. Similar findings have also been observed in giant silk moth H. cecropia (Sun and Faye, 1995) indicating that the duplication of attacin gene as well as gene synteny is conserved within the insect taxa. Kadalayil et al. (1999) showed that the promoters of several inducible insect immune genes possess GATA sites 0 - 12 bp away from NF- kappaB binding site (NF-kB site) in functionally important promoter regions. Clusters of GATA and NF-kB sites are also observed in the promoters of two important mammalian immune genes, namely IL-6 and IL-3. In B. mori also the nucleotide sequence of both the attacin gene 5’-upstream region 221   Fig. 10 Relative gene expression patterns of gene expression in BmNPV infected and uninfected samples of CSR-2 with attacin I. RNA was isolated at 6 hourly intervals from 0 to 30 h. The relative expression levels of each gene at different time points were normalized using the Ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. In each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. All samples were tested in triplicate. The mean value ±SD was used for analysis of relative transcript levels for each time point using the ΔΔCt method. A Non-template control (NTC) sample was also run to detect contamination if any. ■- BmNPV infected □-uninfected. contains a lipopolysaccharide (LPS) response element (NF-kB site), CAAT box and TATA box. The cap site of both attacin I and attacin II genes is located at a position of 52 bp in the upstream region. Tanaka et al. (2008) reported that the cap site is very important for the active transcription of attacin genes thereby indicating that both B. mori attacin I and II genes are actively transcribed after microbial infection. However, there is a variation in the position of different promoter elements of attacin genes which may affect transcription efficiency. Lazzaro and Clark (2001) analyzed natural genetic variation in alleles of D. melanogaster attacins A and B and observed that, the overall level of nucleotide diversity is high in each of these, but there is no excess of amino acid polymorphism. They also observed that, attacins A and B have experienced multiple paralogous gene conversion events. Our results also revealed attacin gene polymorphisms at nucleotide level, but not at amino acid level, in the different silkworm strains studied. Gloverin and lebocin seems to be lepidopteran- specific antibacterial peptides (Axen et al., 1997) and have expression levels that were strongly induced in B. mori larval fat body by Escherichia coli immune challenge. BmNPV infection also caused a strong induction of B. mori gloverin and lebocin gene expressions in larval midguts and this induction occurred in both B. mori strains for gloverin-3 and lebocin genes. The antiviral mechanism that occurs in the resistant B. mori strain is not due to resistance against the BmNPV invasion but rather due to the inhibition of BmNPV proliferation in the larval midgut (Bao et al., 2009). The defense processes against BmNPV infection that occur in the resistant larvae might be regulated via interactions involving multiple genes (Liu et al., 2000). Huang et al. (2013) observed that antimicrobial peptides have an antiviral role in response to Alphavirus replication in arthropods. They focused their study on the antiviral response of D. melanogaster innate immune system induced by RNA replication of Sindbis virus (SINV). Further, they carried out microarray analysis in search for SINV replication sensitive genes. Out of the 95 SINV replication genes identified, two of the genes were found to be antimicrobial peptides viz. attC and dptB. Knocking out these genes either led to an increase in viral RNA synthesis or defects in development in the presence of SINV replication complex. These findings clearly demonstrate the antiviral role of attacin in Drosophila. Choi et al. (2012) also demonstrated that, genes like attacin were significantly up regulated during viral infection. In the present study, similar attempts have been made to identify immune response genes in BmNPV infected silkworms using microarray techniques. Attacin I and II genes of B.mori were found to be differentially expressed after BmNPV infection thereby proving these genes to be BmNPV responsive. Further, the work carried out by Huang et al. (2013) also demonstrates the phenomenon of allelic 222   Fig. 11 Relative gene expression patterns of gene expression in BmNPV infected and uninfected samples of CSR-2 with attacin II. RNA was isolated at 6 hourly intervals from 0 to 30 h. The relative expression levels of each gene at different time points were normalized using the Ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. In each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. All samples were tested in triplicate. The mean value ± SD was used for analysis of relative transcript levels for each time point using the ΔΔCt method. A Non-template control (NTC) sample was also run to detect contamination if any. ■- BmNPV infected □-uninfected. divergence and the functional diversity of attacin genes. Drosophila consists of four attacin genes. The attaA, attB and attC genes are located on chromosome 2 while attD is located on chromosome 3. The AttA and AttB proteins share 98% identity with each other while AttC is 73% identical to AttA and AttB. In spite of such close similarity the expressions of attA, attB and attD were not found to be SINV replication sensitive. Among the four paralogous attacin genes only attC was found to be SINV replication responsive which suggests that only a single gene (attC) acquired the antiviral role while the other genes remained devoid of such function. In our study it was found that attacin I gene specifically was upregulated during BmNPV infection, in the resistant race. On the contrary attacin II was found to be devoid of such expression. A list of immune protein genes has been identified from the microarray analysis that is BmNPV responsive and regulated by the innate immune pathways of B. mori. Among these genes attacin I was found to demonstrate an anti-viral role which otherwise has always been reported for anti- bacterial activity. Among the two reported attacin paralogous genes, attacin I acquired antiviral role which is unique when compared to its ancestral gene. 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