Genome-Wide Transcriptional Response of Silkworm (<i>Bombyx mori)</i> to Infection by the Microsporidian <i>Nosema bombycis</i>

<div><p>Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on <i>Nosema ceranae</i>, few works have focused on elucidating the mechanism in host response to microsporidia infection. <i>Nosema bombycis</i> is a pathogen of silkworm pébrine that causes great economic losses to the silkworm industry. Detailed understanding of the host (<i>Bombyx mori</i>) response to infection by <i>N. bombycis</i> is helpful for prevention of this disease. A genome-wide survey of the gene expression profile at 2, 4, 6 and 8 days post-infection by <i>N. bombycis</i> was performed and results showed that 64, 244, 1,328, 1,887 genes were induced, respectively. Up to 124 genes, which are involved in basal metabolism pathways, were modulated. Notably, <i>B. mori</i> genes that play a role in juvenile hormone synthesis and metabolism pathways were induced, suggesting that the host may accumulate JH as a response to infection. Interestingly, <i>N. bombycis</i> can inhibit the silkworm serine protease cascade melanization pathway in hemolymph, which may be due to the secretion of serpins in the microsporidia. <i>N. bombycis</i> also induced up-regulation of several cellular immune factors, in which CTL11 has been suggested to be involved in both spore recognition and immune signal transduction. Microarray and real-time PCR analysis indicated the activation of silkworm Toll and JAK/STAT pathways. The notable up-regulation of antimicrobial peptides, including gloverins, lebocins and moricins, strongly indicated that antimicrobial peptide defense mechanisms were triggered to resist the invasive microsporidia. An analysis of <i>N. bombycis-</i>specific response factors suggested their important roles in anti-microsporidia defense. Overall, this study primarily provides insight into the potential molecular mechanisms for the host-parasite interaction between <i>B. mori</i> and <i>N. bombycis</i> and may provide a foundation for further work on host-parasite interaction between insects and microsporidia.</p></div

Real-time quantitative PCR was used to validate the differential expression of genes measured by microarray.

<p>A total of 9 genes, including peptidoglycan recognition protein-short 3 (<i>PGRP-S3</i>), <i>PGRP-S4</i>, β-glucan recognition protein 2 (<i>β-GRP2</i>), out domain containing protein (<i>OTC</i>), serpin12 (<i>SPN12</i>), C-type lectin 11 (<i>CTL11</i>), defense protein (<i>DEP</i>), adenylate cyclase (<i>ADC</i>) and alkaline phosphatase (<i>ALP</i>), at four time points was selected for primer design. The detailed sequences of the related primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084137#pone.0084137.s005" target="_blank">Table S1</a>.</p

Diagram showing the induced biosynthesis, metabolism and signal transduction related genes of juvenile hormone in the silkworm.

<p>(A) Observation of silkworm infected by <i>N. bombycis</i> at 8 dpi. Cont: Uninfected silkworm larvae. Exp: Silkworm oral infected by <i>N. bombycis</i> spores. (B)The expression pattern for each gene is indicated near the gene name and the probe ID of BmMDB is shown aside. Diagram of the pathway is referenced from Daojun Cheng, 2008. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084137#pone.0084137.s008" target="_blank">Table S4</a> for a detailed view of the cluster ratios.</p

<i>N. bombycis</i> induced a silkworm cellular immune response.

<p>(A) Cluster of cellular immune response families. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084137#pone.0084137.s012" target="_blank">Table S8</a> for a detailed view of the cluster ratios. (B,C) The phylogenetic analysis and domain prediction of <i>N. bombycis</i> induced BmCTLs and βGRPs. The up-regulated genes were indicated by the arrows pointing upward. The arrows pointing downward showed the down-regulated genes. If the genes were not induced in our data, horizontal arrows were used to represent them.</p

<i>N. bombycis</i> perturbed the silkworm serine protease cascade melanization pathway.

<p>(A) Cluster diagram of the induced genes involved in the serine protease cascade melanization pathway. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084137#pone.0084137.s008" target="_blank">Table S4</a> for a detailed view of the cluster ratios. (B) Measurement of the melanization speed of silkworm plasma. The silkworms were oral-infected by <i>N. bombycis</i> and hemolymph was collected at 10 dpi. The cell-free plasma was obtained and three replicates were performed. The averages represented the three replicates from one experiment and the error bars in the graph indicated the standard deviation (SD) value. Cont: Uninfected silkworm larvae. Exp: Silkworm treated with <i>N. bombycis</i> spores. Pht: phenylthiourea. (C) Transcriptional analysis of of <i>N.b.serpins</i> using RT-PCR. <i>Nbserpin2</i> (SilkPathDB No. NBO_18g0004), <i>Nbserpin6</i> (NBO_34g0030), <i>Nbserpin9</i> (NBO_39i001), <i>Nbserpin13</i> (NBO_124g0001), <i>Nbserpin14</i> (NBO_372g0002), <i>Nbserpin19</i> (NBO_1570g0002).</p