Several rounds of amplifications of MITE families <i>in N</i>. <i>bombycis</i>.

<p>(A) The distribution profile of genetic distance for copies of MITE families. X-axis represents the interval of genetic distance; Y-axis represents the numbers of any pairwise copies. (B) The tree diagram for the copies of <i>Nbh2</i> families with neighbor-joining (NJ) method.</p

MITE-derived small RNAs in <i>Nosema bombycis</i>.

<p>(A) Length distribution of small RNAs generated by MITE sequences. (B) Density (sense, black; antisense, red) of small RNA tags assigned to MITE sequences. Frequency is shown along the Y-axis. Relative nucleotide position within the consensus sequence is indicated along the X-axis.</p

Presence/absence of MITEs polymorphisms in four geographical strains of <i>N</i>. <i>bombycis</i>.

<p>(A) The product of <i>NbS3</i>-inserted PCR amplification in four strains, SD1 and SD2 represent one pair of segmental duplication in <i>N</i>. <i>bombycis</i>. (B) The product of <i>NbS24</i>-inserted PCR amplification in three strains and the illustration. M, DNA marker, CQ1: Chongqing isolate, YN: Yunnan isolate, GD: Guangdong isolate, GX: Guangxi isolate. Black arrowhead: MITE-inserted PCR-amplified products. Gray arrowheads: PCR-amplified product without MITEs insertion. Rectangular arrowheads: genes in the syntenic region among several <i>N</i>. <i>bombycis</i> isolates and <i>N</i>. <i>antheraeae</i>. Same color rectangles correspond to homologous genes.</p

Expression of <i>β</i>-Tubulin.

<p>(A) Validation of pCold I-<i>β-tubulin</i> vector by PCR and Bam HI/Sal I enzyme digestion. ~1300 bp products were amplified by PCR or cleaved from recombinant vector. (B) SDS-PAGE of proteins expressed in <i>Escherichia coli</i> Rosetta. Recombinant <i>β-</i>Tubulin protein was induced to express at 37°C and 16°C. pCold I vector transformed <i>E</i>. <i>coli</i> Rosetta were induced for expression at 37°C as a control. (C) Immunoblot for <i>β-</i>Tubulin in total protein of <i>Nosema bombycis</i> mature spore. The antibody recognized a 50 kDa band which was consistent with prediction.</p

Immunofluorescence localization of NbSWP12 in the intracellular parasite.

<p><i>N</i>. <i>bombycis</i>-infected BmE cells were incubated with anti-NbSWP12 (A2-1, A2-2) coupled with Alexa Fluo 488 labeled secondary antibody and anti-<i>β-</i>Tubulin (A3-1, A3-2) coupled with Alexa Fluo 594 labeled secondary antibody. Red fluorescence of <i>β-</i>Tubulin indicated the proliferative phase of <i>N</i>. <i>bombycis</i>. Blue fluorescence of Calcofluor White M2R-stained chitin displayed the sporogonic phase of <i>N</i>. <i>bombycis</i>. Overlapping red and green signals (A1-1) indicated that NbSWP12 was partly co-localized with microtubules in the meront. Overlapping green and blue signals (A1-2) in some early sporonts indicated that NbSWP12 was gradually transferred to the spore wall. (Bars = 5 μm).</p

Location of <i>β</i>-Tubulin in intracellular microsporidian <i>N</i>. <i>bombycis</i>.

<p>Images were taken by laser scanning confocal microscopy using filter sets for Alexa fluo 488 labeling <i>β-</i>Tubulin proteins and DAPI staining nucleus. Immunofluorescence assay with <i>β-</i>Tubulin antiserum demonstrated that the membrane and cell plasma location contained in <i>N</i>. <i>bombycis</i> cells in the proliferative phase. (Bars = 5 μm)</p

Labeling the two different intracellular phase of microsporidian <i>N</i>. <i>bombycis</i> by immunofluorescence histochemistry in intestinal tissue slices.

<p><i>β-</i>Tubulin antibody coupled with Alexa Fluo 488 (green) labeled secondary antibody were used to label the proliferative phase of microsporidia. Calcofluor White M2R (blue) were used to stain the chitin layer of sporogony phase. (Bars = 10 μm)</p

Labeling the merogony phase (red) and the sporogony phase (blue) of microsporidian in <i>N</i>. <i>bombycis</i>-infected BmE culture cells.

<p>Based on the absence or presence of chitin, parasites in proliferative phase were labeled by <i>β-</i>Tubulin using an indirect immunofluorescence assay, while cells in sporogonic phase were marked by chitin using 0.1 μg/mL Calcofluor White M2R. <i>N</i>. <i>bombycis</i> infected culture cells were incubated with negative serum used as a negative control. (Bars = 10 μm).</p