10.1371/journal.pone.0078376.g005 Toru Kariu Toru Kariu Alexis Smith Alexis Smith Xiuli Yang Xiuli Yang Utpal Pal Utpal Pal <i>IsCDA</i> knockdown failed to interfere with formation of the PM or the persistence of <i>B. burgdorferi</i> within <i>I. scapularis</i>. Public Library of Science 2013 knockdown failed pm persistence 2013-10-17 04:35:35 Figure https://plos.figshare.com/articles/figure/_IsCDA_knockdown_failed_to_interfere_with_formation_of_the_PM_or_the_persistence_of_B_burgdorferi_within_I_scapularis_/826877 <p>(A) Schematic representation of <i>IsCDA</i> open reading frame showing regions targeted for RNA interference studies. Out of the four dsRNA constructs generated and tested (relative positions are indicated by straight black lines), dsRNA3 (spanning nucleotide positions 385-1192) was most effective and used for subsequent studies. Location of primers that were used to assess the efficacy of <i>IsCDA</i> knockdown via injection of dsRNA3 is indicated by the red line. (B) Knockdown of <i>IsCDA</i> transcripts induced by RNA interference. Nymphal ticks (10/group) were injected with <i>IsCDA</i> dsRNA or control <i>GFP</i> dsRNA and fed on naïve mice to repletion, and isolated guts were processed for quantitative RT-PCR using primers against <i>IsCDA</i> and the results normalized against tick <i>β-actin</i>. Each diamond represents an individual tick that was processed and analyzed separately. <i>IsCDA</i> transcripts levels in <i>IsCDA</i> dsRNA-injected ticks were reduced by 1000-fold or more compared to <i>GFP</i>-injected control ticks (p <0.05). (C) Injection of <i>IsCDA</i> dsRNA failed to influence the formation of the PM in fed ticks. Guts from fed nymphal ticks were cryosectioned, labeled with WGA-FITC (green) and PI (red), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078376#pone-0078376-g001" target="_blank">Figure 1B</a>, and imaged using a confocal microscope. Note that for a global and comparative assessment of the PM in the gut diverticula of <i>IsCDA</i>-knockdown or control ticks, the gut samples were imaged under lower magnification, which renders the PM, appearing as green fluorescent lines (arrows), and luminal spaces (arrowheads), relatively inconspicuous and intermittent across the cryosectioned gut diverticulum. Scale bar, 20 μm. (D) <i>IsCDA</i> knockdown failed to interfere with persistence of spirochetes in fed ticks during their acquisition from infected mice. Mice were infected with <i>B. burgdorferi</i> and parasitized by naïve nymphal <i>I. scapularis</i> that were injected with either <i>IsCDA</i> dsRNA or control <i>GFP</i> dsRNA. Replete ticks were collected, and <i>B. burgdorferi</i> levels were determined using quantitative RT-PCR targeting <i>flaB</i> and normalized against tick <i>β-actin</i> levels. The difference in spirochete number between the <i>IsCDA</i> dsRNA-treated and <i>GFP</i> dsRNA-treated groups was nonsignificant, p > 0.05. (E) <i>IsCDA</i> knockdown did not influence persistence of <i>B. burgdorferi</i> in feeding ticks during pathogen migration to naïve hosts. <i>IsCDA</i> dsRNA or control <i>GFP</i> dsRNA-injected spirochete-infected ticks were allowed to engorge on naïve mice, and <i>B. burgdorferi</i> levels were determined in replete ticks, as detailed in panel C. The difference in pathogen level between the <i>IsCDA</i> dsRNA-treated and <i>GFP</i> dsRNA-treated groups was nonsignificant, p >0.05.</p>