IsCDA knockdown failed to interfere with formation of the PM or the persistence of B. burgdorferi within I. scapularis.

(A) Schematic representation of IsCDA 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 IsCDA knockdown via injection of dsRNA3 is indicated by the red line. (B) Knockdown of IsCDA transcripts induced by RNA interference. Nymphal ticks (10/group) were injected with IsCDA dsRNA or control GFP dsRNA and fed on naïve mice to repletion, and isolated guts were processed for quantitative RT-PCR using primers against IsCDA and the results normalized against tick β-actin. Each diamond represents an individual tick that was processed and analyzed separately. IsCDA transcripts levels in IsCDA dsRNA-injected ticks were reduced by 1000-fold or more compared to GFP-injected control ticks (p <0.05). (C) Injection of IsCDA 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 Figure 1B, and imaged using a confocal microscope. Note that for a global and comparative assessment of the PM in the gut diverticula of IsCDA-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) IsCDA knockdown failed to interfere with persistence of spirochetes in fed ticks during their acquisition from infected mice. Mice were infected with B. burgdorferi and parasitized by naïve nymphal I. scapularis that were injected with either IsCDA dsRNA or control GFP dsRNA. Replete ticks were collected, and B. burgdorferi levels were determined using quantitative RT-PCR targeting flaB and normalized against tick β-actin levels. The difference in spirochete number between the IsCDA dsRNA-treated and GFP dsRNA-treated groups was nonsignificant, p > 0.05. (E) IsCDA knockdown did not influence persistence of B. burgdorferi in feeding ticks during pathogen migration to naïve hosts. IsCDA dsRNA or control GFP dsRNA-injected spirochete-infected ticks were allowed to engorge on naïve mice, and B. burgdorferi levels were determined in replete ticks, as detailed in panel C. The difference in pathogen level between the IsCDA dsRNA-treated and GFP dsRNA-treated groups was nonsignificant, p >0.05.

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