IsCDA antibodies influence the persistence of <i>B. burgdorferi</i> in feeding ticks.

<p>(A) Passive transfer of antibodies raised against IsCDA interferes with spirochete persistence in fed ticks during their acquisition from infected hosts. Naïve nymphal ticks were microinjected with equal amounts of antibodies against IsCDA or control (normal mouse serum, NMS). <i>B. burgdorferi</i> burdens in replete ticks were assessed by qRT-PCR analyses by measuring copies of <i>B. burgdorferi </i><i>flaB</i> RNA and normalized against tick <i>β-actin</i> levels. Each diamond represents an individual tick that was processed and analyzed separately. The difference in spirochete number between ticks injected with anti-IsCDA antibodies and control (NMS) was significant, p < 0.01. (B) Passive transfer of IsCDA antibodies influences persistence of <i>B. burgdorferi</i> in feeding ticks during pathogen transmission to naïve hosts. Spirochete-infected nymphal ticks were microinjected with antibodies, and <i>B. burgdorferi</i> burdens in replete ticks were assessed, as detailed in panel A. Each diamond represents an individual tick that was processed and analyzed separately. The difference in spirochete number between ticks injected with anti-IsCDA antibodies and control (NMS) was significant, p < 0.05.</p

Temporal and spatial distribution of IsCDA in tick gut.

<p>(A) The highest levels of <i>IsCDA</i> expression are noted during early tick attachment on the host. Ticks were collected at various times of attachment to the murine hosts, and <i>IsCDA</i> expression was measured using quantitative RT-PCR. (B) Antisera generated against recombinant IsCDA detect a native protein in the PM. The PM structure from the tick gut was isolated and immunoblotted with IsCDA antibodies, which recognized native IsCDA with an approximate MW of 60 kDa (arrow). Migration of the protein MW marker is indicated on the left (in kDa). (C) Cellular localization of IsCDA in the tick gut tissue. Cryosections from unfixed tick gut collected at 24 h of feeding were labeled with antibodies against IsCDA and Alexa 488-labeled secondary antibodies and processed for confocal immunofluorescence microscopy. The tick tissues were labeled with a nuclear stain (DAPI). Scale bar, 10 μm.</p

Analysis and isolation of peritrophic membrane in <i>I. scapularis</i>.

<p>(A) Transmission electron microscopic detection of peritrophic membrane (PM). While undetectable in the gut of unfed ticks (left panel), a PM structure is conspicuous in ticks and was isolated and processed at 36 h of feeding on mice (L, lumen; EC, gut epithelial cell). Arrows point to the PM. Scale bar, 5 μm. (B) Immunofluorescence localization of PM. Cryosections from unfixed tick gut samples were labeled with WGA-FITC (green) and propidium iodide (PI, red) and imaged using a confocal microscope. Arrows point to the green PM, while the nuclei (N) of gut epithelial cells are labeled red. Scale bar, 10 μm. (C) Isolation of an intact PM structure. The PM was isolated from the dissected gut of nymphal ticks and viewed under a binocular dissecting microscope. Scale bar, 100 μm. (D) Relative purity of the extracted PM. The PM, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078376#pone-0078376-g001" target="_blank">Figure 1C</a>, was stained with WGA-FITC (green) and PI (red) and imaged using a confocal microscope. Lack of red fluorescence indicated absence of contaminating gut cells. Scale bar, 20 μm. </p

Amino acid sequence alignment of potential catalytic domains of CDA with orthologs from other insect species.

<p>Partial amino acid sequences (annotations are shown according to NCBI reference sequences) from <i>Anopheles gambiae</i> (XP_316929), <i>Culex quinquefasciatus</i> (XP_001848571 and XP_001868428), <i>Drosophila melanogaster</i> (NP_001097045), and <i>Tribolium castaneum</i> (NP_001103739) were aligned with <i>IsCDA</i> using the GENETYX software. The chitin-binding and catalytic domains are indicated with dark lines, while CDA motifs are denoted by gray boxes.</p

Identification of tick PM proteins.

<p>(A) Western blot analysis of PM proteins from the tick gut. Gut samples dissected from unfed and fed ticks were immunoblotted using antibodies generated against the PM. Specificity of anti-PM antisera was tested against normal mouse serum (present in fed ticks) or by replacing the primary antibody with normal mouse serum. Arrows denote proteins (between 46-175 kDa) that are preferentially recognized by the anti-PM antibodies, suggesting specific association with the PM in the fed vector gut. (B) Identification of abundant PM proteins. PM samples were isolated from fed ticks and extensively rinsed with water. Proteins in the solubilized PM fraction were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Clearly resolved and detectable protein bands, as indicated by arrows (1-3), were excised and analyzed by LC-MS/MS spectrometry for identification of the proteins. Migration of the protein MW marker is indicated on the left (in kDa). Asterisks indicate the IsCDA protein that is the focus of the present work.</p

Contig assembly error identification through genome map comparison.

<p>The top line represents the <i>in silico</i> map for the original sequence assembly, the majority of which is covered by a single sequence contig. The genome map matches on the left and right sides of the contig (shown with shaded boxes). ∼3 kb of sequence was incorrectly inserted into the contig during assembly.</p

Deletion of incorrect contigs in genome map-guided <i>de novo</i> sequence assembly.

<p>The original assembly contained two Nt.BspQI sites and ∼8 kb of sequence that were absent from the genome map. The top image is output from gsAssembler and shows the scaffolding of contigs using paired-end reads. The green line represents the sequence coverage for each region. Paired-end reads are represented by pink (high coverage) and aqua (low coverage) carrots (□). The three contigs with red bars beneath them contain the extra sequence motifs and total sequence consistent with the predicted incorrect scaffold. They also contain weak paired-end data indicating that the contigs are misplaced. The bottom line shows the sequence assembly after deletion of the three contigs with red bars.</p

Comparison of the final genome map guided sequence assembly to the genome map.

<p>The final sequence assembly almost completely spans the genome map. Gaps in the genome map are denoted with asterisks.</p

Comparison of the sequence assembly scaffold to the genome map.

<p>The sequence assembly is shown with dark grey boxes representing sequence contigs. Contigs were bridged by paired-end sequence reads. The genome map is represented by light grey boxes. Shaded boxes around regions of both maps denote regions where the sequence assembly matches the genome map well. Regions where there are significant discrepancies are numbered and discussed in the results section. The two gaps in the genome map are denoted with asterisks.</p

Identification and assembly of repeat sequences in genome map-guided <i>de novo</i> sequence assembly.

<p>Panel A shows the strip diagram for one of the clones that covers the high density region, each line represents a different molecule and the spots are the location of green (Nt.BspQI) labels. Two high-density label regions are marked “label repeats,” and they do not cluster into discrete peaks. Panel B shows a pairwise alignment of the high-density region after reassembly based on the genome map. The alignment shows two blocks of direct repeats which are inverted with respect to one another. Panel C shows the original assembly on the top, the genome map in the middle and the final assembly on the bottom, containing the repeat sequence as predicted by the genome map.</p