Temporal expression of housekeeping genes.

<p>Raw Ct values for the mRNA of seven <i>A</i>. <i>americanum</i> housekeeping genes from tick salivary glands (A) and midgut (B) throughout a blood meal (unfed–168 h). Each gene was amplified from each sample and the point at which the amplification curve crosses the threshold is considered the Ct.</p

Temporal expression of immunity-related genes.

<p>Notably, gene AamerSigP-41992 was not expressed, according to qRT–PCR.</p

Temporal expression of seven tick-specific genes with unknown functions.

<p>AamerSigp-22563 was not expressed, according to qRT–PCR.</p

Larval hatching of tick eggs after ds-Reprolysin multiple gene knockdown.

<p>Depletion of MP transcripts negatively affected the hatching of larvae, whereas no changes in the control samples were observed.</p

Temporal gene expression of a miscellaneous pool of protein families.

<p>Aam-5252 was not expressed, according to qRT–PCR.</p

Temporal expression of glycine-rich protein genes.

<p>Temporal expression of glycine-rich protein genes.</p

Analysis of microbial load in female salivary glands.

<p>The salivary glands of ticks treated with both Rep-Family RNAi and Aam-41580 RNAi were assessed for their total bacterial loads on day 8 of feeding. MP depletion in the salivary glands caused a significant reduction in the total microbial load in ticks in which both the MP family and single genes were knocked down. Bacterial loads after both types of knockdown were 5–12-fold lower than those of the Lac-Z-RNAi-treated and no-treatment control salivary glands. *P < 0.05.</p

Engorgement weights of female ticks.

<p>Tick engorgement weights (mg) from both Rep-MP RNAi and Aa-41580 RNAi ticks fed on sheep. Am-41580 RNAi knockdown caused a significant reduction in tick weights (P < 0.05) compared with the (No Treatment) control; weights were measured on day 10. The size of the ds-Lac-Z (irrelevant RNA)-treated sample was significantly reduced during feeding and was not included in the analysis. RNAi knockdown of the reprolysin family significantly reduced tick weight (P < 0.05) compared with both controls.</p

Multiple sequence alignment of reprolysin MP nucleotide sequences identified for multiple-gene RNAi.

<p>Knockdown was observed in four MPs. Nucleotides with more than 60% homology are highlighted in blue. Red underlining shows the locations of the primers. Actual primer sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147966#pone.0147966.s003" target="_blank">S3 Table</a>.</p

Temporal expression of lipocalin genes.

<p>AamerSigP-18604 was not expressed at any time point, according to qRT–PCR.</p