Knockdown of the <i>Rhipicephalus microplus</i> Cytochrome <i>c</i> Oxidase Subunit III Gene Is Associated with a Failure of <i>Anaplasma marginale</i> Transmission

<div><p><i>Rhipicephalus microplus</i> is an obligate hematophagous ectoparasite of cattle and an important biological vector of <i>Anaplasma marginale</i> in tropical and subtropical regions. The primary determinants for <i>A. marginale</i> transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of <i>A. marginale</i> to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of <i>A. marginale</i> to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to <i>A. marginale</i> infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome <i>c</i> oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional <i>in vivo</i> genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on <i>A. marginale</i> acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit <i>A. marginale</i> to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of <i>A. marginale</i> transmission.</p></div

qRT-PCR of infestin.

<p>Adult insects infected with <i>T. cruzi</i> and uninfected <i>T. infestans</i> were used for analysis (three biological samples were used for both the uninfected and infected groups). All data were normalized to 18S ribosomal RNA, representing the mean (n = 3) of identical triplicates ± standard deviation. An unpaired <i>t</i> test was performed for statistical analysis.</p

A pie chart showing the species distribution of BLASTx hits of the <i>T. infestans</i> clusters for several organisms.

<p>A pie chart showing the species distribution of BLASTx hits of the <i>T. infestans</i> clusters for several organisms.</p

A list of contigs with unchanged expression that are important for metabolic processes or defense in <i>T. infestans</i>.

<p>FABL – Fatty acid binding lipocalin.</p><p>JHBP – Juvenile hormone binding protein.</p

Length distribution of <i>T. infestans</i> clusters.

<p>Length distribution of <i>T. infestans</i> clusters.</p

qRT-PCR of four transcripts upregulated upon <i>T. cruzi</i> infection.

<p>Amounts of mRNA from nitrophorin-like protein (A), 14 kDa protein (B), lysozyme (C) and cathepsin D (D) were obtained by relative quantification. Adult insects infected with <i>T. cruzi</i> and uninfected <i>T. infestans</i> were used for analysis (three biological samples were used for both the uninfected and infected groups). All data were normalized to 18S ribosomal RNA, representing the mean (n = 3) of identical triplicates ± standard deviation. An unpaired <i>t</i> test was performed for statistical analysis, and differences were considered significant at P<0.05. Asterisks represent significant differences (** P<0.01; *** P<0.001).</p

Functional classification of transcripts that originated from downregulated contigs.

<p>Functional classification of transcripts that originated from downregulated contigs.</p

Functional classification of transcripts from all clusters.

<p>Functional classification of transcripts from all clusters.</p

Functional classification of transcripts that originated from upregulated contigs.

<p>Functional classification of transcripts that originated from upregulated contigs.</p

Relative gene expression in gut and salivary glands of ticks infected with <i>A. marginale</i>.

<p>The expression of the cytochrome <i>c</i> oxidase sub III (COXIII), glutathione S-transferase (GST), synaptobrevin (SYN), dynein (DYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS) genes in guts and salivary glands from <i>R. microplus</i> males fed for 8 days on either one uninfected calf (C38080) or one <i>A. marginale</i>-infected calf (C37837) was assessed by RT-qPCR. Threshold values were normalized according to the Ct of the reference gene (tubulin). The relative expression level of each gene in infected ticks in relation to uninfected ticks (control) was calculated using the Delta Delta Ct method. The data represent the mean ± S.D. of four pools of 5 guts and salivary glands. An asterisk (*) represent data with differences statistically significant with respect to control (<i>P</i>≤0.05).</p