Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

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Differential expression profiles in the midgut of Triatoma infestans infected with Trypanosoma cruzi.

Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs) from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp). Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification) showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP), which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease

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

Functional classification of transcripts that originated from upregulated contigs.

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

Functional classification of transcripts from all clusters.

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