Transcriptome Sequencing and Developmental Regulation of Gene Expression in <i>Anopheles aquasalis</i>

<div><p>Background</p><p><i>Anopheles aquasalis</i> is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to <i>Plasmodium vivax</i> and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of <i>An. aquasalis</i> genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the <i>An. aquasalis</i> transcriptome.</p><p>Methodology/Principal Findings</p><p>A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥2 sequences, and 1999 singletons. The majority of the <i>An. aquasalis</i> transcripts encode proteins with their closest counterparts in another neotropical malaria vector, <i>An. darlingi</i>. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced <i>An. aquasalis</i> proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed.</p><p>Conclusions/Significance</p><p>This study represents the first large-scale effort to sequence the transcriptome of <i>An. aquasalis</i>. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The <i>An. aquasalis</i> transcriptome is accessible at <a href="http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx" target="_blank">http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx</a>.</p></div

Comparisons of developmental changes in gene expression between <i>An. aquasalis</i> and <i>An. gambiae</i>.

<p>Developmental gene regulations [up(U) or down(D)-regulation] between larvae and sugar fed females (L-S all) or between sugar fed females and blood fed females (S-B all) of <i>An. aquasalis</i> transcripts that have a homolog <i>An. gambiae</i> (best Blast match) represented in the GeneChip <i>Plasmodium</i>/<i>Anopheles</i> Genome Array <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003005#pntd.0003005-Marinotti1" target="_blank">[42]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003005#pntd.0003005-Marinotti4" target="_blank">[95]</a> were compared. The pairwise comparisons including all <i>An. aquasalis</i>/<i>An. gambiae</i> homologous pairs of genes demonstrated a lack of conservation of developmental changes in transcript abundance between the two mosquito species. Similar analyses restricting the transcript list to putative 1∶1 ortholog pairs, defined by reciprocal blast and only those significantly regulated in <i>An. aquasalis</i>, with at least 3 fold change between two compared samples (L-S 1∶1 ort or S-B 1∶1 ort) showed that 75% the transcripts regulated by blood feeding were consistently up or down regulated in both species. Using the same restricted list of transcripts, only 49% of the transcripts were consistently up- or down-regulated between L-S in both species. Genes up-regulated or down-regulated in both species are indicated by (UU) or (DD), respectively. Transcripts differentially regulated between the two species are indicated by (UD/DU).</p

B and T cells are increased in the BAL of mice sensitized by mosquito bites followed by SGE challenge.

<p>BAL collection from control and sensitized groups was performed 24 h after the last challenge with PBS or SGE. Differential analyses of lymphocytes were performed by flow cytometry in cells stained with florescence-conjugated antibodies for cell surface markers. <b>(A)</b> Sensitization protocol; <b>(B)</b> total number of CD19⁺ cells, <b>(C)</b> total number of CD4⁺ cells; <b>(D)</b> total number of CD8⁺ cells. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 when compared with PBS group; ^<i>#</i><i>p</i> < 0.05 when compared with the 1x group.</p

Mice sensitized by mosquito bites develop eosinophilic airway inflammation after intranasal SGE challenge.

<p>BAL collection from control and sensitized groups was performed 24 h after the last challenge with PBS or SGE. Total cell number and differential cell counts were analyzed by microscopic evaluation of cytocentrifuged slides. <b>(A)</b> Sensitization protocol; <b>(B)</b> total cells; <b>(C)</b> eosinophils; <b>(D)</b> neutrophils; <b>(E)</b> macrophages; <b>(F)</b> lymphocytes. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 when compared with PBS group; ^<i>#</i><i>p</i> < 0.05 when compared with the 1x group.</p

Th2 cytokine levels are upregulated in BAL of sensitized mice in response to SGE challenge.

<p>BAL of control and sensitized mice was collected 24 h after the last challenge with PBS or SGE, and the cytokine levels in the free-cell supernatant were determined by ELISA. <b>(A)</b> Sensitization protocol; <b>(B)</b> IL-4; <b>(C)</b> IL-5; <b>(D)</b> IL-13; <b>(E)</b> IFN-γ; <b>(F)</b> IL-17. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 compared with PBS group; ^<i>#</i><i>p</i> < 0.05 compared with SGE group.</p

Respiratory pattern and tracheal responsiveness were not affected in SGE-challenged sensitized mice.

<p>Twenty-four hours after the last challenge, the respiratory pattern was assessed in control and mosquito bite-sensitized mice using whole-body plethysmography, as described in Material and Methods. Airway sensitivity was tested in the presence of increasing concentration of methacholine and maximal resistance values was recorded after 5 minutes. <b>(A)</b> Sensitization protocol; <b>(B)</b> results expressed as Penh. For the tracheal reactivity assay, mice were sensitized or not by mosquito bites and challenged twice with PBS or SGE. Basal values were obtained from the control group and concentration-effect curves for methacholine were constructed using an organ bath system. <b>(C)</b> Sensitization protocol; <b>(D)</b> trachea responsiveness. Results are expressed as mean ± SEM (n = 6–8). *<i>p</i> < 0.05 when compared with PBS group; ^#<i>p</i> < 0.05 when compared with SGE group.</p

Distribution of the best matches of all <i>An. aquasalis</i> predicted proteins by organisms: Andar- <i>An. darlingi</i>; Angam- <i>An. gambiae</i>; Aeaeg, <i>Ae. aegypti</i>; Cuqui, <i>Cu. quinquefasciatus</i>; Drmel, <i>D. melanogaster</i>; Other <i>Anopheles</i> species; Other insects- not of the <i>Anopheles</i> genus; Other- non-insect organisms.

<p>Distribution of the best matches of all <i>An. aquasalis</i> predicted proteins by organisms: Andar- <i>An. darlingi</i>; Angam- <i>An. gambiae</i>; Aeaeg, <i>Ae. aegypti</i>; Cuqui, <i>Cu. quinquefasciatus</i>; Drmel, <i>D. melanogaster</i>; Other <i>Anopheles</i> species; Other insects- not of the <i>Anopheles</i> genus; Other- non-insect organisms.</p

Number of sequences composing the assembled <i>An. aquasalis</i> contigs.

<p>A total of 7544 contigs were assembled from ≥2 sequences. The number of sequences that compose each contig varies from 2 to 5,207, with an average of 35 sequences per contig. Forty-three percent of the assembled contigs contained 10 or more sequences.</p