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

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