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

Bootstrapped phylogram of <i>Rhodnius prolixus</i> midgut lipocalins aligned with their best matches to the NR database.

<p>Bootstrap values above 50% are shown on the branches. The bottom line indicates 20% amino acid sequence divergence between the proteins. <i>R. prolixus</i> sequences are shown by the notation RP followed by a unique number. The remaining sequences, obtained from GenBank, are annotated with the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank GI number. One thousand replicates were done for the bootstrap test using the neighbor joining test.</p

Functional classification of AM-overexpressed transcripts (>10× compared to posterior) from <i>Rhodnius prolixus</i>.

<p>Functional classification of AM-overexpressed transcripts (>10× compared to posterior) from <i>Rhodnius prolixus</i>.</p

Functional classification of gut-overexpressed transcripts (>10× compared to whole body) from <i>Rhodnius prolixus</i>.

<p>Functional classification of gut-overexpressed transcripts (>10× compared to whole body) from <i>Rhodnius prolixus</i>.</p

Cladogram of insect Lysozymes from glycoside hydrolase Family 22.

<p>The <i>R. prolixus</i> sequences are shown by the notation RP- followed by a unique number. The remaining proteins were obtained from GenBank and they are annotated with accession number followed by species name. The dendrogram was generated with the UPGMA algorithm. The branches were statistically supported by bootstrap analysis (cut-off 40) based on 1,000 replicates.</p

Bootstrapped phylogram of <i>Rhodnius prolixus</i> and other aspartyl proteinases.

<p>Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. <i>R. prolixus</i> sequences are shown by the notation RP followed by a unique number and have a red circle preceding their names. The <i>Triatoma infestans</i> sequences from Balczun et. al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002594#pntd.0002594-Balczun1" target="_blank">[2]</a> have a green marker. The remaining sequences were obtained from GenBank and are annotated with the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank GI number. One thousand replicates were done for the bootstrap test using the neighbor joining test.</p

Bootstrapped phylogram of <i>Rhodnius prolixus</i> and other insect peritrophin annotated as Group IV peritrophin in Fig. 1.

<p>Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. <i>R. prolixus</i> sequences are shown by the notation RP followed by a unique number. The remaining protein sequences were obtained from GenBank and are annotated with the first three letters of the genus name followed by the first three letters of the species name followed by their GenBank GI number. All non-<i>Rhodnius</i> sequences derive mostly from mosquitoes, with one deriving from a flea and another from a sand fly. Roman numerals indicate clades with mixed mosquito genera. Ten thousand replicates were done for the bootstrap test using the neighbor joining method.</p