An insight into the salivary gland and fat body transcriptome of <i>Panstrongylus lignarius</i> (Hemiptera: Heteroptera), the main vector of Chagas disease in Peru

<div><p>Triatomines are hematophagous arthropod vectors of <i>Trypanosoma cruzi</i>, the causative agent of Chagas Disease. <i>Panstrongylus lignarius</i>, also known as <i>Panstrongylus herreri</i>, is considered one of the most versatile triatomines because it can parasitize different hosts, it is found in different habitats and countries, it has sylvatic, peridomestic and domestic behavior and it is a very important vector of Chagas disease, especially in Peru. Molecules produced and secreted by salivary glands and fat body are considered of important adaptational value for triatomines because, among other functions, they subvert the host haemostatic, inflammatory and immune systems and detoxify or protect them against environmental aggressors. In this context, the elucidation of the molecules produced by these tissues is highly valuable to understanding the ability of this species to adapt and transmit pathogens. Here, we use high-throughput sequencing techniques to assemble and describe the coding sequences resulting from the transcriptome of the fat body and salivary glands of <i>P</i>. <i>lignarius</i>. The final assembly of both transcriptomes together resulted in a total of 11,507 coding sequences (CDS), which were mapped from a total of 164,676,091 reads. The CDS were subdivided according to their 10 folds overexpression on salivary glands (513 CDS) or fat body (2073 CDS). Among the families of proteins found in the salivary glands, lipocalins were the most abundant. Other ubiquitous families of proteins present in other sialomes were also present in <i>P</i>. <i>lignarius</i>, including serine protease inhibitors, apyrase and antigen-5. The unique transcriptome of fat body showed proteins related to the metabolic function of this organ. Remarkably, nearly 20% of all reads mapped to transcripts coded by Triatoma virus. The data presented in this study improve the understanding on triatomines’ salivary glands and fat body function and reveal important molecules used in the interplay between vectors and vertebrate hosts.</p></div

Classification of all coding sequences (CDS) with putative housekeeping function extracted from the transcriptome of <i>P</i>.<i>lignarius</i>.

<p>Classification of all coding sequences (CDS) with putative housekeeping function extracted from the transcriptome of <i>P</i>.<i>lignarius</i>.</p

Coding sequences (CDS) 10X overexpressed in fat body compared to salivary glands from transcriptome of <i>P</i>. <i>lignarius</i>.

<p>Coding sequences (CDS) 10X overexpressed in fat body compared to salivary glands from transcriptome of <i>P</i>. <i>lignarius</i>.</p

Phylogram of the lipocalin family proteins from <i>P</i>. <i>lignarius</i> and their best matches.

<p>The optimal tree with the sum of branch length = 123.81570372 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The bar at the center of the graph indicates a value of 0.5. The analysis involved 330 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 423 positions in the final dataset. The values near the branches represent the percentage of bootstrap support. Values below 50% are not shown. For more details, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006243#sec002" target="_blank">Material and Methods</a>.</p

Phylogram of the apyrase/5’-nucleotidase family of proteins from <i>P</i>. <i>lignarius</i> and their best matches.

<p>The optimal tree with the sum of branch length = 15.40595903 is shown. The values near the branches represent the percentage of bootstrap support. Values below 75% are not shown. The analysis involved 41 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 689 positions in the final dataset. For more details, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006243#sec002" target="_blank">Material and Methods</a>.</p

Validation by qPCR of the differential expression of transcripts between salivary gland and fat body libraries.

<p>Relative gene expression was calculated by ΔΔCT method using PhSigP-51408_FR4_55–276 as reference gene (similar expression in salivary gland and fat body). Scatter plot presenting the SG:FB expression ratio of FB overexpressed, similarly expressed and SG overexpressed genes. The Y axis is the log2 of the observed ratio between SG and FB by qPCR on three groups of arbitrarily selected transcripts that are overexpressed in either tissue, or similarly expressed. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006243#pntd.0006243.s003" target="_blank">S1 Table</a> provides for the transcript names and primer sequences used in this experiment.</p

Phylogram of the vitellogenin protein of <i>P</i>. <i>lignarius</i> and their best matches.

<p>The optimal tree with the sum of branch length = 11.41384103 is shown. The values near the branches represent the percentage of bootstrap support. Values below 50% are not shown. The analysis involved 45 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1713 positions in the final dataset. For more details, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006243#sec002" target="_blank">Material and Methods</a>. The <i>Panstrongylus lignarius</i> sequence is shown with a red marker.</p

Classification of all coding sequences (CDS) from the fat body transcriptome of <i>P</i>. <i>lignarius</i>.

<p>Classification of all coding sequences (CDS) from the fat body transcriptome of <i>P</i>. <i>lignarius</i>.</p

Phylogram of the Kazal-domain containing family of proteins from <i>P</i>. <i>lignarius</i> and their best matches.

<p>The optimal tree with the sum of branch length = 14.99541793 is shown. The values near the branches represent the percentage of bootstrap support. Values below 50% are not shown. The analysis involved 43 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 969 positions in the final dataset. For more details, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006243#sec002" target="_blank">Material and Methods</a>. Robust clades are named I-IV (where no member has been functionally characterized); Infestin and Rhodnius indicate clades containing previously functionally characterized proteins.</p

Venn diagram of SG and FB transcriptomes of <i>P</i>. <i>lignarius</i>.

<p>SG10x, 10-fold overexpressed transcripts in the salivary gland compared to fat body; FB10x, 10-fold overexpressed transcripts in the fat body compared to the salivary gland; and, Not Enriched, transcripts expressed in both SG and FB with less than a 10-fold variation.</p