An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus

Abstract

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

Figure 1. Cladogram of Rhodnius prolixus peritrophins.

The dendrogram was generated with the neighbor-joining algorithm. Branches were statistically supported by bootstrap analysis (cut-off 45) based on ten thousand replicates. The Roman numerals indicate the perithrophin's group classification.

Figure 2. Bootstrapped phylogram of Rhodnius prolixus and other insect peritrophin annotated as Group IV peritrophin in Fig. 1.

Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. R. prolixus 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-Rhodnius 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.

Figure 3. Cladogram of insect Lysozymes from glycoside hydrolase Family 22.

The R. prolixus 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.

Figure 4. Bootstrapped phylogram of Rhodnius prolixus and other aspartyl proteinases.

Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. R. prolixus sequences are shown by the notation RP followed by a unique number and have a red circle preceding their names. The Triatoma infestans sequences from Balczun et. al. 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.

Figure 5. Bootstrapped phylogram of Rhodnius prolixus and other cysteinyl proteinases.

Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. R. prolixus sequences are shown by the notation RP followed by a unique number and have a red circle preceding their names. 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.

Figure 6. Bootstrapped phylogram of Rhodnius prolixus and other carboxypeptidases.

Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. R. prolixus sequences are shown by the notation RP followed by a unique number and have a red circle preceding their names. 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.

Figure 7. Bootstrapped phylogram of Rhodnius prolixus midgut lipocalins aligned with their best matches to the NR database.

Bootstrap values above 50% are shown on the branches. The bottom line indicates 20% amino acid sequence divergence between the proteins. R. prolixus 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.

Figure 8. Bootstrapped phylogram of Rhodnius prolixus midgut Takeout-JHBP, Odorant Binding Protein and Chemosensorial Protein.

Amino acid sequences of 46 contigs were combined to create an entry file for phylogenetic analysis in MEGA 4.0.2. An unrooted consensus neighbor joining tree was generated based on ten thousand bootstrap replicates with pairwise gap deletions using neighbor joining method. Bootstrap values lower than 50% are not shown. Red boxes indicate the over expressed proteins. JHBP: Juvenile hormone binding proteins. OBP: Odorant binding proteins. CSP: Chemosensorial proteins. For more details, see text.

Figure 9. Bootstrapped phylogram of Rhodnius prolixus midgut lectins aligned with their best matches to the NR database.

Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. R. prolixus sequences are shown by the notation RP followed by a unique number. 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.