RNA-seq de novo Assembly Reveals Differential Gene Expression in Glossina palpalis gambiensis Infected with Trypanosoma brucei gambiense vs. Non-Infected and Self-Cured Flies

Abstract

Trypanosoma brucei gambiense (Tbg), causing the sleeping sickness chronic form, completes its developmental cycle within the tsetse fly vector Glossina palpalis gambiensis (Gpg) before its transmission to humans. Within the framework of an anti-vector disease control strategy, a global gene expression profiling of trypanosome infected (susceptible), non-infected, and self-cured (refractory) tsetse flies was performed, on their midguts, to determine differential genes expression resulting from in vivo trypanosomes, tsetse flies (and their microbiome) interactions. An RNAseq de novo assembly was achieved. The assembled transcripts were mapped to reference sequences for functional annotation. Twenty-four percent of the 16,936 contigs could not be annotated, possibly representing untranslated mRNA regions, or Gpg- or Tbg-specific ORFs. The remaining contigs were classified into 65 functional groups. Only a few transposable elements were present in the Gpg midgut transcriptome, which may represent active transpositions and play regulatory roles. One thousand three hundred and seventy three genes differentially expressed (DEGs) between stimulated and non-stimulated flies were identified at day-3 post-feeding; 52 and 1025 between infected and self-cured flies at 10 and 20 days post-feeding, respectively. The possible roles of several DEGs regarding fly susceptibility and refractoriness are discussed. The results provide new means to decipher fly infection mechanisms, crucial to develop anti-vector control strategies.

Comparative Genomics of Glossina palpalis gambiensis and G. morsitans morsitans to Reveal Gene Orthologs Involved in Infection by Trypanosoma brucei gambiense

“…The different steps are described in the corresponding report (Hamidou Soumana et al, 2015), as well as pro parte in reports related to the differential expression of S. glossinidius and W. glossinidia genes (Hamidou Soumana et al, 2014a,b). Sample processing is summarized in Figure 1.…”

“…Analyzing the two annotation processes of the Gpg DEGs consisted in comparing the list of the “best hits” resulting from the Gpg DEG annotation on the Gmm database with the list resulting from the Gpg DEG annotation previously performed on a set of other databases ( Ceratitis capitata, Drosophila melanogaster, D. willistoni, D. virilis, D. mojavensis, Acyrthosiphon pisum, Hydra magnipapillata, Anopheles sp., Bombyx sp., Aedes sp., and Glossina morsitans ; Hamidou Soumana et al, 2015). The first step consisted in mixing the DEGs identified at the three experimental times (3, 10, and 20 days) and removing the duplicates, so as to take into account all recorded DEGs except for one of each.…”

“…The aim of this previous work was to identify the genes that are differentially expressed in trypanosome infected vs. non-infected or self-cured (refractory) flies and that, consequently, can be suspected to positively or negatively control fly infection. Similarly, using the RNA-seq de novo assembly approach, we investigated the differential expression of G. p. gambiensis genes in flies challenged or not with trypanosomes (Hamidou Soumana et al, 2015). Furthermore, transcriptome profiling of T. b. brucei development in Gmm has recently been reported (Savage et al, 2016).…”

“…As Gpg and Gmm are two separate Glossina species, their genomes should display some differences between each other. Furthermore, the Gmm genome and the sequences of the Gpg RNA-seq de novo assembled genes have been annotated with reference to two distinct database sets: the first set comprises Drosophila melanogaster, Aedes aegypti, Anopheles gambiae, Culex quinquefasciatus , and Phlebotomus papatasi (International Glossina Genome Initiative, 2014), whereas the second set comprises Ceratitis capitata, Drosophila melanogaster, D. willistoni, D. virilis, D. mojavensis, Acyrthosiphon pisum, Hydra magnipapillata, Anopheles sp., Bombyx sp., Aedes sp., and Glossina morsitans (data that were available before the publication of the whole genome sequence; Hamidou Soumana et al, 2015). This indicates that only the D. melanogaster database was common to the two database sets used to annotate the differentially expressed Gpg genes and the Gmm genome, respectively.…”

“…Thus, for the present study, it was necessary to map the sequences of the Gpg RNA-seq de novo assembled genes on the Gmm genome and annotate them on the corresponding database. This has been achieved, and the Gpg genes that were previously shown to be differentially expressed (i.e., stimulated vs. non-stimulated flies, and infected vs. non-infected flies; Hamidou Soumana et al, 2015) were annotated on the Gmm database. Finally, the data resulting from the best hits annotation, which provide a translation product for each gene (and thus its potential biological function and physiological role), were compared with data resulting from the previous annotation of the same genes on the set of above-mentioned databases.…”