Computational protocol: A fine tuned vector parasite dialogue in tsetse's cardia determines peritrophic matrix integrity and trypanosome transmission success

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Protocol publication

[…] Using CLC Genomics Workbench 8 (Qiagen), transcriptome reads were first trimmed and filtered to remove ambiguous nucleotides and low-quality sequences. The remaining reads were mapped to Glossina morsitans morsitans reference transcriptome GmorY1.5 (VectorBase.org). Reads aligning uniquely to Glossina transcripts were used to calculate differential gene expression using EdgeR package in R software [].Significance was determined using EdgeR exact test for the negative binomial distribution, corrected with a False Discovery Rate (FDR) at P<0.05.Identified genes were functionally annotated by BlastX, with an E-value cut-off of 1e-10 and bit score of 200, and peptide data available from D. melanogaster database (FlyBase.org). Blast2GO was utilized to identify specific gene ontology (GO) terms that were enriched between treatments based on a Fisher’s Exact Test []. [...] To assess the PM integrity, we applied a host survival assay following per os treatment of each group with Serratia marcescens as described in [, ]. We provided to three groups of 8 day-old flies (in their 4th blood meal) either cardia extracts obtained from challenged flies that cleared the trypanosomes and are subsequently recovered from initial infection (rec-/-), or a cardia extract from inf+/- flies, or a cardia extract from inf+/+ flies. We included a fourth group of 8-day old flies that received an untreated blood meal.Cardia extract was obtained by dissecting, in PBS, the cardia from 40 days-old infected as described above. Approximately fifty cardia from either rec-/-, inf+/- or inf+/+ flies were pooled together, and then gently homogenized. Parasites were counted from the homogenates of inf+/- and inf+/+ using a hemocytometer. The three cardia homogenates were then heated at 100°C for 10 minutes. inf+/- and inf+/+ extracts were provided to reach a concentration of 5×105 parasites per ml of blood. As inf+/- cardia contain fewer parasites than inf+/+ cardia, the volume of the inf+/+ extract provided was adjusted by dilution in PSG buffer to be equal to inf+/- volume. Rec-/- extract was provided at an equal volume than infected extracts to ensure the presence of a similar quantity of extract molecules coming from the cardia in these groups. 48 hours after the flies received blood meal supplemented with the different extracts, all flies were provided a blood meal supplemented with 1,000 CFU/ml of S. marcescens strain Db11. Thereafter, flies were maintained on normal blood every other day, while their mortality was recorded every day for 30 days. Precise counting data are indicated in .Statistical analysis was carried out using the R software for macOS (version 3.3.2). We used an accelerated failure time model (Weibull distribution) where survival was analyzed as a function of the extract received (survreg() function of "survival" package). Pairwise tests were generated using Tukey contrasts on the survival model (glht() function of "multcomp" package). Precise statistical results are indicated in . […]

Pipeline specifications

Software tools CLC Genomics Workbench, edgeR, BLASTX, Blast2GO, multcomp
Databases FlyBase VectorBase
Applications RNA-seq analysis, GWAS
Organisms Toxoplasma gondii, Drosophila melanogaster, Trypanosoma brucei, Homo sapiens
Diseases Esophageal Neoplasms, Infection
Chemicals Oxygen