Comparative transcriptomics of neuropeptides in vectors of Chagas disease

TRAVERSO LUCILA; SIERRA IVANA; ONS SHEILA

Lovaina

Conferencia; 28th Conference of European Comparative Endocrinologists; 2016

p { margin-bottom: 0.25cm; direction: ltr; line-height: 120%; text-align: left; }Introduction:Chagas' disease is a neglected tropical disease which is moreprevalent in the Americas, besides the movement of human populationshas spread the disease to others regions in the world. The causativeagent is the protozoan parasite Trypanosoma cruzi,Transmittedby triatomine insects (Figure 1). Despite the actions performed toeliminate the vectors, intheGran Chaco ecoregion the disease is still prevalent, and theresistance to insecticidesseemsto be one of the main causes of the failure in its elimination. Asthe involvement ofneuropeptidesin the response to insecticides is a poorly explored research fieldto date,inthis work the identification of neuropeptide precursors in T.infestans, T. dimidiata and T.pallidipennistranscriptomes is presented, along with peptidomic analysis in T.infestans. Furthermore, we compare gene expressionof neuropeptidesprecursor genes in T. infestans populations sensitive and resistantto pyrethroids.MyM: For neuropeptide precursor genes identification, iterative BLASTXsearches wereperformedusing local BLAST on a R. prolixus neuropeptide precursor genesdatasetdescribedearlier, along with other insect species. Furthermore, an onlineTBLASTNsearchwas performed in the salivary gland transcriptome from T. infestansavailable inVectorBase.For peptidomic analysis, central nervous system (CNS) from male andfemale adult T. infestansweredissected, separated in brain (B), suboesophageal ganglion (SOG),prothoracicganglion(PRO) and meso-metathoracic ganglion (MTGM) and analyzed in anon-linenano-LC-Electrospray(ESI)-Orbi-Trap Mass Spectrometer. The software Peaks Studio7.0was used for peptide identificaton. Unfed 5th instarnymphsfrom the pyrethroid-sensitive and resistant T. infestans strains wereused for transcriptabundancecomparisons by qRT-PCR.Results:Thesequences of 20 neuropeptide precursor genes in T. infestans, 16 inT. dimidiataand13 in T. pallidipenis were detected in the transcriptomic databasesanalyzed. A totalof59 neuropeptides were identified by liquid chromatography-tandemmassspectrometryin brain and nervous ganglia from T. infestans (Table 1), revealingthe existence ofdifferentialpost-translational modifications, extended and truncated forms.Examples of the expectra obtained are shown in Figure 1. Taking intoaccounttranscriptomic and peptidomic data, this work provides sequenceinformationandevidence of expression of 26 neuropeptide precursor genes in T.infestans. Wecompared the expression of selected neuropeptide precursor genes inpyrethroid sensitive and resistant populations (Figure 3). Theexpressionlevelsof CCHa are almost duplicated and the ones of OKB are triplicated intheresistant population, even though this differences didn?t reachstatistical significance (n=4). The other genes studied did not showrelevant differences of expression between populations. Incomparisonto the 41 neuropeptides found in the exhaustive analysis of R.prolixusgenome,it can be estimated that this set covers almost 75% of theneuropeptideprecursorspresent in T. infestansFigure2 leyenda: MS/MSspectra from T. infestans CNS. (A) (pyro)QDIDHVF(M-OH)RF(amide), amodified form of theS core peptide encoded in Myosuppressinprecursor; (B) a core peptide encoded in RY-amide precursor; (C ) aspacer peptide encoded in the Myo inhibitory peptide precursor.Figura3 leyenda: Relativeexpression of neuropeptide precursor genes in twopopulationsof Triatoma infestans. Black bar: pyrethroid sensitivepopulation;grey bar: pyrethroid resistant population. The values areexpressedas mean +/- s.e.m (n=4). The genes studied were: Adipokinetichormone(AKH); CCH-amide (CCH); ITG-like (ITG); Neuropeptide-likeprecursor1 (NPLP-1); NVP-Like (NVP); F: Orcokinin B (OKB). Despite thatthedifferences found didn't reach statistical significance, theexpressionlevelsof CCHa are almost duplicated and the ones of OKB are triplicated intheresistant population. These results should be cautiously considered,buttheycould help in the formulation of hypothesis for further research.Conclusion:Theresults suggest a high sequence conservation in some neuropeptidesystemsin triatomines, whereas remarkable differences occur in the coremotifs inseveralothers. Incomparisonto the 41 neuropeptides found in the exhaustive analysis of R.prolixusgenome,it can be estimated that this set covers almost 75% of theneuropeptideprecursorspresent in T. infestans. Even though the results should be cautiouslyconsidered, they point to a higher expression or OKB and CCHaneuropeptides in insecticide resistant population. Given that both ofthem are poorly studied neuropeptides, our results could help in theformulation of hypothesis for further research. The informationprovided here contributes to create conditions forwidelyextend functional and genetic studies involving neuropeptides inthese medical relevant species.