Comparative proteomic analysis of the hemolymph and salivary glands of Rhodnius prolixus and R. colombiensis reveals candidates associated with differential lytic activity against Trypanosoma cruzi Dm28c and T. cruzi Y.

BACKGROUND: Immune response of triatomines plays an important role in the success or failure of transmission of T. cruzi. Studies on parasite-vector interaction have shown the presence of trypanolytic factors and have been observed to be differentially expressed among triatomines, which affects the transmission of some T. cruzi strains or DTUs (Discrete Typing Units). METHODOLOGY/PRINCIPAL FINDINGS: Trypanolytic factors were detected in the hemolymph and saliva of R. prolixus against epimastigotes and trypomastigotes of the Y strain (T. cruzi II). To identify the components of the immune response that could be involved in this lytic activity, a comparative proteomic analysis was carried out, detecting 120 proteins in the hemolymph of R. prolixus and 107 in R. colombiensis. In salivary glands, 1103 proteins were detected in R. prolixus and 853 in R. colombiensis. A higher relative abundance of lysozyme, prolixin, nitrophorins, and serpin as immune response proteins was detected in the hemolymph of R. prolixus. Among the R. prolixus salivary proteins, a higher relative abundance of nitrophorins, lipocalins, and triabins was detected. The higher relative abundance of these immune factors in R. prolixus supports their participation in the lytic activity on Y strain (T. cruzi II), but not on Dm28c (T. cruzi I), which is resistant to lysis by hemolymph and salivary proteins of R. prolixus due to mechanisms of evading oxidative stress caused by immune factors. CONCLUSIONS/SIGNIFICANCE: The lysis resistance observed in the Dm28c strain would be occurring at the DTU I level. T. cruzi I is the DTU with the greatest geographic distribution, from the south of the United States to central Chile and Argentina, a distribution that could be related to resistance to oxidative stress from vectors. Likewise, we can say that lysis against strain Y could occur at the level of DTU II and could be a determinant of the vector inability of these species to transmit T. cruzi II. Future proteomic and transcriptomic studies on vectors and the interactions of the intestinal microbiota with parasites will help to confirm the determinants of successful or failed vector transmission of T. cruzi DTUs in different parts of the Western Hemisphere.

Nuevo polimorfismo en el gen del canal del sodio de la garrapata Rhipicephalus microplus (Ixodida: Ixodidae) resistente a piretroides

Para el control de Rhipicephalus microplus, garrapata común de los bovinos, se usan compuestos piretroides entre otros. El estudio de los mecanismos de resistencia hacia piretroides en esta garrapata ha demostrado la presencia de mutaciones en el gen del canal del sodio, sitio blanco del acaricida. El presente estudio tuvo como objetivo principal detectar el polimorfismo T2134A en el gen del canal del sodio que ha sido relacionado con resistencia a piretroides en individuos portadores. Para ello se recolectaron 182 garrapatas R. microplus en dos fincas con historial de resistencia a este tipo de acaricidas y se sometieron a una prueba de inmersión de hembras en una solución de cipermetrina preparada a la dosis recomendada para uso en campo, obteniendo seis individuos susceptibles, 92 resistentes y 84 medianamente resistentes. Se extrajo el ADN de todos los especímenes utilizando QIAamp DNA Mini Kit® y se procesó por PCR convencional empleando cebadores alelo específicos para amplificar un segmento del dominio IIIS6 del gen del canal del sodio. Los amplicones resultantes se detectaron por electroforesis en geles de agarosa al 2 % teñidos con GelRed® y se visualizaron con luz ultravioleta. De igual manera se efectuó el secuenciamiento de 20 muestras obtenidas en los tres fenotipos susceptible, medianamente resistente y resistente y las secuencias resultantes fueron alineadas mediante el programa Mega 7 y comparadas con secuencias almacenadas en el GenBank®. Los análisis mostraron que la población de R. microplus estudiada no presentaba la mutación esperada sino una mutación diferente en el sitio 2 134. Se observó la sustitución de una timina pero por una citosina, T2134C, que a la traducción genera el cambio de una fenilalanina por una leucina, a diferencia de la mutación previamente identificada, que genera el cambio de una fenilalanina por una isoleucina. Esto demuestra la presencia de un polimorfismo que no ha sido reportado en esta especie de garrapata y que generaría un nuevo marcador molecular de resistencia a piretroides para poblaciones de este ácaro.

For the control of Rhipicephalus microplus, cattle tick, pyrethroid compounds are used among others. The study of the mechanisms of resistance to pyrethroids in this tick has shown the presence of mutations in the sodium channel gen, the target site of acaricide. The main objective of the present study was to detect the T2134A polymorphism in the sodium channel gene that has been related to pyrethroid resistance in carriers. To do so, 182 ticks R. microplus were collected in two farms with a history of resistance to this type of acaricide and were subjected to a dip test of females in a cypermethrin solution prepared at the recommended dose for field use, obtaining six susceptible, 92 resistants and 84 moderately resistants. DNA is extracted from all specimens using QIAamp DNA Mini Kit® and processed by conventional PCR using allele-specific primers to amplify a segment of domain IIIS6 of the sodium channel gene. The resulting amplicons were detected by electrophoresis in 2 % agarose gels stained with GelRed® and visualized with ultraviolet light. In the same way, 20 samples were sequenced of the three susceptible phenotypes, moderately resistant and resistant and the resulting sequences were aligned through the Mega 7 program and compared with the sequences stored in the GenBank®. The information of the population of R. Microplus studied did not show the expected mutation but a different mutation in the site 2134. We also observed the substitution of a thymine but by a cytosine, T2134C, which changes to a phenylalanine by a leucine, a difference from the previously identified mutation, that the change from phenylalanine to isoleucine. This shows the presence of a polymorphism that has not been reported in this species of tick and that would generate a new molecular marker of pyrethroid resistance for populations of this mite.