RESUMEN
Background and Aim: Organophosphate pesticides (OPs) used in agricultural production pose environmental and public health risks whenever non-target organisms are exposed to them. Oxon-type OPs, such as trichlorfon (TCF) and chlorpyrifos (CPF), are frequently used in Colombia and have been detected in water bodies in the vicinity of croplands; however, their effect on aquatic organisms, especially fish, is largely unknown. The neurotoxicity of OPs includes inhibition of esterase enzymes, neuronal damage, and increased glial reactivity. This study aimed to assess the astrocytic response in the brain tissue of juvenile red-bellied pacu (Piaractus brachypomus) exposed to TCF and CPF. Materials and Methods: A 25-day subchronic assay was conducted in which juvenile red-bellied pacu were exposed to CPF and TCF. After 25 days of exposure, the fish were killed and brain samples were collected and processed for immunohistochemistry to assess the morphology and reactivity of astrocytes; glial acidic fibrillary protein was used as a biomarker. Results: The brain samples from animals under subchronic exposure to OPs for 25 days showed higher cellular density as well as changes in astrocyte phenotype characterized by shortening of cytoplasmic projections, hypertrophy, and ameboid morphology compared to those from nonexposed animals. Similarly, astrocyte hyperreactivity was detected in the optic tectum and medial longitudinal fasciculus of the exposed group. Conclusion: Immunoreactivity of brain glial cells under subchronic exposure to OPs measured through immunohistochemical tests as well as OPs-induced neuropathology may be useful as a biomarker for monitoring environmental pollution. The results also indicate that P. brachypomus is a suitable biomonitoring model for studying neurotoxicological and neurodegenerative diseases.
RESUMEN
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.
