Transcriptome comparison of dengue-susceptible and -resistant field derived strains of Colombian Aedes aegypti using RNA-sequencing

BACKGROUND Forty percent of the world's population live in areas where they are at risk from dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dengue viruses are transmitted primarily by the mosquito Aedes aegypti. In Cali, Colombia, approximately 30% of field collected Ae. aegypti are naturally refractory to all four dengue serotypes. OBJECTIVES Use RNA-sequencing to identify those genes that determine refractoriness in feral mosquitoes to dengue. This information can be used in gene editing strategies to reduce dengue transmission. METHODS We employed a full factorial design, analyzing differential gene expression across time (24, 36 and 48 h post bloodmeal), feeding treatment (blood or blood + dengue-2) and strain (susceptible or refractory). Sequences were aligned to the reference Ae. aegypti genome for identification, assembled to visualize transcript structure, and analyzed for dynamic gene expression changes. A variety of clustering techniques was used to identify the differentially expressed genes. FINDINGS We identified a subset of genes that likely assist dengue entry and replication in susceptible mosquitoes and contribute to vector competence. MAIN CONCLUSIONS The differential expression of specific genes by refractory and susceptible mosquitoes could determine the phenotype, and may be used to in gene editing strategies to reduce dengue transmission.

Efecto tóxico de los extractos de semillas de Annona muricata potenciados con dimetilsulfóxido sobre larvas IV y pupas de Aedes aegypti / Toxic effect of Annona muricata seed extracts potentiated with dimethyl sulfoxide on IV larvae and pupae of Aedes aegypti

Resumen El objetivo de este estudio fue demostrar que los principios activos de las semillas de Annona muricata combinados con el extracto etanólico y dimetilsulfóxido (EE-DMSO), incrementan la mortalidad de larvas IV y pupas de Aedes aegypti con relación a extractos acuosos (EA) y extractos etanólicos (EE). Las bioactividades se calcularon por comparación de los porcentajes de mortalidad a las 6, 12, 24 y 48 horas in vitro y campo simulado. Los resultados indicaron mortalidad progresiva dependiente de las concentraciones y tiempos de exposición en larvas y reacción knock-down en pupas. In vitro a 5 mg.L-1, EA y EE ejercieron 100% de mortalidad larvaria en 24 horas de exposición (CL50=46.16 y 19.28 mg.L-1 respectivamente), en contraste con EE-DMSO, que inició sobre 62% con 0.5 mg.L-1 a las 6 horas (CL50=20.33 mg.L-1). La acción pupicida de EA y EE reveló 100% de mortalidad desde 24 horas en todas las concentraciones, a diferencia de EE-DMSO que se alcanzó entre 6 y 12 horas. En campo simulado, EA y EE ejercieron 100% de mortalidad a las 24 horas en larvas (16.91 y 21.21 mg.L-1 ), mientras que en pupas (20.44 y 23.03 mg.L-1) ocurrió a las 12 horas, entretanto, la actividad pupicida de EE-DMSO fue 100% en 6 horas. Los efectos comparativos in vitro y campo simulado denotaron patrones similares de respuestas larvicida y pupicida, pero con mayor sensibilidad en pupas. Los principios activos de las semillas de A. muricata combinados con EE-DMSO potencian la respuesta mortal de larvas y pupas de A. aegypti in vitro y campo simulado.

Abstract The objective of this study was to demonstrate that active ingredients of Annona muricata seeds can be enhanced as a result of mixture of both ethanolic extract of A. muricata seeds and Dimethylsulfoxide (EE-DMSO). Percentage mortalities at 6, 12, 24 and 48 hours on fourth instar larvae and pupae of Aedes aegypti were calculated in order to compare bioactivities of aqueous (AE), ethanolic extracts (EE) and EE-DMSO under laboratory and simulated field conditions. Results showed larval mortality concentration- and time-dependent, and knock-down responses in pupae. In laboratory, AE and EE exerted 100% larval mortality at 5 mg.L-1 after 24 hours (LC50= 46.16 and 19.28 mg.L-1). Conversely, EE-DMSO showed between 62 - 100% mortality at 0.5 mg.L-1 for over 6 hours (LC50= 20.33 mg.L-1). Pupicidal effects in AE and EE revealed 100% mortality at 24 hours employing all concentrations, except in EE-DMSO which commenced when individuals were exposed between 6 and 12 hours. In simulated field, AE and EE provoked 100% larval mortality at 24 hours (16.91 y 21.21 mg.L-1) while pupal mortality at 12 hours (20.44 y 23.03 mg.L-1). Percentage mortality of pupae was 100% using EE-DMSO even before 6 hours. Comparative toxic effects of laboratory and simulated-field systems have shown to maintain a similar pattern of larval mortality and more sensitive responses in pupae. Accordingly, larval and pupal mortality responses of A. aegypti were enhanced with the use of EE-DMSO and active ingredients of A. muricata seeds under laboratory and simulated field conditions.