RESUMO
Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR-Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.
Assuntos
Biolística , Controle de Mosquitos/métodos , Mosquitos Vetores/genética , Transformação Genética , Aedes/genética , Animais , Anopheles/genéticaRESUMO
Aedes aegypti (Linnaeus) (Diptera: Culicidae) is the main vector of viruses causing dengue, chikungunya, Zika, and yellow fever, worldwide. This report focuses on immuno-blocking four critical proteins in the female mosquito when fed on blood containing antibodies against ferritin, transferrin, one amino acid transporter (NAAT1), and acetylcholinesterase (AchE). Peptides from these proteins were selected, synthetized, conjugated to carrier proteins, and used as antigens to immunize New Zealand rabbits. After rabbits were immunized, a minimum of 20 female mosquitos were fed on each rabbit, per replicate. No effect in their viability was observed after blood-feeding; however, the number of infertile females was 20% higher than the control when fed on AchE-immunized rabbits. The oviposition period was significantly longer in females fed on immunized rabbits than those fed on control (non-immunized) rabbits. Fecundity (eggs/female) of treated mosquitoes was significantly reduced (about 50%) in all four treatments, as compared with the control. Fertility (hatched larvae) was also significantly reduced in all four treatments, as compared with the control, being the effect on AchE and transferrin the highest, by reducing hatching between 70 and 80%. Survival to the adult stage of the hatched larvae showed no significant effect, as more than 95% survival was observed in all treatments, including the control. In conclusion, immuno-blocking of these four proteins caused detrimental effects on the mosquito reproduction, being the effect on AchE the most significant.