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
Recent studies have suggested that courtship trait expression indicates immune strength. However, most studies have measured only one immune parameter, have not assessed individual differences in immune ability according to time and have not controlled for ecological differences among individuals after an immune challenge. In this work, we tested this hypothesis and controlled for these factors using males of the American rubyspot damselfly which bear a wing red spot whose size is evolutionarily maintained via male-male territorial competition. Our general hypothesis was that territorial, large-spotted males, had a better immune ability compared to nonterritorial, small-spotted males. We expected that the following variables were greater in territorial males compared to nonterritorial males: spot size, phenoloxidase (PO) and hydrolytic enzymatic (HE) activity in males challenged and nonchallenged with a nylon implant, PO and HE activity rate; PO activity after a Serratia marcescens challenge, and survival after a nylon challenge controlling for activity and feeding differences. We found that territorial males showed larger spot areas, greater PO and HE activity (independently of whether they were challenged or not), a higher rate of PO and HE activity (but only expressed at 8h), greater PO production after the bacterial challenge, and a higher survival after the challenge. These results corroborate that males with more pronounced sexual traits have a superior immune function.