59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
49
Gene duplication in the voltage gated sodium
channel gene of the dengue vector Aedes
aegypti
Brito, LP1; Martins, AJ1,2; Rivas, GB3; Bruno, RV2,3; Valle, D1,3; Peixoto, AA†
Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz – FIOCRUZ / Laboratório de
Entomologia, Instituto de Biologia do Exército, RJ – Brasil; 2Laboratório de Biologia Molecular de Insetos/ Instituto
Oswaldo Cruz – FIOCRUZ, RJ – Brasil; 3Instituto Nacional de Ciência e Tecnologia – Entomologia Molecular – Brazil; † – in
memoriam
1
[email protected]
Keywords: Gene duplication, voltage gated sodium channel, Aedes aegypti.
The Voltage gated sodium channel is a conserved protein responsible for the propagation of nerve impulse by the axons
of neurons. It is composed of four highly conserved homologous domains, each with six transmembrane segments.
Beyond the physiological importance, it is the target molecule of pyrethroid insecticides, widely used to control
pests and vectors in public health, veterinary medicine and agriculture. Some mutations in the sodium channel
gene (NaV) are related to pyrethroid resistance at homologous position in a series of insect species. In Aedes aegypti,
however, these mutations lay in different sites. In addition, here we show a series of evidence that strongly suggest
the occurrence of a NaV duplication in Ae. aegypti natural populations. First of all, although one of the mutant alleles
is highly disseminated, we found no one homozygous individual for this locus (1011 site, in the NaV exon 20) in all
populations examined so far, by allele-specific PCR. Following, we cloned and sequenced the region spanning the NaV
exons 20-21, revealing three different haplotypes in some individuals, raising the hypothesis of duplication. We then
performed crosses between homo (1011 Ile/Ile) and heterozygous (1011 Ile/Met) and genotyped their offspring. Some
crosses resulted in 1:1 Ile/Ile : Ile/Met, whilst others gave 100% Ile/Met, supporting the duplication hypothesis. It
also suggests that the duplication is composed of a wild-type (1011 Ile) and a mutant (1011 Met) copy, what explains
why we never found homozygous for the mutation (1011 Met/Met). It is of note that Ile/Ile and Ile/Met parental
individuals of these crosses belonged, respectively, to the laboratory reference strain and to a lineage selected in the
laboratory for pyrethroid resistance. Finally, a customized copy number variation analysis, based in TaqMan real time
PCR, was performed in order to compare the number of sodium channel genes among the laboratory reference strain
and the supposed ‘duplicated lineage’. This assay revealed that the duplicated linage had five more copies of NaV than
the reference strain. We are now investigating the NaV copy number variation as a polymorphic trait in Ae. aegypti
natural populations, its possible relationship with pyrethroid resistance and the fitness cost.