Molecular genetic of the circadian rhythms in mosquitoes vectors (Diptera: Culicidae).
Biological clocks are endogenous time keeping systems that can be found in animals, plants, fungi and even unicellular organisms. In the Drosophila model, the molecularmechanism that controls the circadian rhythm (~24 hours) works through negative feedback loops composed of a number of genes, among which the principal genes are: period, timeless,cycle and Clock. The homologues of these genes have been found in many animal species and their comparison points to a common ancestral origin. Mosquitoes are vectors of severalorganisms and viruses that cause diseases in humans and other animals, however despite their epidemiological relevance not much is known about the molecular mechanisms controlling theiractivity rhythms and bloodfeeding behaviour. The purpose of the present work was to investigate the molecular regulation of the biological clock in mosquitoes, using as models thediurnal species Aedes aegypti and the nocturnal species Culex quinquefasciatus. Analysis of the mosquitoes? activity/rest behaviour was performed in parallel to the molecular studies. The first stage of this research was to obtain, in Aedes aegypti, the full coding sequence of the genehomologue to the timeless gene of Drosophila melanogaster and to perform comparative analysis between them. The results showed high similarities between the timeless gene of thetwo species, mainly in regions known, in Drosophila, to have a crucial involvement in the operation of the biological clock, thus suggesting function conservation of the gene inmosquitoes. The second stage of the research involved the determination of the patterns of expression of the clock genes period, timeless, cycle and Clock in mosquitoes. After preliminarystudies of male Ae. aegypti, in which the temporal pattern of the expression of timeless and cycle was determined, the circadian expression of period, timeless, cycle and Clock was characterised in the heads of females of the species Ae. aegypti and Cx. quinquefasciatus. The results suggestthat the central clock works in a very similar way in both species. The expression of the same four genes in the beheaded bodies of females of Ae. aegypti, however, showed a pattern different from that of the head. The effects of insemination and blood meal intake upon the expression of clock genes in Ae. aegypti were also analysed. In spite of the data in the literature showing that insemination drives important changes in the behaviour of the female mosquito, inthis study only the intake of blood seemed to cause significant changes in the expression of the clock genes. To finalize the study of the biological clock in mosquitoes, silencing of the timeless gene in the head and body of Ae. aegypti mosquitoes was performed using an interference RNAtechnique in which the thoraxes of adult females were injected with timeless double-stranded RNA. Silencing of gene expression was achieved on the fourth day after injection, the same dayin which a significant alteration of the activity pattern of these females was detected. This result confirms the role of timeless in the control of activity behaviour in mosquitoes.
Advisor:Alexandre Afranio Peixoto
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Culicidae Drosophila Circadian Rhythm Biological Clocks
Date of Publication:03/08/2007