Molecular phylogeny and evolution of internal fertilization in South American seasonal cynopoeciline killifishes.

Internal fertilization is a widespread mode of reproduction in chondrichthyans and tetrapods, but uncommon in actinopterygian fishes. In killifishes of the suborder Aplocheiloidei, internal fertilization is restricted to two genera, Campellolebias and Cynopoecilus, both containing species adapted to life in seasonal pools of subtropical South America and exhibiting elaborated inseminating structures. Phylogenetic studies involving these genera are scarce and limited to morphological characters and fragments of mitochondrial DNA sequences available for a few taxa, providing incongruent results and thus impeding hypotheses on the evolution of insemination and related morphological traits. We analyzed three nuclear loci (GLYT1, ENC1, Rho) for 13 aplocheiloid taxa obtaining the first well-supported phylogeny for cynopoecilines, thus providing a significant background to interpret evolutionary changes within the group. Like in killifishes of the suborder Cyprinodontoidei, the evolution of internal fertilization in aplocheiloids is associated with deep changes in the structure of male anal fin. The phylogenetic analyses indicate that internal fertilization corresponds to a single evolutionary event during the evolution of aplocheiloid killifishes. The analyses also indicate that male specialized muscle characters, comprising a muscular ejaculatory pump in the urogenital region and hypertrophied inclinatores and depressores anales, arose in the ancestor of the clade comprising Campellolebias and Cynopoecilus. On the other hand, anal fin specialized structures including the male inseminating tube of Campellolebias and the male inseminating fan of Cynopoecilus evolved independently in each genus.

Assessing the effects of Aedes aegypti kdr mutations on pyrethroid resistance and its fitness cost.

Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference.

Mosquitoes are the culprits of some of the most important vector borne diseases. A species' potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female's physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti. We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila, clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference

Mosquitoes are the culprits of some of the most important vector borne diseases. A species’ potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female’s physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti . We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila , clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti .