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Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.

Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening a S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.