Project description:Female Aedes aegypti mosquitoes impose a severe global public health burden as primary vectors of multiple viral and parasitic pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges, like intense droughts and intermittent precipitation, which create unpredictable and suboptimal conditions for the egg-laying step of their reproductive cycle. Aedes aegypti mosquitoes nonetheless show remarkable reproductive resilience, but how they achieve this is unknown. Here we show that under drought-like conditions simulated in the laboratory, mated, blood-fed Aedes aegypti females carrying mature eggs retain them in their ovaries for extended periods, while maintaining the viability of these eggs until they can be deposited in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes – here named tweedledee and tweedledum – that show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. Using loss-of-function mutagenesis to disrupt both genes, we show that, tweedledee and tweedledum, which encode secreted proteins, are specifically required for extended retention of viable eggs, such as during intermittent precipitation or drought. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with “insurance” to, when contextually appropriate, flexibly extend their reproductive sequence without losing reproductive capacity, thus allowing this species to exploit diverse and unpredictable habitats.

Project description:The genomes of three major mosquito vectors of human diseases, including Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus, have been previously sequenced. C. p. quinquefasciatus has the largest number of predicted protein-coding genes, which partially results from the expansion of three detoxification gene families: cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST), and carboxylcholinesterases (CCE). However, unlike A. gambiae and A. aegypti, which have large amounts of gene expression data, C. p. quinquefasciatus has limited transcriptomic resources. Knowledge of complete gene expression information is very important for the exploration of the functions of genes involved in specific biological processes. In the present study, the three detoxification gene families of C. p. quinquefasciatus were analyzed for phylogenetic classification and compared with those of three other dipteran insects. Gene expression during various developmental stages and the differential expression responsible for parathion resistance were profiled using the digital gene expression (DGE) technique. Results: A total of 291 detoxification genes were found in C. p. quinquefasciatus, including 70 CCE, 186 P450, and 35 GST genes. Compared with three other dipteran species, gene expansion in Culex mainly occurred in the CCE and P450 families, where the genes of M-NM-1-esterases, juvenile hormone esterases, and CYP325 of the CYP4 subfamily showed the most pronounced expansion on the genome. A total of 13314 genes were expressed in five DGE libraries. Genes with signal transduction and odorant binding functions were prominently expressed during egg development. Genes involved in proteolysis, glycosphingolipid biosynthesis, and purine metabolism were preferentially expressed at the larval stage. Seventy five percent of the detoxification genes were found to be expressed. One fourth of the CCE and P450 genes were expressed at unique stages, indicating their developmentally regulated expression. Fifteen detoxification genes, including 2 CCEs, 6 GSTs, and 7 P450s, were expressed at higher levels in a parathion-resistant strain than in a susceptible strain. Conclusion: The results of the present study provide new insights into the functions and evolution of three detoxification gene families in mosquitoes and comprehensive transcriptomic resources for C. p. quinquefasciatus, which will facilitate the elucidation of molecular mechanisms underlying the different biological characteristics of the three major mosquito vectors. Raw data were deposited in SRA and assigned accession number SRA049959: http://www.ncbi.nlm.nih.gov/sra?term=SRA049959 Five DGE libraries were sequenced: the egg, third instar larval, pupal, and adult stages of the SG strain, and the third instar larval stage of the S-lab strain.

Project description:Female Aedes aegypti mosquitoes impose a severe global public health burden as primary vectors of multiple viral and parasitic pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges, like intense droughts and intermittent precipitation, which create unpredictable and suboptimal conditions for the egg-laying step of their reproductive cycle. Aedes aegypti mosquitoes nonetheless show remarkable reproductive resilience, but how they achieve this is unknown. Here we show that under drought-like conditions simulated in the laboratory, mated, blood-fed Aedes aegypti females carrying mature eggs retain them in their ovaries for extended periods, while maintaining the viability of these eggs until they can be deposited in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes – here named tweedledee and tweedledum – that show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. Using loss-of-function mutagenesis to disrupt both genes, we show that, tweedledee and tweedledum, which encode secreted proteins, are specifically required for extended retention of viable eggs, such as during intermittent precipitation or drought. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with “insurance” to, when contextually appropriate, flexibly extend their reproductive sequence without losing reproductive capacity, thus allowing this species to exploit diverse and unpredictable/chaotic/changing habitats.

Project description:Background: The piRNA pathway has been shown in model organisms to be involved in silencing of transposons thereby providing genome stability. In D. melanogaster the majority of piRNAs map to these sequences. The medically important mosquito species Aedes aegypti has a large genome size, a high transposon load which includes Miniature Inverted repeat Transposable Elements (MITES) and an expansion of the piRNA biogenesis genes. Studies of transgenic lines of Ae. aegypti have indicated that introduced transposons are poorly remobilized and we sought to explore the basis of this. We wished to analyze the piRNA profile of Ae. aegypti and thereby determine if it be responsible for transposon silencing in this mosquito. Results: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism. Conclusions: Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster. 5 small RNA libraries were generated from total RNA of whole adult Aedes aegypti tissues, two of these libraries were sequenced twice (technical replicates). 1 small RNA library was generated from total RNA of a whole adult Drosophila melanogaster tissue.

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De novo assembly of Aedes aegypti using Hi-C yields chromosome-length scaffolds

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