Project description:We custom-built a bioinformatics pipeline to search for 20E-modifying enzymes in the accessory glands of Anopheles gambiae males, searching for ecdysteroid kinases (EcK), ecdysone oxidases (EO), and ecdysteroid-phosphate phosphatases (EPP). To this end, we generated RNAseq datasets of different An. gambiae tissues dissected from virgin and mated females and males, and produced similar datasets for Anopheles albimanus, a South American species that does not synthetize and transfer ecdysteroids during mating. These analyses led to the identification of one candidate EPP and two potential EcKs (EcK1 and EcK2), which we demonstrated are involved in the activity of a male-specific oxidized ecdysteroid (3D20E). We further determined that 3D20E is specifically produced by the An. gambiae male accessory glands and is transferred to females during copulation, where it triggers a series of post-mating responses.
Project description:Anopheles mosquitoes are a potential transmitter of viruses and parasites that cause human diseases. An. albimanus is among the main malaria vectors in Mexico and Latin America. Mosquitoes' ability to transmit pathogens (vectorial competence) is directly related to their genetics and immunity. The innate immunity system is the first line of defense against non-self microbial invaders. The mosquito midgut is a crucial entry point for such microbial invaders and for mosquito immune interactions with them. Here, we provide evidence that the lola gene expression is upregulated by immune challenge in three mosquito tissues and cells: the midgut, fat body, and hemocytes. We applied an efficient method for double-stranded RNA (dsRNA)-mediated silencing of this gene expression in vivo as assessed by RT-qPCR. A transcriptome analysis was conducted to investigate the potential involvement of the Lola transcription factor in regulating gene expression. We combined RNAseq with reverse genetics, and our transcriptome analysis revealed that Lola participates in mosquito defense mechanisms. The knockdown of lola showed that it regulates the expression of genes associated with multiple functions, especially those related to detoxification and immunity. Here, we show for the first time a list of mosquito immunity genes that require Lola expression. These lola-dependent genes include antimicrobial peptides, C-lectins, pattern recognition receptors, Clip-domain serine proteases, ML-domain-containing proteins, enzymes that catalyze the generation and detoxification of reactive oxygen species, and components of the JNK immune signaling pathways in the midgut of An. albimanus. Our studies provide direct evidence of a functional link between JNK signaling and Lola factor in the mosquito midgut. Dysregulation of this pathway may contribute to increased pathogen invasion.
Project description:Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and, although P. vivax causes 80-300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquito's luminal midgut brush border. While the genome of the "model" African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, enabling technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published which address this issue. To bolster our understanding of P. vivax-An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus. Summary from: http://www.mcponline.org/content/early/2012/10/17/mcp.M112.019596.long The An. albimanus transcriptome dataset is available at http://funcgen.vectorbase.org/RNAseq/Anopheles_albimanus/INSP/v2
