Functional genomic analyses of Enterobacter, Anopheles and Plasmodium reciprocal interactions that impact vector competence.

BACKGROUND: Malaria exerts a tremendous socioeconomic impact worldwide despite current control efforts, and novel disease transmission-blocking strategies are urgently needed. The Enterobacter bacterium Esp_Z, which is naturally harboured in the mosquito midgut, can inhibit the development of Plasmodium parasites prior to their invasion of the midgut epithelium through a mechanism that involves oxidative stress. Here, a multifaceted approach is used to study the tripartite interactions between the mosquito, Esp_Z and Plasmodium, towards addressing the feasibility of using sugar-baited exposure of mosquitoes to the Esp_Z bacterium for interruption of malaria transmission. METHODS: The ability of Esp_Z to colonize Anopheles gambiae midguts harbouring microbiota derived from wild mosquitoes was determined by qPCR. Upon introduction of Esp_Z via nectar feeding, the permissiveness of colonized mosquitoes to Plasmodium falciparum infection was determined, as well as the impact of Esp_Z on mosquito fitness parameters, such as longevity, number of eggs laid and number of larvae hatched. The genome of Esp_Z was sequenced, and transcriptome analyses were performed to identify bacterial genes that are important for colonization of the mosquito midgut, as well as for ROS-production. A gene expression analysis of members of the oxidative defence pathway of Plasmodium berghei was also conducted to assess the parasite's oxidative defence response to Esp_Z exposure. RESULTS: Esp_Z persisted for up to 4 days in the An. gambiae midgut after introduction via nectar feeding, and was able to significantly inhibit Plasmodium sporogonic development. Introduction of this bacterium did not adversely affect mosquito fitness. Candidate genes involved in the selection of a better fit Esp_Z to the mosquito midgut environment and in its ability to condition oxidative status of its surroundings were identified, and parasite expression data indicated that Esp_Z is able to induce a partial and temporary shutdown of the ookinetes antioxidant response. CONCLUSIONS: Esp_Z is capable of inhibiting sporogonic development of Plasmodium in the presence of the mosquito's native microbiota without affecting mosquito fitness. Several candidate bacterial genes are likely mediating midgut colonization and ROS production, and inhibition of Plasmodium development appears to involve a shutdown of the parasite's oxidative defence system. A better understanding of the complex reciprocal tripartite interactions can facilitate the development and optimization of an Esp_Z-based malaria control strategy.

A maleness gene in the malaria mosquito Anopheles gambiae.

The molecular pathways controlling gender are highly variable and have been identified in only a few nonmammalian model species. In many insects, maleness is conferred by a Y chromosome-linked M factor of unknown nature. We have isolated and characterized a gene, Yob, for the M factor in the malaria mosquito Anopheles gambiae Yob, activated at the beginning of zygotic transcription and expressed throughout a male's life, controls male-specific splicing of the doublesex gene. Silencing embryonic Yob expression is male-lethal, whereas ectopic embryonic delivery of Yob transcripts yields male-only broods. This female-killing property may be an invaluable tool for creation of conditional male-only transgenic Anopheles strains for malaria control programs.

MicroRNA-regulation of Anopheles gambiae immunity to Plasmodium falciparum infection and midgut microbiota.

Invasion of the malaria vector Anopheles gambiae midgut by Plasmodium parasites triggers transcriptional changes of immune genes that mediate the antiparasitic defense. This response is largely regulated by the Toll and Immune deficiency (IMD) pathways. To determine whether A. gambiae microRNAs (miRNAs) are involved in regulating the anti-Plasmodium defense, we showed that suppression of miRNA biogenesis results in increased resistance to Plasmodium falciparum infection. In silico analysis of A. gambiae immune effector genes identified multiple transcripts with miRNA binding sites. A comparative miRNA microarray abundance analysis of P. falciparum infected and naïve mosquito midgut tissues showed elevated abundance of miRNAs aga-miR-989 and aga-miR-305 in infected midguts. Antagomir inhibition of aga-miR-305 increased resistance to P. falciparum infection and suppressed the midgut microbiota. Conversely, treatment of mosquitoes with an artificial aga-miR-305 mimic increased susceptibility to P. falciparum infection and resulted in expansion of midgut microbiota, suggesting that aga-miR-305 acts as a P. falciparum and gut microbiota agonist by negatively regulating the mosquito immune response. In silico prediction of aga-miR-305 target genes identified several anti-Plasmodium effectors. Our study shows that A. gambiae aga-miR-305 regulates the anti-Plasmodium response and midgut microbiota, likely through post-transcriptional modification of immune effector genes.

The mosquito microbiota influences vector competence for human pathogens.

The midgut of insect vectors of human disease contains not only pathogens harmful to human health, but also a diverse microbiota. This microbiota can influence insects' susceptibility to human pathogens, and the capacity to transmit them, through different mechanisms. Understanding the interaction between the vector, its microbiota and transmitted pathogens will provide novel opportunities to limit disease transmission.