Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development before invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.

Here, we show that the major mosquito vector for dengue virus uses the JAK-STAT pathway to control virus infection. Dengue virus infection in Aedes aegypti mosquitoes activates the JAK-STAT immune signaling pathway. The mosquito's susceptibility to dengue virus infection increases when the JAK-STAT pathway is suppressed through RNAi depletion of its receptor Domeless (Dome) and the Janus kinase (Hop), whereas mosquitoes become more resistant to the virus when the negative regulator of the JAK-STAT pathway, PIAS, is silenced. The JAK-STAT pathway exerts its anti-dengue activity presumably through one or several STAT-regulated effectors. We have identified, and partially characterized, two JAK-STAT pathway-regulated and infection-responsive dengue virus restriction factors (DVRFs) that contain putative STAT-binding sites in their promoter regions. Our data suggest that the JAK-STAT pathway is part of the A. aegypti mosquito's anti-dengue defense and may act independently of the Toll pathway and the RNAi-mediated antiviral defenses.

Aedes aegypti is the primary transmitter of the four viruses that have had the greatest impact on human health, the viruses causing yellow fever, dengue fever, chikungunya, and Zika fever. Because this mosquito is easy to rear in the laboratory and these viruses grow in laboratory tissue culture cells, many studies have been performed testing the relative competence of different populations of the mosquito to transmit many different strains of viruses. We review here this large literature including studies on the effect of the mosquito microbiota on competence. Because of the heterogeneity of both mosquito populations and virus strains used, as well as methods measuring potential to transmit, it is very difficult to perform detailed meta-analysis of the studies. However, a few conclusions can be drawn: (1) almost no population of Ae. aegypti is 100% naturally refractory to virus infection. Complete susceptibility to infection has been observed for Zika (ZIKV), dengue (DENV) and chikungunya (CHIKV), but not yellow fever viruses (YFV); (2) the dose of virus used is directly correlated to the rate of infection; (3) Brazilian populations of mosquito are particularly susceptible to DENV-2 infections; (4) the Asian lineage of ZIKV is less infective to Ae. aegypti populations from the American continent than is the African ZIKV lineage; (5) virus adaptation to different species of mosquitoes has been demonstrated with CHIKV; (6) co-infection with more than one virus sometimes causes displacement while in other cases has little effect; (7) the microbiota in the mosquito also has important effects on level of susceptibility to arboviral infection; (8) resistance to virus infection due to the microbiota may be direct (e.g., bacteria producing antiviral proteins) or indirect in activating the mosquito host innate immune system; (9) non-pathogenic insect specific viruses (ISVs) are also common in mosquitoes including genome insertions. These too have been shown to have an impact on the susceptibility of mosquitoes to pathogenic viruses. One clear conclusion is that it would be a great advance in this type of research to implement standardized procedures in order to obtain comparable and reproducible results.

Dengue virus is one of the most important arboviral pathogens and the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. It is transmitted between humans by the mosquitoes Aedes aegypti and Aedes albopictus, and at least 2.5 billion people are at daily risk of infection. During their lifecycle, mosquitoes are exposed to a variety of microbes, some of which are needed for their successful development into adulthood. However, recent studies have suggested that the adult mosquito's midgut microflora is critical in influencing the transmission of human pathogens. In this study we assessed the reciprocal interactions between the mosquito's midgut microbiota and dengue virus infection that are, to a large extent, mediated by the mosquito's innate immune system. We observed a marked decrease in susceptibility to dengue virus infection when mosquitoes harbored certain field-derived bacterial isolates in their midgut. Transcript abundance analysis of selected antimicrobial peptide genes suggested that the mosquito's microbiota elicits a basal immune activity that appears to act against dengue virus infection. Conversely, the elicitation of the mosquito immune response by dengue virus infection itself influences the microbial load of the mosquito midgut. In sum, we show that the mosquito's microbiota influences dengue virus infection of the mosquito, which in turn activates its antibacterial responses. Dengue virus is transmitted by Aedes mosquitoes. During their lifecycle, mosquitoes are exposed to a variety of microbes, and many of them inhabit the mosquito midgut, thereby sharing the same environment with ingested pathogens. The mosquito midgut is the site of multiple reciprocal interactions between the mosquito, its commensal bacteria, and ingested pathogens that will ultimately influence the level of pathogen infection and transmission. In this study the authors addressed the reciprocal interactions between the Aedes immune system, dengue virus and mosquito midgut microbiota using molecular and microbiological assays. The study showed that certain field-derived bacterial isolates of the mosquito midgut exert a detrimental effect on dengue virus infection. This effect is at least partly manifested through the action of the mosquito immune system which is activated by microbes. Conversely, dengue virus infection induces immune responses in the mosquito midgut tissue that act against the natural mosquito midgut microbiota. This study contributes to our understanding of dengue virus infection in Aedes mosquitoes, which may aid towards the development of novel biocontrol strategies to halt dengue transmission.

Dengue fever is endemic in many countries in South and Southeast Asia, Africa and South America, and elsewhere it is an emerging threat. It is caused by one of four dengue viruses — DENV-1, 2, 3 and 4 — transmitted by Aedes sp. mosquitoes; there is no approved vaccine and no effective specific therapy. The dengue viruses are compact flaviviruses likely to require a large number of host factors, and knowledge of those factors could lead to the discovery of potential targets for drugs and new vector control strategies. Now by using a high-throughput RNAi screening approach in DENV-2-infected Drosophila cells — Drosophila is related to the vector species and more amenable to the tools of genomics — more than a hundred candidate dengue virus host factors have been identified, many of them also acting as host factors in human cells. This report identifies host factors required for Dengue virus propagation by using a high throughput genome-wide RNA interference screening approach in Drosophila cells. Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world’s population at risk of infection1. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat1,2. Given their compact genomes, dengue viruses (DENV-1–4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified3,4,5,6,7,8,9,10. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and α-glucosidases)3,4,5,7,9,10, most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

A causative agent of human malaria, Plasmodium falciparum, is transmitted by Anopheles mosquitoes. The malaria parasite is under intensive attack from the mosquito's innate immune system during its sporogonic development. We have used genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes through blood meal-inducible expression of a transgene encoding the IMD pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and microbial infection as a result of timely concerted tissue-specific immune attacks involving multiple effectors. The relatively weak impact of this genetic modification on mosquito fitness under laboratory conditions encourages further investigation of this approach for malaria control. Malaria is caused by the Plasmodium parasites which are transmitted by the Anopheles mosquitoes, and the mosquito's innate immune system plays an important role in blocking the parasite at several sporogonic stages. Our previous studies have shown that one of the major mosquito immune pathways, IMD, is important in the defense against the human malaria parasite in three major malaria vectors, Anopheles gambiae, A. stephensi, and A. albimanus. The transcription factor Rel2 is regulated by the IMD pathway and controls the expression of several potent anti-Plasmodium immune factors. In this study, we have used germ-line transformation technology to generate transgenic Anopheles stephensi mosquitoes with blood meal-inducible expression of Rel2 transcripts in both midgut and fat-body tissues. These transgenic mosquitoes were close to completely resistant to human malaria parasites as a result of the timely concerted tissue-specific expression of anti-Plasmodium effector genes. Under laboratory conditions, the fitness cost of this genetic modification was minimal, suggesting that this system could be utilized for the development of a malaria control strategy.