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Background Malaria vector control and research rely heavily on monitoring mosquito populations for the development of resistance to public health insecticides. One standard method for determining resistance in adult mosquito populations is the World Health Organization test (WHO bioassay). The WHO bioassay kit consists of several acrylic pieces that are assembled into a unit. Parts of the kit commonly break, reducing the capacity of insectaries to carry out resistance profiling. Since there is at present only a single supplier for the test kits, replacement parts can be hard to procure in a timely fashion. Methods Using computer-aided design software and widely available polylactic acid (PLA) filament as a printing material, we 3D designed and printed replacement parts for the WHO bioassay system. We conducted a comparison experiment between original WHO bioassay kits and 3D printed kits to assess congruence between results. The comparison experiment was performed on two Kenyan laboratory strains of Anopheles gambiae ( s.s. ), Kilifi and Mbita. Studentʼs t-tests were used to assess significant differences between tube types. Finally, we exposed the PLA filament to common solutions used with the bioassay kit. Results We were able to design and print functional replacements for each piece of the WHO bioassay kit. Replacement parts are functionally identical to and interchangeable with original WHO bioassay parts. We note no significant difference in mortality results obtained from PLA printed tubes and WHO acrylic tubes. Additionally, we observed no degradation of PLA in response to prolonged exposure times of commonly used cleaning solutions. Conclusions Our designs can be used to produce replacement parts for the WHO bioassay kit in any facility with a 3D printer, which are becoming increasingly widespread. 3D printing technologies can affordably and rapidly address equipment shortages and be used to develop bespoke equipment in laboratories.

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Elucidating the genetic basis of metabolic resistance to insecticides in malaria vectors is crucial to prolonging the effectiveness of insecticide-based control tools including long lasting insecticidal nets (LLINs). Here, we show that cis -regulatory variants of the cytochrome P450 gene, CYP6P9b , are associated with pyrethroid resistance in the African malaria vector Anopheles funestus . A DNA-based assay is designed to track this resistance that occurs near fixation in southern Africa but not in West/Central Africa. Applying this assay we demonstrate, using semi-field experimental huts, that CYP6P9b-mediated resistance associates with reduced effectiveness of LLINs. Furthermore, we establish that CYP6P9b combines with another P450, CYP6P9a , to additively exacerbate the reduced efficacy of insecticide-treated nets. Double homozygote resistant mosquitoes (RR/RR) significantly survive exposure to insecticide-treated nets and successfully blood feed more than other genotypes. This study provides tools to track and assess the impact of multi-gene driven metabolic resistance to pyrethroids, helping improve resistance management. Bed nets treated with insecticides have been instrumental in reducing malaria mortality, but insecticide resistance is on the rise. Here, Mugenzi et al. identify genetic variants in the P450 gene CYP6P9b of Anopheles funestus that associate with insecticide resistance and develop a PCR-based diagnostic assay to help identify pyrethroid-resistant strains.

Insecticidal fungi represent a promising alternative to chemical pesticides for disease vector control. Here, we show that the pathogenic fungus Beauveria bassiana exports a microRNA-like RNA (bba-milR1) that hijacks the host RNA-interference machinery in mosquito cells by binding to Argonaute 1 (AGO1). bba-milR1 is highly expressed during fungal penetration of the mosquito integument, and suppresses host immunity by silencing expression of the mosquito Toll receptor ligand Spätzle 4 (Spz4). Later, upon entering the hemocoel, bba-milR1 expression is decreased, which avoids induction of the host proteinase CLIPB9 that activates the melanization response. Thus, our results indicate that the pathogen deploys a cross-kingdom small-RNA effector that attenuates host immunity and facilitates infection. Fungi that infect insects can potentially be exploited for disease vector control. Here the authors show that the fungus Beauveria bassiana exports a microRNA-like RNA into mosquito cells that modulates host immunity by suppressing expression of Toll receptor ligand Spätzle 4.

Climate change will have profound impacts on the burden of viruses transmitted by mosquitoes. While we know how changes in temperature impact mosquito physiology and dynamics of viral replication within the mosquito, there is a complete lack of knowledge in how low humidity, or drought tolerance, will impact interactions between mosquitoes and arboviruses. Understanding how drought tolerance will alter mosquito infection with arboviruses is critical in predicting and preventing the impact that climate change will have on mosquito-borne viruses. This work demonstrates a functional link between dehydration tolerance and midgut infection. This knowledge significantly enhances our understanding of how the predicted increase in droughts could impact the dynamics of mosquito-borne viruses.

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

Increasing insecticide resistance in malaria vectors threatens the efficacy of current control tools, however knowledge of metabolic and cuticular mechanisms is widely lacking in Ghana. We examined the metabolic and cuticular resistance mechanisms in Anopheles gambiae mosquitoes from coastal and sahel zones of Ghana. WHO susceptibility tests and synergist assays were performed on F0 field collected An. gambiae s.l. Gene expression profiles of eight key metabolic and cuticular genes were determined using qRT-PCR. Moderate to high pyrethroid resistance (< 70%) were observed across all the sites. Piperonyl butoxide significantly increased susceptibility to pyrethroids across all sites and insecticides, implicating P450s. Gene expression analysis revealed overexpression of metabolic and cuticular resistance genes in field An. gambiae populations compared to the susceptible Kisumu strain. CYP6M2 and CYP6P3 were the most overexpressed metabolic genes in pyrethroid-resistant mosquitoes, compared to the pyrethroid susceptible mosquitoes in the coastal (FC: 122.28 and 231.86, p  < 0.05) and sahel (FC: 344.955 and 716.37, p  < 0.001) zones respectively. CYP4G16 (previously associated with cuticular resistance) was significantly overexpressed in only resistant mosquitoes (FC: 3.32–30.12, p  < 0.05). Overexpression of metabolic and cuticular resistance genes in local malaria vectors highlights the need to intensify insecticide resistance management strategies to control malaria in Ghana.

Aedes aegypti is an important vector of dengue virus and other arboviruses that affect human health. After being ingested in an infectious bloodmeal, but before being transmitted from mosquito to human, dengue virus must disseminate from the vector midgut into the hemocoel and then the salivary glands. This process, the extrinsic incubation period, typically takes 6–14 days. Since older mosquitoes are responsible for transmission, understanding the age structure of vector populations is important. Transcriptional profiling can facilitate predictions of the age structures of mosquito populations, critical for estimating their potential for pathogen transmission. In this study, we utilized a two-gene transcript model to assess the age structure and daily survival rates of three populations (Key West, Marathon, and Key Largo) of Ae . aegypti from the Florida Keys, United States, where repeated outbreaks of autochthonous dengue transmission have recently occurred. We found that Key Largo had the youngest Ae . aegypti population with the lowest daily survival rate, while Key West had the oldest population and highest survival rate. Across sites, 22.67% of Ae . aegypti females were likely old enough to transmit dengue virus (at least 15 days post emergence). Computed estimates of the daily survival rate (0.8364 using loglinear and 0.8660 using non-linear regression), indicate that dengue vectors in the region experienced relatively low daily mortality. Collectively, our data suggest that Ae . aegypti populations across the Florida Keys harbor large numbers of older individuals, which likely contributes to the high risk of dengue transmission in the area.

Hemocytes play several key roles in the mosquito’s immune response. Despite most of our understanding regarding their immunological role concerns their responses against bacteria, fungi, and Plasmodium, our knowledge of hemocyte’s role in antiviral defense is poorly understood. We performed a comprehensive comparative transcriptomic analysis between the dengue vector Aedes aegypti’s two major immune cell types, hemocytes and fat body, revealing a plethora of differentially expressed immune genes that indicates a high level of functional specialization as well as complementation between the two immune cell types. Our transcriptomic approach yielded molecular insights into the antiviral immune response of Ae. aegypti hemocytes during systemic infection. In fact, hemocytes showed abundant expression of RNAi pathway genes under naive conditions and upregulated many of these upon dengue virus (DENV) infection. Furthermore, chemical depletion of phagocytic hemocytes resulted in a higher DENV systemic infection. Our results suggest that hemocytes possess mechanisms to control systemic viral infections.

Zika virus (ZIKV), a flavivirus transmitted primarily by Aedes aegypti, has recently spread globally in an unprecedented fashion, yet we have a poor understanding of host-microbe interactions in this system. To gain insights into the interplay between ZIKV and the mosquito, we sequenced the small RNA profiles in ZIKV-infected and non-infected Ae. aegypti mosquitoes at 2, 7 and 14 days post-infection. ZIKA induced an RNAi response in the mosquito with virus-derived short interfering RNAs and PIWI-interacting RNAs dramatically increased in abundance post-infection. Further, we found 17 host microRNAs (miRNAs) that were modulated by ZIKV infection at all time points. Strikingly, many of these regulated miRNAs have been reported to have their expression altered by dengue and West Nile viruses, while the response was divergent from that induced by the alphavirus Chikungunya virus in mosquitoes. This suggests that conserved miRNA responses occur within mosquitoes in response to flavivirus infection. This study expands our understanding of ZIKV-vector interactions and provides potential avenues to be further investigated to target ZIKV in the mosquito host.