This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

AbstractThis work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the bloodmeals was achieved through recombinant DNA-taggedAsaiabacteria that successfully colonizedAedesandAnophelesmosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles and neonicotinoids were enriched for compounds with systemic adulticide activity againstAnopheles. Using a luminescentPlasmodium falciparumreporter strain, barcoded screens identified 48 drug-like transmission blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects, and identifies novel candidate small molecules for disease control.

Isoxazolines are oral insecticidal drugs currently licensed for ectoparasite control in companion animals. Here we propose their use in humans for the reduction of vector-borne disease incidence. Fluralaner and afoxolaner rapidly killed Anopheles, Aedes, and Culex mosquitoes and Phlebotomus sand flies after feeding on a drug-supplemented blood meal, with IC50 values ranging from 33 to 575 nM, and were fully active against strains with preexisting resistance to common insecticides. Based on allometric scaling of preclinical pharmacokinetics data, we predict that a single human median dose of 260 mg (IQR, 177-407 mg) for afoxolaner, or 410 mg (IQR, 278-648 mg) for fluralaner, could provide an insecticidal effect lasting 50-90 days against mosquitoes and Phlebotomus sand flies. Computational modeling showed that seasonal mass drug administration of such a single dose to a fraction of a regional population would dramatically reduce clinical cases of Zika and malaria in endemic settings. Isoxazolines therefore represent a promising new component of drug-based vector control.

BACKGROUND:With the increasing interest in vaccines to interrupt malaria transmission, there is a demand for harmonization of current methods to assess Plasmodium transmission in laboratory settings. Potential vaccine candidates are currently tested in the standard membrane feeding assay (SMFA) that commonly relies on Anopheles stephensi mosquitoes. Other mosquito species including Anopheles gambiae are the dominant malaria vectors for Plasmodium falciparum in sub-Saharan Africa.METHODS:Using human serum and monoclonal pre-fertilization (anti-Pfs48/45) and post-fertilization (anti-Pfs25) antibodies known to effectively inhibit sporogony, we directly compared SMFA based estimates of transmission-reducing activity (TRA) for An. stephensi and An. gambiae mosquitoes.RESULTS:In the absence of transmission-reducing antibodies, average numbers of oocysts were similar between An. gambiae and An. stephensi. Antibody-mediated TRA was strongly correlated between both mosquito species, and absolute TRA estimates for pre-fertilisation monoclonal antibodies (mAb) showed no significant difference between the two species. TRA estimates for IgG of naturally exposed individuals and partially effective concentrations of anti-Pfs25 mAb were higher for An. stephensi than for An. gambiae.CONCLUSION:Our findings support the use of An. stephensi in the SMFA for target prioritization. As a vaccine moves through product development, better estimates of TRA and transmission-blocking activity (TBA) may need to be obtained in epidemiologically relevant parasite-species combination.

Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum , although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3–5 strain of A. gambiae . All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites.

Plasmodium falciparum gametocytes, specifically the mature stages, are the only malaria parasite stage in humans transmissible to the mosquito vector. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria and tools allowing the screening of large compound libraries with high predictive power are needed to identify new candidates. As gametocytes are not a replicative stage it is difficult to apply the same drug screening methods used for asexual stages. Here we propose an assay, based on high content imaging, combining "classic" gametocyte viability readout based on gametocyte counts with a functional viability readout, based on gametocyte activation and the discrimination of the typical gamete spherical morphology. This simple and rapid assay has been miniaturized to a 384-well format using acridine orange staining of wild type P. falciparum 3D7A sexual forms, and was validated by screening reference antimalarial drugs and the MMV Malaria Box. The assay demonstrated excellent robustness and ability to identify quality hits with high likelihood of confirmation of transmission reducing activity in subsequent mosquito membrane feeding assays.

Mosquito feeding assays are important in evaluations of malaria transmission-reducing interventions. The proportion of mosquitoes with midgut oocysts is commonly used as an outcome measure, but in natural low intensity infections the effect of oocyst non-rupture on mosquito infectivity is unclear. By identifying ruptured as well as intact oocysts, we show that in low intensity P. falciparum infections i) 66.7–96.7% of infected mosquitoes experienced oocyst rupture between 11–21 days post-infection, ii) oocyst rupture led invariably to sporozoite release, iii) oocyst rupture led to salivary gland infections in 97.8% of mosquitoes and iv) 1250 (IQR 313-2400) salivary gland sporozoites were found per ruptured oocyst. These data show that infectivity can be predicted with reasonable certainty from oocyst prevalence in low intensity infections. High throughput methods for detecting infection in whole mosquitoes showed that 18s PCR but not circumsporozoite ELISA gave a reliable approximation of mosquito infection rates on day 7 post-infection.

Malaria is a human parasitic disease that is transmitted by a mosquito vector. Plasmodium parasites, the causative agents, differ in their infectivity and virulence to the mammalian host, but the mechanistic underpinnings of this variation remain unknown. As mosquitoes provide a nutrient-rich niche for development of transmissible stages, we examined the role of lipids in parasite development and infectivity by disrupting lipid trafficking in mosquito adults. We show that depleting the major mosquito lipoprotein lipophorin deprives parasites of neutral lipids, arrests oocysts growth and impairs sporozoite formation. Importantly, lipid deficiency decreases parasite mitochondrial membrane potential and severely compromises sporozoite infectivity and virulence in the mammalian host. Our findings demonstrate the requirement of mosquito lipids for Plasmodium metabolism, and uncover the mitochondrial contribution to parasite infectivity and virulence. By drawing a connection between vector nutrition and malaria virulence, our results redefine the paradigm of vector-host-pathogen interactions.