An assay to probe <i>Plasmodium falciparum</i> growth, transmission stage formation and early gametocyte development

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d

Temperature affects viral kinetics and vectorial capacity of Aedes aegypti mosquitoes co-infected with Mayaro and Dengue viruses

Abstract Background Increasing global temperatures and unpredictable climatic extremes have contributed to the spread of vector-borne diseases. The mosquito Aedes aegypti is the main vector of multiple arboviruses that negatively impact human health, mostly in low socioeconomic areas of the world. Co-circulation and co-infection of these viruses in humans have been increasingly reported; however, how vectors contribute to this alarming trend remains unclear. Methods Here, we examine single and co-infection of Mayaro virus (D strain, Alphavirus) and dengue virus (serotype 2, Flavivirus) in Ae. aegypti adults and cell lines at two constant temperatures, moderate (27 °C) and hot (32 °C), to quantify vector competence and the effect of temperature on infection, dissemination and transmission, including on the degree of interaction between the two viruses. Results Both viruses were primarily affected by temperature but there was a partial interaction with co-infection. Dengue virus quickly replicates in adult mosquitoes with a tendency for higher titers in co-infected mosquitoes at both temperatures, and mosquito mortality was more severe at higher temperatures in all conditions. For dengue, and to a lesser extent Mayaro, vector competence and vectorial capacity were higher at hotter temperature in co- vs. single infections and was more evident at earlier time points (7 vs. 14 days post infection) for Mayaro. The temperature-dependent phenotype was confirmed in vitro by faster cellular infection and initial replication at higher temperatures for dengue but not for Mayaro virus. Conclusions Our study suggests that contrasting kinetics of the two viruses could be related to their intrinsic thermal requirements, where alphaviruses thrive better at lower temperatures compared to flaviviruses. However, more studies are necessary to clarify the role of co-infection at different temperature regimes, including under more natural temperature settings. Graphical Abstrac

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential.

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite's extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R0, and find the average R0 is higher (range: 10.1%-12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions

A mating-induced reproductive gene promotes Anopheles tolerance to Plasmodium falciparum infection

International audienceAnopheles mosquitoes have transmitted Plasmodium parasites for millions of years, yet it remains unclear whether they suffer fitness costs to infection. Here we report that the fecundity of virgin and mated females of two important vectors— Anopheles gambiae and Anopheles stephensi —is not affected by infection with Plasmodium falciparum , demonstrating that these human malaria parasites do not inflict this reproductive cost on their natural mosquito hosts. Additionally, parasite development is not impacted by mating status. However, in field studies using different P . falciparum isolates in Anopheles coluzzii , we find that Mating-Induced Stimulator of Oogenesis ( MISO ), a female reproductive gene strongly induced after mating by the sexual transfer of the steroid hormone 20-hydroxyecdysone (20E), protects females from incurring fecundity costs to infection. MISO- silenced females produce fewer eggs as they become increasingly infected with P . falciparum , while parasite development is not impacted by this gene silencing. Interestingly, previous work had shown that sexual transfer of 20E has specifically evolved in Cellia species of the Anopheles genus, driving the co-adaptation of MISO. Our data therefore suggest that evolution of male-female sexual interactions may have promoted Anopheles tolerance to P . falciparum infection in the Cellia subgenus, which comprises the most important malaria vectors

Additional information on <i>P</i>. <i>falciparum</i> isolates.

MSP1-genotyping results for P. falciparum isolates (p1-p6) used in infections with An. gambiae (An. gam), An. coluzzii (An. col), and/or VK5-collected An. coluzzii (VK5-An. col) (See also Methods). (XLSX)</p

Gametocytemia and COI for <i>P</i>. <i>falciparum</i> field isolates.

Gametocytemia and COI for P. falciparum field isolates.</p

Egg development for individual infections.

(A-B) The effects of EcR-silencing on egg development across individual infections for (A) An. coluzzii (unpaired t-test and Mann-Whitney) and (B) An. gambiae (unpaired t-test and Mann-Whitney) compared to controls (Cntrl). N = sample size. p# = parasite isolate. (TIF)</p

<i>P</i>. <i>falciparum</i> oocyst growth is negatively linked to egg development.

(A) In An. coluzzii controls (dsGFP-injected), egg numbers are negatively associated with oocyst size, but this association is lost following dsEcR treatment (GLMM, LRT). (B) In An. gambiae, egg numbers are negatively associated with oocyst size in both control and dsEcR conditions (GLMM, LRT), but (C) this association differentially varies across oocyst density in control and dsEcR females (3-way interaction, treatment*egg#*oocyst#, GLMM, LRT, X21 = 8.57, p = 0.003). Lines across egg numbers and oocyst size graphically represent the model-based analysis that was performed, which used nested individual oocyst measurements. Shading shows 95% confidence interval. N = sample size, or number of mosquitoes. Number of individual oocyst measurements including in analysis were: An. coluzzii controls = 926, An. coluzzii dsEcR = 1003, An. gambiae controls = 669, An. gambiae dsEcR = 760.</p

Additional infection data for colony mosquitoes and VK5-<i>An</i>. <i>coluzzii</i>.

(A) The majority of VK5-An. coluzzii females that were provided a parasite-infected (p1-p6) blood meal, failed to develop any eggs. (B) Oocyst prevalence (P) and intensity for individual infections with VK5-An. coluzzii. (C) Mean oocyst size per VK5-An. coluzzii female for each infection with a different parasite isolate (P#). Mean oocyst sizes are shown for simplicity, but all analyses were done with all individual oocyst measurements nested by mosquito. N = sample size. P# = parasite isolate. (TIF)</p

Full-factor GLMM Output for VK5 <i>An</i>. <i>coluzzii</i>.

Effect of egg development (eggs vs no eggs), oocyst number, and their interactions on oocyst size. In this model, egg development and oocyst number were considered as fixed effects whereas parasite isolate, mosquito generation, and mosquito ID were set as random effects. Significant effects are in bold. (XLSX)</p