The circadian clock modulates Anopheles gambiae infection with Plasmodium falciparum

Abstract

Key behaviours, physiologies and gene expressions in Anopheles mosquitoes impact the transmission of Plasmodium. Such mosquito factors are rhythmic to closely follow diel rhythms. Here, we set to explore the impact of the mosquito circadian rhythm on the tripartite interaction between the vector, the parasite and the midgut microbiota, and investigate how this may affect the parasite infection outcomes. We assess Plasmodium falciparum infection prevalence and intensity, as a proxy for gametocyte infectivity, in Anopheles gambiae mosquitoes that received a gametocyte-containing bloodfeed and measure the abundance of the midgut microbiota at different times of the mosquito rearing light-dark cycle. Gametocyte infectivity is also compared in mosquitoes reared and maintained under a reversed light-dark regime. The effect of the circadian clock on the infection outcome is also investigated through silencing of the CLOCK gene that is central in the regulation of animal circadian rhythms. The results reveal that the A. gambiae circadian cycle plays a key role in the intensity of infection of P. falciparum gametocytes. We show that parasite gametocytes are more infectious during the night-time, where standard membrane feeding assays (SMFAs) at different time points in the mosquito natural circadian rhythm demonstrate that gametocytes are more infectious when ingested at midnight than midday. When mosquitoes were cultured under a reversed light/dark regime, disrupting their natural physiological homeostasis, and infected with P. falciparum at evening hours, the infection intensity and prevalence were significantly decreased. Similar results were obtained in mosquitoes reared under the standard light/dark regime upon silencing of CLOCK, a key regulator of the circadian rhythm, highlighting the importance of the circadian rhythm for the mosquito vectorial capacity. At that time, the mosquito midgut microbiota load is significantly reduced, while the expression of lysozyme C-1 (LYSC-1) is elevated, which is involved in both the immune response and microbiota digestion. We conclude that the tripartite interactions between the mosquito vector, the malaria parasite and the mosquito gut microbiota are finely tuned to support and maintain malaria transmission. Our data add to the knowledge framework required for designing appropriate and biologically relevant SMFA protocols.

Fig 1. Plasmodium infectivity at day-time vs night time.

(A) Oocyst intensity in mosquitoes received bloodmeal containing gametocyte from same culture flask at day-time (7 ZT) vs night-time (14 ZT); (B) Oocyst infection prevalence in day-time and night-time. The data in A&C were from five replicates while B were from two replicates. Midguts dissection for oocyst examination was carried out on day-7 post infection (pi). (C) number of RBCs (millions) in the blood bolus isolated from freshly fed mosquitoes; Box represents the 25th to 75th percentiles and whiskers represent the mininum and maximum values. (D) Oocyst infection intensity in mosquitoes received gametocyte at different ZT.

Fig 2. Diurnal dynamics of mosquito midgut microbiota and the expression of antibacterial gene lysozyme c-1 (LYSC-1).

Floating bars (A) represent maximum to minimum microbiota count at different timepoints, where the horizontal bar represents the median count, and box plot (B) represent the level of relative expression (ng/mosquito) of LYSC-1 determined using RT-qPCR on tRNA extracted from mosquitoes sampled at different ZT.

Fig 3. Mosquito circadian rhythm on the P. falciparum gametocyte infectivity.

(A) Forest plot showing an estimate of effect ratio (± 95% CI) of oocyst intensities between mosquitoes under a LD regime and DL regimes; Squares and diamond show the sample size for each replicate and the total, respectively. (B) Overall oocyst intensities between mosquitoes under a LD and DL regimes; Red line represents the median. (C) Pie-charts showing oocyst infection prevalence in mosquitoes under LD and DL light-dark regime. The plots represent data from three independent replicates. (D) Oocyt intensity in the midguts of mosquitoes reared and maintanied under differnet light/dark regime: (LD) the standard insectary ligh (12h)/dark (12h), (DL) reversed dark/light regime, (LL) constant light or (DD) constant dark. (E) Effect of CLOCK gene silencing on the oocyst intensity in mosquitoes under normal and reversed light/dark cycle. Red lines represent the median.