BACKGROUND : Malaria is a devastating disease, transmitted by female Anopheles mosquitoes infected with Plasmodium parasites. Current insecticide-based strategies exist to control the spread of malaria by targeting vectors. However, the increase in insecticide resistance in vector populations hinder the efficacy of these methods. It is, therefore, essential to develop novel vector control methods that efficiently target transmission reducing factors such as vector density and competence. A possible vector control candidate gene, the ecdysone receptor, regulates longevity, reproduction, immunity and other physiological processes in several insects, including malaria vectors. Anopheles funestus is a prominent vector in sub-Saharan Africa, however, the function of the ecdysone receptor in this mosquito has not previously been studied. This study aimed to determine if the ecdysone receptor depletion impacts An. funestus longevity, reproduction and susceptibility to Plasmodium falciparum infection.
METHODS : RNA interference was used to reduce ecdysone receptor expression levels in An. funestus females and investigate how the above-mentioned phenotypes are influenced. Additionally, the expression levels of the ecdysone receptor, and reproduction genes lipophorin and vitellogenin receptor as well as the immune gene, leucine rich immune molecule 9 were determined in ecdysone receptor-depleted mosquitoes using quantitative polymerase chain reaction.
RESULTS : Ecdysone receptor-depleted mosquitoes had a shorter lifespan, impaired oogenesis, were less fertile, and had reduced P. falciparum infection intensity.
CONCLUSIONS : Overall, this study provides the first experimental evidence that supports ecdysone receptor as a potential target in the development of vector control measures targeting An. funestus.Additional file 1: Figure S1.
Relative EcR expression levels in dsEcR injected An. funestus females compared to dsGFP injected An. funestus females. The EcR gene was knocked down in dsEcR injected An. funestus females as EcR expression levels were drastically reduced compared to the GFP control. Statistically significant knockdown was evident in dsEcR injected An. funestus females 24, 48 and 72 h after injection as EcR expression in dsEcR injected An. funestus females was 0.11 ± 0.006 (p < 0.05), 0.01 ± 0.001 (p < 0.01) and 0.2 ± 0.06 (p < 0.05) respectively when compared to the GFP injected control of 1. This data confirmed EcR knockdown in An. funestus females injected with dsEcR. Data is representative of 2 biological replicates and normalised using an average of RPS7 and RPL19 reference genes. Expression levels calculated using relative quantification method (∆∆Ct). At each time point statistical significance was assessed with the unpaired student’s t-test. *p < 0.05, **p < 0.01. Error bars represent standard deviation.Additional file 2: Figure S2.
The highest mating success rate was achieved when An. funestus males and females are combined for 12 days after which no further increases are observed. The percentage mating success rate increased progressively until it reached its highest value of 62.2% after 12 days of mating. After this point, the mating success rate reached a plateau until day 20. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc analysis to correct for multiple comparison. Data represents the means of 3 biological replicates. Error bars represent standard deviation of means. ns = not statistically significant p > 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. (n) = number of females per time point across 3 biological replicates.Additional file 3: Figure S3.
Blood feeding rates did not differ between treatment groups. Insignificant differences amongst treatment groups confirmed that the blood feeding rates did not influence any changes observed in the phenotypes of dsEcR treated An. funestus females (p > 0.05). Statistical significance calculated using an unpaired student’s t-test. Error bars represent standard deviation. ns = not statistically significant p > 0.05.Additional file 4: Table S1.
Statistical significance between dsGFP and uninjected controls from the various biological assays conducted.The Department of Science and Innovation (DSI); the National Research Foundation (NRF) South African Research Chairs Initiative; the NRF Communities of Practice and the South African Medical Research Council Strategic Health Innovation Partnerships (SHIP) with funds from DSI.https://malariajournal.biomedcentral.comhj2022BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC
