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Abstract Background Malaria vector mosquitoes acquire midgut microbiota primarily from their habitat. The homeostasis of these microbial communities plays an essential role in the mosquito longevity, the most essential factor in the mosquito vectorial capacity. Our recent study revealed that silencing genes involved in regulation of the midgut homeostasis including FN3D1, FN3D3 and GPRGr9 reduced the survival of female adult Anopheles arabiensis mosquitoes. In the present study, we investigate the stability of the gene silencing efficiency of mosquitoes reared in three different breeding conditions representing distinct larval habitat types: town brick pits in Jimma, flood pools in the rural land of Asendabo and roadside pools in Wolkite. Methods First-instar larvae of An. arabiensis mosquitoes were reared separately using water collected from the three breeding sites. The resulting adult females were micro-injected with dsRNA targeting the FN3D1 gene (AARA003032) and their survival was monitored. Control mosquitoes were injected with dsRNA Lacz. In addition, the load of midgut microbiota of these mosquitoes was determined using flow cytometry. Results Survival of naïve adult female mosquitoes differed between the three sites. Mosquitoes reared using water collected from brick pits and flood pools survived longer than mosquitoes reared using water collected from roadside. However, the FN3D1 gene silencing effect on survival did not differ between the three sites. Conclusions The present study revealed that the efficacy of FN3D1 gene silencing is not affected by variation in the larval habitat. Thus, silencing this gene has potential for application throughout sub-Saharan Africa.

Abstract Background Vector control remains the most important tool to prevent malaria transmission. However, it is now severely constrained by the appearance of physiological and behavioral insecticide resistance. Therefore, the development of new vector control tools is warranted. Such tools could include immunization of blood hosts of vector mosquitoes with mosquito proteins involved in midgut homeostasis (anti-mosquito vaccines) or genetic engineering of mosquitoes that can drive population-wide knockout of genes producing such proteins to reduce mosquito lifespan and malaria transmission probability. Methods To achieve this, candidate genes related to midgut homeostasis regulation need to be assessed for their effect on mosquito survival. Here, different such candidate genes were silenced through dsRNA injection in the naturally occurring Anopheles arabiensis mosquitoes and the effect on mosquito survival was evaluated. Results Significantly higher mortality rates were observed in the mosquitoes silenced for FN3D1 (AARA003032), FN3D3 (AARA007751) and GPRGr9 (AARA003963) genes as compared to the control group injected with dsRNA against a non-related bacterial gene (LacZ). This observed difference in mortality rate between the candidate genes and the control disappeared when gene-silenced mosquitoes were treated with antibiotic mixtures, suggesting that gut microbiota play a key role in the observed reduction of mosquito survival. Conclusions We demonstrated that interference with the expression of the FN3D1, FN3D3 or GPRGr9 genes causes a significant reduction of the longevity of An. arabiensis mosquito in the wild.

Abstract Background The Anopheles gambiae complex consists of species that vary greatly in their capacity to transmit malaria. The mosquito immune system has been identified as a key factor that can influence whether Plasmodium infection establishes within the mosquito vector. This study was designed to investigate the immune responses of An. coluzzii, An. arabiensis and An. quadriannulatus mosquitoes. The first two mosquito species are major vectors of malaria in sub-Saharan Africa, while the third is thought to be a non-vector. Methods All three mosquito species were reared in mixed cultures. Their capacity to eliminate P. berghei and regulate midgut bacteria was examined. Results Our results revealed large differences in mosquito resistance to P. berghei. In all three mosquito species, immune reactions involving the complement system were triggered when the number of parasites that mosquitoes were challenged with exceeded a certain level, i.e. immune tolerance threshold. This threshold was markedly lower in An. quadriannulatus compared to An. coluzzii and An. arabiensis. We also demonstrated that the level of immune tolerance to P. berghei infection in the haemolymph is inversely correlated with the level of immune tolerance to microbiota observed in the midgut lumen after a blood meal. The malaria non-vector mosquito species, An. quadriannulatus was shown to have a much higher level of tolerance to microbiota in the midgut than An. coluzzii. Conclusions We propose a model whereby an increased tolerance to microbiota in the mosquito midgut results in lower tolerance to Plasmodium infection. In this model, malaria non-vector mosquito species are expected to have increased immune resistance in the haemocoel, possibly due to complement priming by microbiota elicitors. We propose that this strategy is employed by the malaria non-vector mosquito, An. quadriannulatus, while An. coluzzii has reduced tolerance to bacterial infection in the midgut and consequently reduced immune resistance to Plasmodium infection at the haemocoel level. An in-depth understanding of the molecular mechanisms regulating immune tolerance versus resistance in different mosquito vectors of malaria could guide the design of new vector and disease control strategies.

Abstract Background Mosquito infection with malaria parasites depends on complex interactions between the mosquito immune response, the parasite developmental program and the midgut microbiota. Simultaneous monitoring of the parasite and bacterial dynamics is important when studying these interactions. PCR based methods of genomic DNA (gDNA) have been widely used, but their inability to discriminate between live and dead cells compromises their application. The alternative method of quantification of mRNA mainly reports on cell activity rather than density. Method Quantitative real-time (qrt) PCR in combination with Propidium Monoazide (PMA) treatment (PMA-qrtPCR) has been previously used for selectively enumerating viable microbial cells. PMA penetrates damaged cell membranes and intercalates in the DNA inhibiting its PCR amplification. Here, we tested the potential of PMA-qrtPCR to discriminate between and quantify live and dead Plasmodium berghei malarial parasites and commensal bacteria in the midgut of Anopheles coluzzii Coetzee & Wilkerson 2013 (formerly An. gambiae M-form). Results By combining microscopic observations with reverse transcriptase PCR (RT-PCR) we reveal that, in addition to gDNA, mRNA from dead parasites also persists inside the mosquito midgut, therefore its quantification cannot accurately reflect live-only parasites at the time of monitoring. In contrast, pre-treating the samples with PMA selectively inhibited qrtPCR amplification of parasite gDNA, with about 15 cycles (Ct-value) difference between PMA-treated and control samples. The limit of detection corresponds to 10 Plasmodium ookinetes. Finally, we show that the PMA-qrtPCR method can be used to quantify bacteria that are present in the mosquito midgut. Conclusion The PMA-qrtPCR is a suitable method for quantification of viable parasites and bacteria in the midgut of Anopheles mosquitoes. The method will be valuable when studying the molecular interactions between the mosquito, the malaria parasite and midgut microbiota.

Abstract Background Herbal remedies are widely used in many malaria endemic countries to treat patients, in particular in the absence of anti-malarial drugs and in some settings to prevent the disease. Herbal medicines may be specifically designed for prophylaxis and/or for blocking malaria transmission to benefit both, the individual consumer and the community at large. Neem represents a good candidate for this purpose due to its inhibitory effects on the parasite stages that cause the clinical manifestations of malaria and on those responsible for infection in the vector. Furthermore, neem secondary metabolites have been shown to interfere with various physiological processes in insect vectors. This study was undertaken to assess the impact of the standardised neem extract NeemAzal® on the fitness of the malaria vector Anopheles stephensi following repeated exposure to the product through consecutive blood meals on treated mice. Methods Batches of An. stephensi mosquitoes were offered 5 consecutive blood meals on female BALB/c mice treated with NeemAzal® at an azadirachtin A concentration of 60, 105 or 150 mg/kg. The blood feeding capacity was estimated by measuring the haematin content of the rectal fluid excreted by the mosquitoes during feeding. The number of eggs laid was estimated by image analysis and their hatchability assessed by direct observations. Results A dose and frequency dependent impact of NeemAzal® treatment on the mosquito feeding capacity, oviposition and egg hatchability was demonstrated. In the 150 mg/kg treatment group, the mosquito feeding capacity was reduced by 50% already at the second blood meal and by 50 to 80% in all treatment groups at the fifth blood meal. Consequently, a 50 – 65% reduction in the number of eggs laid per female mosquito was observed after the fifth blood meal in all treatment groups. Similarly, after the fifth treated blood meal exposure, hatchability was found to be reduced by 62% and 70% in the 105 and 150 mg/kg group respectively. Conclusions The findings of this study, taken together with the accumulated knowledge on neem open the challenging prospects of designing neem-based formulations as multi-target phytomedicines exhibiting preventive, parasite transmission-blocking as well as anti-vectorial properties.

Abstract Background Targeting the stages of the malaria parasites responsible for transmission from the human host to the mosquito vector is a key pharmacological strategy for malaria control. Research efforts to identify compounds that are active against these stages have significantly increased in recent years. However, at present, only two drugs are available, namely primaquine and artesunate, which reportedly act on late stage gametocytes. Methods In this study, we assessed the antiplasmodial effects of 5 extracts obtained from the neem tree Azadirachta indica and Guiera senegalensis against the early vector stages of Plasmodium falciparum, using field isolates. In an ex vivo assay gametocytaemic blood was supplemented with the plant extracts and offered to Anopheles coluzzii females by membrane feeding. Transmission blocking activity was evaluated by assessing oocyst prevalence and density on the mosquito midguts. Results Initial screening of the 5 plant extracts at 250 ppm revealed transmission blocking activity in two neem preparations. Up to a concentration of 70 ppm the commercial extract NeemAzal® completely blocked transmission and at 60 ppm mosquitoes of 4 out of 5 replicate groups remained uninfected. Mosquitoes fed on the ethyl acetate phase of neem leaves at 250 ppm showed a reduction in oocyst prevalence of 59.0% (CI95 12.0 - 79.0; p