Innate immunity against malaria parasites in Anopheles gambiae

Malaria continues to exert a huge toll in the world today, causing approximately 400 million cases and killing between 1-2 million people annually. Most of the malaria burden is borne by countries in Africa. For this reason, the major vector for malaria in this continent, Anopheles gambiae, is under intense study. With the completion of the draft sequence of this important vector, efforts are underway to develop novel control strategies. One promising area is to harness the power of the innate immunity of this mosquito species to block the transmission of the malaria parasites. Recent studies have demonstrated that Toll and Imd signaling pathways and other immunity-related genes （encoding proteins possibly function in recognition or as effector molecules） play significant roles in two different arms of innate immunity： level of infection intensity and melanization of Plasmodium oocysts. The challenges in the future are to understand how the functions of these different genes are coordinated in defense against malaria parasites, and if different arms of innate immunity are cross-regulated or coordinated.     

Anopheles gambiae miRNAs as actors of defence reaction against Plasmodium invasion

The path Plasmodium takes across the Anopheles midgut constitutes the major bottleneck during the malaria transmission cycle. In the present study, using a combination of shot-gun cloning and bioinformatic analysis, we have identified 18 miRNAs from Anopheles gambiae including three miRNAs unique to mosquito. Twelve of them are expressed ubiquitously across the body, independently of gender, while the other six exhibited an expression pattern restricted to the digestive system. Strikingly, the expression patterns of four miRNAs, including the three unique to mosquito, are affected by the presence of Plasmodium. We also show that knocking down Dicer1 and Ago1 mRNAs led to an increased sensitivity to Plasmodium infection. Altogether, these data support an involvement of miRNAs as new layers in the regulation of Anopheles defence reaction.     

Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium

The involvement of reactive oxygen species (ROS) in mosquito immunity against bacteria and Plasmodium was investigated in the malaria vector Anopheles gambiae. Strains of An. gambiae with higher systemic levels of ROS survive a bacterial challenge better, whereas reduction of ROS by dietary administration of antioxidants significantly decreases survival, indicating that ROS are required to mount effective antibacterial responses. Expression of several ROS detoxification enzymes increases in the midgut and fat body after a blood meal. Furthermore, expression of several of these enzymes increases to even higher levels when mosquitoes are fed a Plasmodium berghei-infected meal, indicating that the oxidative stress after a blood meal is exacerbated by Plasmodium infection. Paradoxically, a complete lack of induction of catalase mRNA and lower catalase activity were observed in P. berghei-infected midguts. This suppression of midgut catalase expression is a specific response to ookinete midgut invasion and is expected to lead to higher local levels of hydrogen peroxide. Further reduction of catalase expression by double-stranded RNA-mediated gene silencing promoted parasite clearance by a lytic mechanism and reduced infection significantly. High mosquito mortality is often observed after P. berghei infection. Death appears to result in part from excess production of ROS, as mortality can be decreased by oral administration of uric acid, a strong antioxidant. We conclude that ROS modulate An. gambiae immunity and that the mosquito response to P. berghei involves a local reduction of detoxification of hydrogen peroxide in the midgut that contributes to limit Plasmodium infection through a lytic mechanism.     

Repellency of essential oils of some Kenyan plants against Anopheles gambiae

Essential oils of six plants growing in Kenya were screened for repellent activities against Anopheles gambiae sensu stricto. The oils of Conyza newii (Compositeae) and Plectranthus marrubioides (Labiateae) were the most repellent (RD50=8.9 x 10(-5) mg cm(-2), 95% CI) followed by Lippia javanica (Verbenaceae), Lippia ukambensis (Verbenaceae), Tetradenia riparia, (Iboza multiflora) (Labiateae) and Tarchonanthus camphoratus (Compositeae). Eight constituents of the different oils (perillyl alcohol, cis-verbenol, cis-carveol, geraniol, citronellal, perillaldehyde, caryophyllene oxide and a sesquiterpene alcohol) exhibited relatively high repellency. Four synthetic blends of the major components (present in > or = 1.5%) of the essential oils were found to exhibit comparable repellent activity to the parent oils.     

Essential oils enhance the toxicity of permethrin against Aedes aegypti and Anopheles gambiae

Insecticide resistance and growing public concern over the safety and environmental impacts of some conventional insecticides have resulted in the need to discover alternative control tools. Naturally occurring botanically‐based compounds are of increased interest to aid in the management of mosquitoes. Susceptible strains of Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Anopheles gambiae (Meigen) (Diptera: Culicidae) were treated with permethrin, a common type‐I synthetic pyrethroid, using a discriminate dose that resulted in less than 50% mortality. Piperonyl butoxide (PBO) and 35 essential oils were co‐delivered with permethrin at two doses (2 and 10 µg) to determine if they could enhance the 1‐h knockdown and the 24‐h mortality of permethrin. Several of the tested essential oils enhanced the efficacy of permethrin equally and more effectively than piperonyl butoxide PBO, which is the commercial standard to synergize chemical insecticide like pyrethroids. PBO had a strikingly negative effect on the 1‐h knockdown of permethrin against Ae. aegypti, which was not observed in An. gambiae. Botanical essential oils have the capability of increasing the efficacy of permethrin allowing for a natural alternative to classic chemical synergists, like PBO.     

Confirmation that Plasmodium falciparum has aperiodic infectivity to Anopheles gambiae

Abstract. In preparation for field studies of transmission-blocking malaria vaccines, a study was carried out to determine whether P. falciparum infections obtained in An. gambiae blood-fed at 16.00 hours were quantitatively similar to infections obtained at 23.00 hours. Using a group of children aged 5-12 years from villages at Ahero, near Kisumu in Kenya, 71/74 (96%) of whom were found to be positive for P. falciparum parasitaemia, one batch of fifty colony-bred An. gambiae females were fed on volunteers at 16.00 hours and another batch at 23.00 hours. No statistically significant differences were found in the proportions of mosquitoes becoming infected, the numbers of children infecting mosquitoes or the mean numbers of malaria oocysts developing in mosquitoes blood-fed at the different times. Because mosquito infections obtained by day (16.00 hours) are equivalent in quantity to those obtained at night (23.00 hours), experimental infections can be carried out in the afternoon, when it is most convenient, rather than during the night.     

Unexpected high losses of Anopheles gambiae larvae due to rainfall

Background

Immature stages of the malaria mosquito Anopheles gambiae experience high mortality, but its cause is poorly understood. Here we study the impact of rainfall, one of the abiotic factors to which the immatures are frequently exposed, on their mortality.

Methodology/Principal Findings

We show that rainfall significantly affected larval mosquitoes by flushing them out of their aquatic habitat and killing them. Outdoor experiments under natural conditions in Kenya revealed that the additional nightly loss of larvae caused by rainfall was on average 17.5% for the youngest (L1) larvae and 4.8% for the oldest (L4) larvae; an additional 10.5% (increase from 0.9 to 11.4%) of the L1 larvae and 3.3% (from 0.1 to 3.4%) of the L4 larvae were flushed away and larval mortality increased by 6.9% (from 4.6 to 11.5%) and 1.5% (from 4.1 to 5.6%) for L1 and L4 larvae, respectively, compared to nights without rain. On rainy nights, 1.3% and 0.7% of L1 and L4 larvae, respectively, were lost due to ejection from the breeding site.

Conclusions/Significance

This study demonstrates that immature populations of malaria mosquitoes suffer high losses during rainfall events. As these populations are likely to experience several rain showers during their lifespan, rainfall will have a profound effect on the productivity of mosquito breeding sites and, as a result, on the transmission of malaria. These findings are discussed in the light of malaria risk and changing rainfall patterns in response to climate change.     

New repellent effective against African malaria mosquito Anopheles gambiae: implications for vector control

Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is a vector for Plasmodium, the causative agent of malaria. Current control strategies to reduce the impact of malaria focus on reducing the frequency of mosquito attacks on humans, thereby decreasing Plasmodium transmission. A need for new repellents effective against Anopheles mosquitoes has arisen because of changes in vector behaviour as a result of control strategies and concern over the health impacts of current repellents. The response of A. gambiae to potential repellents was investigated through an electroantennogram screen and the most promising of these candidates (1‐allyloxy‐4‐propoxybenzene, 3c {3,6}) chosen for behavioural testing. An assay to evaluate the blood‐host seeking behaviour of A. gambiae towards a simulated host protected with this repellent was then performed. The compound 3c {3,6} was shown to be an effective repellent, causing mosquitoes to reduce their contact with a simulated blood‐host and probe less at the host odour. Thus, 3c {3,6} may be an effective repellent for the control of A. gambiae.     

Distribution of African malaria mosquitoes belonging to the Anopheles gambiae complex

The distribution of malaria vector mosquitoes, especially those belonging to species complexes that contain non-vector species, is important for strategic planning of malaria control programmes. Geographical information systems have allowed researchers to visualize distribution data on maps together with environmental parameters, such as rainfall and temperature. Here, Maureen Coetzee, Marlies Craig and David le Sueur review our current knowledge on the distribution of the members of the Anopheles gambiae complex.     

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.     

Energy metabolism affects susceptibility of Anopheles gambiae mosquitoes to Plasmodium infection

Previous studies showed that Anopheles gambiae L3-5 females, which are refractory (R) to Plasmodium infection, express higher levels of genes involved in redox-metabolism and mitochondrial respiration than susceptible (S) G3 females. Our studies revealed that R females have reduced longevity, faster utilization of lipid reserves, impaired mitochondrial state-3 respiration, increased rate of mitochondrial electron leak and higher expression levels of several glycolytic enzyme genes. Furthermore, when state-3 respiration was reduced in S females by silencing expression of the adenine nucleotide translocator (ANT), hydrogen peroxide generation was higher and the mRNA levels of lactate dehydrogenase increased in the midgut, while the prevalence and intensity of Plasmodium berghei infection were significantly reduced. We conclude that there are broad metabolic differences between R and S An. gambiae mosquitoes that influence their susceptibility to Plasmodium infection.     

Synergy in Efficacy of Fungal Entomopathogens and Permethrin against West African Insecticide-Resistant Anopheles gambiae Mosquitoes

Background

Increasing incidences of insecticide resistance in malaria vectors are threatening the sustainable use of contemporary chemical vector control measures. Fungal entomopathogens provide a possible additional tool for the control of insecticide-resistant malaria mosquitoes. This study investigated the compatibility of the pyrethroid insecticide permethrin and two mosquito-pathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, against a laboratory colony and field population of West African insecticide-resistant Anopheles gambiae s.s. mosquitoes.

Methodology/Findings

A range of fungus-insecticide combinations was used to test effects of timing and sequence of exposure. Both the laboratory-reared and field-collected mosquitoes were highly resistant to permethrin but susceptible to B. bassiana and M. anisopliae infection, inducing 100% mortality within nine days. Combinations of insecticide and fungus showed synergistic effects on mosquito survival. Fungal infection increased permethrin-induced mortality rates in wild An. gambiae s.s. mosquitoes and reciprocally, exposure to permethrin increased subsequent fungal-induced mortality rates in both colonies. Simultaneous co-exposure induced the highest mortality; up to 70.3±2% for a combined Beauveria and permethrin exposure within a time range of one gonotrophic cycle (4 days).

Conclusions/Significance

Combining fungi and permethrin induced a higher impact on mosquito survival than the use of these control agents alone. The observed synergism in efficacy shows the potential for integrated fungus-insecticide control measures to dramatically reduce malaria transmission and enable control at more moderate levels of coverage even in areas where insecticide resistance has rendered pyrethroids essentially ineffective.