Gene silencing of serine proteases affects melanization of Sephadex beads in Anopheles gambiae

Serine proteases play an important role in activation of prophenoloxidase (proPO), a critical enzyme in the production of melanin. We tested the effect of knockdown of gene expression for five clip domain serine proteases on melanization of abiotic targets in Anopheles gambiae. Knockdown of CLIPB4 resulted in a striking lack of melanization of Sephadex beads while knockdown of CLIPB8 caused a strong shift towards incompletely melanized beads. Knockdown of CLIPB1, B9 and B10 had lesser effects. CLIPB4 and CLIPB8 are strong candidates for activating enzymes in the proPO enzymatic cascade.     

A role for lysozyme in melanization of Sephadex beads in Anopheles gambiae

Melanization of foreign targets in the mosquito, Anopheles gambiae, was studied using a model Sephadex bead system. A mosquito factor that was deposited on beads and prevented bead melanization (MPF) was purified. The N-terminal sequence of the factor identified it as lysozyme c-1 (Lys c-1). Gene silencing of Lys c-1 mediated by RNA interference resulted in a significant reduction in the MPF activity compared with controls. The purified Lys c-1 protein reduced dopachrome formation by mosquito hemolymph phenoloxidase in solution assays in vitro. In vivo, Lys c-1 might inhibit melanization of beads by blocking attachment of critical factors to the bead surface or by inhibiting PO directly. This work indicates that insect lysozymes can play unexpected roles in mediating melanization of foreign targets.     

A genetic module regulates the melanization response of Anopheles to Plasmodium

Two modes of refractoriness to Plasmodium, ookinete lysis and melanization, are known in the malaria vector, Anopheles gambiae. Melanization, a potent insect immune response, is manifested in a genetically selected refractory strain and in susceptible mosquitoes that are depleted of specific C-type lectins (CTLs). Here we use a systematic in vivo RNA interference-mediated reverse genetic screen and other recent results to define a melanization-regulating genetic module or network. It encompasses at least 14 genes, including those that encode five Easter-like clip domain serine proteases and four Masquerade-like serine protease homologues of the mosquito CLIPB and CLIPA subfamilies respectively. We show that several but not all CLIPB genes promote Plasmodium melanization, exhibiting partial functional overlap and synergy. We also report that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanization, while three other CLIPAs are novel synergistic inhibitors of this response. Importantly, the roles of certain CLIPAs and CLIPBs are strain specific, indicating that this network may differ between strains. Finally, we provide evidence that in susceptible mosquitoes melanization induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.     

Anopheles gambiae immune responses to Sephadex beads: involvement of anti-Plasmodium factors in regulating melanization

We have performed a global genome expression analysis of mosquito responses to CM-25 Sephadex beads and identified 27 regulated immune genes, including several anti-Plasmodium factors and other components with likely roles in melanization. Silencing of two bead injection responsive genes, TEP1 and LRIM1, which encode proteins known to mediate Plasmodium killing, significantly compromised the ability to melanize the beads. In contrast, silencing of two Plasmodium protective c-type lectins, CTL4 and CTLMA2, did not affect bead melanization. This data suggest that the anti-Plasmodium factors have dual functions, as determinants of both Plasmodium killing and melanization of the parasite and other foreign bodies, while the Plasmodium protective factors are specifically utilized by the parasite for evasion of mosquito defense mechanisms.     

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.     

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.     

Population dynamics of Plasmodium falciparum sporogony in laboratory-infected Anopheles gambiae.

The population dynamics of cultured Plasmodium falciparum parasites was examined during their sporogonic development in Anopheles gambiae mosquitoes. Estimates of absolute densities were determined for each life stage, and life tables were constructed for each of 38 experimental infections. Macrogametocyte and ookinete mortalities contributed equally to the overall mortality. On average, there was a 40-fold decrease in parasite numbers in the transition from the macrogametocyte to the ookinete stage, a 69-fold decrease in the transition from ookinete to oocyst stages, and a total net decrease in parasite numbers from macrogametocyte to oocyst stage of 2,754-fold (i.e., multiplicative). There was no relationship between macrogametocyte and ookinete densities due to the inherent variability in fertility among different gametocyte cultures. There was a curvilinear relationship (r2 = 0.66) between ookinete and oocyst densities. Above a threshold of about 30 ookinetes/mosquito, the oocyst yield per ookinete became increasingly greater with increasing ookinete density. There was a linear relationship (r2 = 0.73) between oocyst and sporozoite densities, with an average of 663 salivary gland sporozoites produced per oocyst. Sporozoite production per oocyst was not affected by oocyst density and virtually all oocyst infections resulted in sporozoite infections of the salivery glands. This quantitative study indicates that the sporogony of cultured P. falciparum in laboratory-infected A. gambiae is an inefficient process and that the ookinete is the key transitional stage affecting the probability of vector infectivity.     

Plasmodium yoelii: contribution of oocysts melanization to natural refractoriness in Anopheles dirus

It is well known that Anopheles dirus is naturally refractory to rodent malaria parasite, Plasmodium yoelii, but the mechanism is still largely unknown. Here, we found that some P. yoelii taken into An. dirus could develop into oocysts, but oocysts were partially melanized at 7 days and completely melanized at 15 days post-infectious blood meal. Transmission electronic microscopy could find the melanized P. yoelii oocysts in An. dirus as early as 5 days post-infection, with a few haemocytes attaching to the melanized oocysts, indicating a typical humoral melanization reaction. Although the change of protein pattern at 24h post-infection suggested that other unknown mechanisms and/or factors might be involved in killing ookinetes, our data implied that oocysts melanization was one of the mechanisms of An. dirus to block P. yoelii development. In addition, activity of phenoloxidase, such as monophenol oxidase and o-diphenoloxidase, in haemolymph of An. dirus fed on infectious blood meal was much higher than that of mosquitoes fed on 5% glucose or normal mouse blood (p<0.05), implying the possible role of PO in oocysts melanization by An. dirus.     

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.     

The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections

The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity.     

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.     

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.     

Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes.

Innate immune-related gene expression in the major disease vector mosquito Anopheles gambiae has been analyzed following infection by the malaria parasite, Plasmodium berghei. Substantially increased levels of mRNAs encoding the antibacterial peptide defensin and a putative Gram-negative bacteria-binding protein (GNBP) are observed 20-30 h after ingestion of an infected blood-meal, at a time which indicates that this induction is a response to parasite invasion of the midgut epithelium. The induction is dependent upon the ingestion of infective, sexual-stage parasites, and is not due to opportunistic co-penetration of resident gut micro-organisms into the hemocoel. The response is activated following infection both locally (in the midgut) and systemically (in remaining tissues, presumably fat body and/or hemocytes). The observation that Plasmodium can trigger a molecularly defined immune response in the vector constitutes an important advance in our understanding of parasite-vector interactions that are potentially involved in malaria transmission, and extends knowledge of the innate immune system of insects to encompass responses to protozoan parasites.     

Genetic correlation between melanization and antibacterial immune responses in a natural population of the malaria vector Anopheles gambiae

The immune system of invertebrates can mount different responses, including melanotic encapsulation and several antibacterial defense mechanisms. Variation of the efficacies of these responses is generally considered to be a product of the evolutionary pressure on each response due to infection by parasites. However, potential interactions and trade-offs among the different responses of the immune system could constrain the evolutionary potential of each response. In a natural population of the mosquito Anopheles gambiae, we measured the genetic association between the melanization response and an antibacterial response in two environmental qualities (well-fed and undernourished larvae). In both environments the two immune responses were positively genetically correlated: in full-sib families that were most likely to melanize a bead, injected bacteria were most likely to be cleared. Thus, our data do not support the idea of a trade-off among different outcomes of the invertebrate immune system, but rather that some families are overall immunologically superior to others.     

Genetic clonality of Plasmodium falciparum affects the outcome of infection in Anopheles gambiae

Mosquito infections with natural isolates of Plasmodium falciparum are notoriously variable and pose a problem for reliable evaluation of efficiency of transmission-blocking agents for malaria control interventions. Here, we show that monoclonal P. falciparum isolates produce higher parasite loads than mixed ones. Induction of the mosquito immune responses by wounding efficiently decreases Plasmodium numbers in monoclonal infections but fails to do so in infections with two or more parasite genotypes. Our results point to the parasites genetic complexity as a potentially crucial component of mosquito-parasite interactions.     

Close association of invading Plasmodium berghei and beta integrin in the Anopheles gambiae midgut

We have used confocal microscopy and an antibody against Anopheles gambiae beta integrin to study this protein's distribution in the mosquito midgut and its relationship to invading Plasmodium berghei parasites. An extensive reorganization of integrin is seen to take place in the midgut epithelial cells following the uptake of either non-infected or parasite-infected blood meal, probably reflecting the reshaping of the gut due to the presence of the food bolus and the peritrophic membrane that surrounds it. Furthermore, malaria parasites are coated with beta integrin immediately upon entry into the epithelium, independent of whether they develop intra- or extracellularly. Although this coat is shed a few days after the invasion, beta integrin remains concentrated in the cells surrounding the maturing oocyst for several days. Finally, the antibody detects a structural change in the midgut epithelial cells in the immediate vicinity of the invading ookinete, which is consistent with Plasmodium-induced apoptosis followed by wound healing. This intimate association suggests a specific role of beta integrin in the invasion process.