Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae

Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.     

Exceptional diversity, maintenance of polymorphism, and recent directional selection on the APL1 malaria resistance genes of Anopheles gambiae

The three-gene APL1 locus encodes essential components of the mosquito immune defense against malaria parasites. APL1 was originally identified because it lies within a mapped QTL conferring the vector mosquito Anopheles gambiae natural resistance to the human malaria parasite, Plasmodium falciparum, and APL1 genes have subsequently been shown to be involved in defense against several species of Plasmodium. Here, we examine molecular population genetic variation at the APL1 gene cluster in spatially and temporally diverse West African collections of A. gambiae. The locus is extremely polymorphic, showing evidence of adaptive evolutionary maintenance of genetic variation. We hypothesize that this variability aids in defense against genetically diverse pathogens, including Plasmodium. Variation at APL1 is highly structured across geographic and temporal subpopulations. In particular, diversity is exceptionally high during the rainy season, when malaria transmission rates are at their peak. Much less allelic diversity is observed during the dry season when mosquito population sizes and malaria transmission rates are low. APL1 diversity is weakly stratified by the polymorphic 2La chromosomal inversion but is very strongly subdivided between the M and S "molecular forms." We find evidence that a recent selective sweep has occurred at the APL1 locus in M form mosquitoes only. The independently reported observation of a similar M-form restricted sweep at the Tep1 locus, whose product physically interacts with APL1C, suggests that epistatic selection may act on these two loci causing them to sweep coordinately.     

Restriction Fragment Length Polymorphism Mapping of Quantitative Trait Loci for Malaria Parasite Susceptibility in the Mosquito Aedes Aegypti

Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F(2) populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs[2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filarial nematode Brugia malayi and the yellow fever virus.     

Mosquito immunity against Plasmodium

Understanding the molecular mechanisms of the innate immune responses of Anopheles gambiae against Plasmodium parasites is of great importance for current efforts to develop novel strategies for malaria disease control. The parasite undergoes substantial stage-specific losses during its development in the mosquito, which in some cases lead to complete refractoriness of the mosquito against the parasite. The underlying genetics of refractoriness are complex and multifactorial. Completion of the genome sequence of An. gambiae 2 years ago, together with the development of DNA microarrays in this species and the extension of the RNAi technique to adult mosquitoes, has allowed comparative and functional genomic approaches of the mosquito innate immune system. A variety of factors were shown to negatively affect the development of Plasmodium parasites in the mosquito, in some cases leading to complete transmission blockage. In addition, mosquito factors have been identified that play positive roles and are required for successful transmission of the parasite. These findings indicate a highly complex interplay between parasite and vector. Research is continuing to identify new factors involved in this interaction and to decipher the interplay of these molecules and their regulation.     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.     

Transmission Blocking Immunity in the Malaria Non-Vector Mosquito Anopheles quadriannulatus Species A

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to control transmission of the disease.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Targeted identification of markers linked to malaria and filarioid nematode parasite resistance genes in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) have been identified for competence of the mosquito Aedes aegypti to transmit the avian malaria parasite Plasmodium gallinaceum and the human filarial parasite Brugia malayi. Efforts towards the map-based cloning of the associated genes are limited by the availability of genetic markers for fine-scale mapping of the QTL positions. Two F2 mosquito populations were subjected to bulked segregant analysis to identify random amplified polymorphic DNA (RAPD)-PCR fragments linked with the major QTL determining susceptibility to both parasites. Individual mosquitoes for the bulks were selected on the basis of their genotypes at restriction fragment length polymorphism (RFLP) loci tightly linked with the QTL. Pool-positive RAPD fragments were cloned and evaluated as RFLP markers. Of the 62 RAPD/RFLP fragments examined, 10 represented low-copy number sequences. Five of these clones were linked with the major QTL for P. gallinaceum susceptibility (pgs1), of which one clone mapped within the flanking markers that define the QTL interval. The remaining five clones were linked with the major QTL for B. malayi susceptibility (fsb1), and again one clone mapped within the flanking markers that define the QTL interval. In addition, nine RAPD/RFLP fragments were isolated that seem to be of non-mosquito origin.     

The malaria parasite,Plasmodium falciparum,increases the frequency of multiple feeding of its mosquito vector,Anopheles gambiae.

It has often been suggested that vector-borne parasites alter their vector''s feeding behaviour to increase their transmission, but these claims are often based on laboratory studies and lack rigorous testing in a natural situation. We show in this field study that the malaria parasite, Plasmodium falciparum, alters the blood-feeding behaviour of its mosquito vector, Anopheles gambiae s.l., in two ways. First, mosquitoes infected with sporozoited, the parasite stage that is transmitted from the mosquito to a human, took up larger blood meals than uninfected mosquitoes. Whereas 72% of the uninfected mosquitoes had obtained a full blood meal, 82% of the infected ones had engorged fully. Second, mosquitoes harbouring sporozoites were more likely to bite several people per night. Twenty-two per cent of the infected mosquitoes, but only 10% of the uninfected mosquitoes, contained blood from at least two people. We conclude that the observed changes in blood-feeding behaviour allow the parasite to spread more rapidly among human hosts, and thus confirm that the parasite manipulates the mosquito to increase its own transmission.     

PbCap380, a novel oocyst capsule protein, is essential for malaria parasite survival in the mosquito

An essential requisite for transmission of Plasmodium, the causative agent of malaria, is the successful completion of a complex developmental cycle in its mosquito vector. Of hundreds of ookinetes that form in the mosquito midgut, only few transform into oocysts, a loss attributed to the action of the mosquito immune system. However, once oocysts form, they appear to be resistant to mosquito defences. During oocyst development, a thick capsule forms around the parasite and appears to function as a protective cover. Little information is available about the composition of this capsule. Here we report on the identification and partial characterization of the first Plasmodium oocyst capsule protein (PbCap380). Genetic analysis indicates that the gene is essential and that PbCap380(-) mutant parasites form oocysts in normal numbers but are gradually eliminated. As a result, mosquitoes infected with PbCap380(-) parasites do not transmit malaria. Targeting of the oocyst capsule may provide a new strategy for malaria control.     

Resistance of early midgut stages of natural Plasmodium falciparum parasites to high temperatures in experimentally infected Anopheles gambiae (Diptera: Culicidae)

We studied the effects of high temperature, 30 and 32 versus 27 C on early Plasmodium falciparum development in Anopheles gambiae experimentally infected with gametocytes from 30 volunteers with mean density of 264.1 gametocytes/microl blood (range: 16-1,536/microl). From several batches of mosquitoes, fed by membrane feeding, midguts of individual mosquitoes were dissected at 24 hr for ookinete enumeration and at 7 days to quantify oocysts. There were temperature-related differences in mean ookinete intensity per mosquito midgut, with 9.71 +/- 1.6 at 27 C, 9.85 +/- 2.32 at 30 C, and 3.89 +/- 0.81 at 32 C. The prevalence of oocyst infection decreased with an increase in temperatures from 15.9 to 8.5 to 6.4% at 27, 30, and 32 C, respectively. The average oocyst intensities for the infected mosquitoes increased with temperatures from 2.9 at 27 C to 3.5 at 30 C, and to 3.3 at 32 C. However, the success of infections was reduced at 30 and 32 C, and resulted in greater losses during consecutive inter-stage parasite development. The most significant impact of high temperatures occurred at the transition between macrogametocytes and ookinetes, whereas the transition between ookinetes and oocysts apparently was not affected. In contrast to other reports, exposure of mosquitoes infected with natural parasites to high temperatures did not eliminate preoocyst stages, as has been observed from laboratory studies using the NF-54 strain of P. falciparum. This observation of parasite resistance to high temperatures is consistent with the natural situation in tropical environments where perennial malaria transmission occurs during hot dry seasons.     

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R₀, and find the average R₀ is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.     

Anopheles gambiae collagen IV genes: cloning,phylogeny and midgut expression associated with blood feeding and Plasmodium infection

A prerequisite for understanding the role that mosquito midgut extracellular matrix molecules play in malaria parasite development is proper isolation and characterisation of the genes coding for components of the basal lamina. Here we have identified genes coding for alpha1 and alpha2 chains of collagen IV from the major malaria vector, Anopheles gambiae. Conserved sequences in the terminal NC1 domain were used to obtain partial gene sequences of this functional region, and full sequence was isolated from a pupal cDNA library. In a DNA-derived phylogeny, the alpha1 and alpha2 chains cluster with dipteran orthologs, and the alpha2 is ancestral. The expression of collagen alpha1(IV) peaked during the pupal stage of mosquito development, and was expressed continuously in the adult female following a blood meal with a further rise detected in older mosquitoes. Collagen alpha1(IV) is also upregulated when the early oocyst of Plasmodium yoelii was developing within the mosquito midgut and may contribute to a larger wound healing response. A model describing the expression of basal lamina proteins during oocyst development is presented, and we hypothesise that the development of new basal lamina between the oocyst and midgut epithelium is akin to a wound healing process.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

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.     

Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes

Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002) Nature, 417, 452-455). Because of the high selection pressure that an effector gene imposes on the parasite population, development of resistant strains is likely to occur. In search of additional antiparasitic effector genes, we have generated transgenic Anopheles stephensi mosquitoes that express the bee venom phospholipase A2 (PLA2) gene from the gut-specific and blood-inducible Anopheles gambiae carboxypeptidase (AgCP) promoter. Northern blot analysis indicated that the PLA2 mRNA is specifically expressed in the guts of transgenic mosquitoes with peak expression at approximately 4 h after blood ingestion. Western blot and immunofluorescence analyses detected PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to 24 h after a blood meal. Importantly, transgene expression reduced Plasmodium berghei oocyst formation by 87% on average and greatly impaired transmission of the parasite to naive mice. The results indicate that PLA2 may be used as an additional effector gene to block the development of the malaria parasite in mosquitoes.