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.     

Anopheles gambiae genome: perspectives for malaria control

Malaria, a disease that infects 300 million people throughout the world and kills more than a million people, mostly children in sub-Saharan Africa, involves three organisms. The human host where the disease is seen, the protozoan Plasmodium parasite and the mosquito. The parasite is transmitted to humans only by the mosquito vector, which in sub-Saharan regions is generally Anopheles gambiae. Malaria along with AIDS and tuberculosis are killing large numbers of people and crippling the economies of the affected African countries. Though an enormous effort has been made during the past twenty years to develop vaccines to block malaria in humans, the incidence of the disease is increasing in Africa. The reasons for this development include a breakdown in mosquito control related to increased insecticide resistance, as well as increased parasite resistance to antimalarial drugs. It is clear that new methods of Anopheles mosquito control are needed to ameliorate the medical and economic situation in sub-Saharan Africa. As a step toward new malaria control methods, the international Plasmodium falciparum and Anopheles gambiae consortia have carried out the full genome sequencing of the most deadly malaria parasite and the most efficient vector. These, combined with the human genome sequence, provide the genomic infrastructure for a better understanding of the complex interactions within the malaria triad. This essay discusses possible strategies as to how the Anopheles genome can contribute to malaria control.     

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.     

Malaria infection increases attractiveness of humans to mosquitoes

Do malaria parasites enhance the attractiveness of humans to the parasite's vector? As such manipulation would have important implications for the epidemiology of the disease, the question has been debated for many years. To investigate the issue in a semi-natural situation, we assayed the attractiveness of 12 groups of three western Kenyan children to the main African malaria vector, the mosquito Anopheles gambiae. In each group, one child was uninfected, one was naturally infected with the asexual (non-infective) stage of Plasmodium falciparum, and one harboured the parasite's gametocytes (the stage transmissible to mosquitoes). The children harbouring gametocytes attracted about twice as many mosquitoes as the two other classes of children. In a second assay of the same children, when the parasites had been cleared with anti-malarial treatment, the attractiveness was similar between the three classes of children. In particular, the children who had previously harboured gametocytes, but had now cleared the parasite, were not more attractive than other children. This ruled out the possibility of a bias due to differential intrinsic attractiveness of the children to mosquitoes and strongly suggests that gametocytes increase the attractiveness of the children.     

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.     

Change in composition of the Anopheles gambiae complex and its possible implications for the transmission of malaria and lymphatic filariasis in north-eastern Tanzania

ABSTRACT: BACKGROUND: A dramatic decline in the incidence of malaria due to Plasmodium falciparum infection in coastal East Africa has recently been reported to be paralleled (or even preceded) by an equally dramatic decline in malaria vector density, despite absence of organized vector control. As part of investigations into possible causes for the change in vector population density, the present study analysed the Anopheles gambiae s.l. sibling species composition in north-eastern Tanzania. METHODS: The study was in two parts. The first compared current species complex composition in freshly caught An. gambiae s.l. complex from three villages to the composition reported from previous studies carried out 2-4 decades ago in the same villages. The second took advantage of a sample of archived dried An. gambiae s.l. complex specimens collected regularly from a fourth study village since 2005. Both fresh and archived dried specimens were identified to sibling species of the An. gambiae s.l. complex by PCR. The same specimens were moreover examined for Plasmodium falciparum and Wuchereria bancrofti infection by PCR. RESULTS: As in earlier studies, An. gambiae s.s., Anopheles merus and Anopheles arabiensis were identified as sibling species found in the area. However, both study parts indicated a marked change in sibling species composition over time. From being by far the most abundant in the past An. gambiae s.s. was now the most rare, whereas An. arabiensis had changed from being the most rare to the most common. P. falciparum infection was rarely detected in the examined specimens (and only in An. arabiensis) whereas W. bancrofti infection was prevalent and detected in all three sibling species. CONCLUSION: The study indicates that a major shift in An. gambiae s.l. sibling species composition has taken place in the study area in recent years. Combined with the earlier reported decline in overall malaria vector density, the study suggests that this decline has been most marked for An. gambiae s.s., and least for An. arabiensis, leading to current predominance of the latter. Due to differences in biology and vectorial capacity of the An. gambiae s.l. complex the change in sibling species composition will have important implications for the epidemiology and control of malaria and lymphatic filariasis in the study area.     

Viral paratransgenesis in the malaria vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.     

Infochemicals in mosquito host selection: human skin microflora and Plasmodium parasites.

The interaction between the African malaria vector Anopheles gambiae and its human host has traditionally been viewed within a bitrophic context, considering only the human and the mosquito. Recently, the influence of the Plasmodium parasite on the interaction has been recognized, because it affects the physiology and/or behaviour of humans and mosquitoes. However, studies on odour-mediated host-seeking behaviour of An. gambiae and other Diptera have provided evidence that a fourth group of organisms should be taken into consideration. Human skin microflora play a role in the production of odorous compounds that might function as kairomones for mosquitoes. Here, Marieta Braks, Rob Anderson and Bart Knols introduce the role of human microflora into the process of odour-mediated host selection and review the interaction in a multipartite context so as to identify research avenues that will enhance our limited knowledge of this aspect of malaria transmission.     

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.     

Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum

Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species. So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies. Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite-containing blood from patients in Cameroon. We found that P.falciparum ingestion triggers a midgut-associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite. Surprisingly, we found a different pattern of expression of these genes in the An.gambiae-P.berghei model. Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei-An.gambiae model in the P.falciparum-An.gambiae system.     

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.     

Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon

Point mutations in the voltage-gated sodium channel gene involved in knockdown resistance to DDT and pyrethroid insecticides have been described in several insect species. In the malaria vector Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) two mutations have been identified. The first, consisting of a leucine-phenylalanine substitution at amino acid position 1014, is widespread in West Africa. The second, a leucine-serine substitution at the same position, has to date only been detected in western Kenya. Analysis of the kdr polymorphism in a sample of 106 An. gambiae s.s. of the rDNA S-form/Type I collected in Libreville (Gabon) surprisingly revealed the presence of both East and West African kdr mutations with frequencies of 63% and 37%, respectively. No wild-type alleles were detected and there was an excess of heterozygous genotypes (P = 0.04). In addition, an inconsistency was found during the kdr genotyping procedures by polymerase chain reaction, which could have lead to an underestimation of resistance alleles. The implications of these findings are discussed.     

What's buzzing? Mosquito genomics and transgenic mosquitoes

Genome projects and associated technologies are now being established for mosquito species that are vectors of human disease. The recent announcement of an award by the National Institute of Allergy and Infectious Diseases (NIAID) to Celera Genomics to sequence the Anopheles gambiae genome will further accelerate the completion of the sequencing of this genome. Completion of the An. gambiae sequence will mean that the genomes of all three organisms involved in the transmission of falciparum malaria--the mosquito, the parasite, and the human--will have been sequenced. This will greatly facilitate the identification of genes and pathways involved in the transmission of malaria. The recent genetic transformation of An. gambiae with the piggyBac transposable element and the transformation of another important malarial vector, Anopheles stephensi using the Minos element, now provide researchers with powerful tools with which to genetically manipulate these medically important vector species. Here we review the recent progress made in the extension of contemporary tools of modern genetics and genomics into these medically important insects.     

Unexpected anthropophagic behaviour in Anopheles quadriannulatus

The strongly anthropophilic behaviour of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae), the most important malaria vector in Africa, has been demonstrated by field and laboratory studies. Other members of the An. gambiae complex express varied degrees of anthropophily. Anopheles quadriannulatus (Theobald) species A and B are more zoophilic members of the complex and hence are considered to be of no medical importance. Olfactometer experiments with An. quadriannulatus species A have demonstrated attraction to both human and cow odour. To extend these olfactometer observations a choice experiment was conducted in an outdoor cage with a human and a calf as baits, using laboratory-reared mosquitoes. Anopheles gambiae s.s. (from Liberia) and two strains of An. quadriannulatus species A (SKUQUA from South Africa, SANGQUA from Zimbabwe), marked with different coloured fluorescent powders for identification purposes, were released simultaneously and given an equal opportunity to feed on either host. The experiment was repeated six times. Bloodmeals were identified using the precipitin technique. Anopheles gambiae s.s. showed highly anthropophagic behaviour, taking 88% of bloodmeals from the human host. In contrast, both strains of An. quadriannulatus fed with equal frequency on the human or the calf; the response to either host was not significantly different. These results confirm the olfactometer findings and demonstrate anthropophagic behaviour not previously recorded in this species. This finding has implications for prospective manipulation of host preference for genetic control purposes.     

Protocol for RNAi assays in adult mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.     

How does Anopheles gambiae kill malaria parasites?

The malaria parasite's lifecycle in the mosquito vector Anopheles gambiae involves several translocations within and between tissues during which large parasite losses have been documented. Interestingly, during the critical transition stages of midgut invasion and relocation of sporozoites from the oocysts to the salivary glands the mosquito innate immune system is activated. These defense reactions could, at least partially, be responsible for the parasite killing in the mosquito. This important question is now being approached by the dissection of the mosquito innate immune system as well as genetic and genomic studies.