Direct and indirect immunosuppression by a malaria parasite in its mosquito vector

Malaria parasites develop as oocysts within the haemocoel of their mosquito vector during a period that is longer than the average lifespan of many of their vectors. How can they escape from the mosquito''s immune responses during their long development? Whereas older oocysts might camouflage themselves by incorporating mosquito-derived proteins into their surface capsule, younger stages are susceptible to the mosquito''s immune response and must rely on other methods of immune evasion. We show that the malaria parasite Plasmodium gallinaceum suppresses the encapsulation immune response of its mosquito vector, Aedes aegypti, and in particular that the parasite uses both an indirect and a direct strategy for immunosuppression. Thus, when we fed mosquitoes with the plasma of infected chickens, the efficacy of the mosquitoes to encapsulate negatively charged Sephadex beads was considerably reduced, whether the parasite was present in the blood meal or not. In addition, zygotes that were created ex vivo and added to the blood of uninfected chickens reduced the efficacy of the encapsulation response. As dead zygotes had no effect on encapsulation, this result demonstrates active suppression of the mosquito''s immune response by malaria parasites.     

The complex interplay between mosquito positive and negative regulators of Plasmodium development

The malaria parasite, Plasmodium, requires sexual development in the mosquito before it can be transmitted to the vertebrate host. Mosquito genes are able to substantially modulate this process, which can result in major decreases in parasite numbers. Even in susceptible mosquitoes, haemolymph proteins implicated in systemic immune reactions, together with local epithelial responses, cause lysis of more than 80% of the ookinetes that cross the mosquito midgut. In a refractory mosquito strain, immune responses lead to melanisation of virtually all parasites. Conversely, certain mosquito genes have an opposite effect: they are used by the parasite to evade defence reactions. Detailed understanding of the interplay between positive and negative regulators of parasite development could lead to the generation of novel approaches for malaria control through the vector.     

Immune stimulation and malaria infection impose reproductive costs in Anopheles gambiae via follicular apoptosis

The employment of defense mechanisms is recognized as a costly life-history trait. In the malaria vector Anopheles gambiae, reproductive costs have been associated with both humoral and cellular innate immune responses and also with malaria infection. The resorption of developing oocytes associated with malaria infection is preceded by the programmed cell death, or apoptosis, of follicular cells. Here we demonstrate that apoptosis in ovarian follicular epithelial cells also occurs when mosquitoes are subjected to artificial immune-elicitors that induce a melanization response or humoral antimicrobial activity. Caspases are key cysteine proteases involved in apoptosis. Caspase-like activity was detected in epithelial cells in approximately 4.0% of the developing ovarian follicles of untreated, blood-fed, mosquitoes. Lipopolysaccharide injection resulted in a significant increase in anti-Micrococcus luteus humoral activity and a significant increase of 257.7% of follicles exhibiting apoptosis compared to results after saline injections. Melanization also triggered follicular apoptosis, which increased by 106.25% or 134.37% in Sephadex C-25 or G-25 bead-inoculated mosquitoes, respectively, compared to that in sham-injected ones. Ovaries from Plasmodium yoelii nigeriensis-infected mosquitoes exhibited a significant increase in follicular apoptosis of 440.9% compared to non-infected ones. Thus, at the time point investigated, infection had a much greater effect than artificial immune-elicitors. Death of follicular epithelial cells has been shown to lead to follicle resorption and hence a decrease in egg production. We propose the trade-off between reproductive fitness and immune defense in A. gambiae operates via the induction of apoptosis in ovarian follicles and that different immune responses impose costs via the same pathway.     

The multifaceted mosquito anti-Plasmodium response

Plasmodium development within its mosquito vector is an essential step in malaria transmission, as illustrated in world regions where malaria was successfully eradicated via vector control. The innate immune system of most mosquitoes is able to completely clear a Plasmodium infection, preventing parasite transmission to humans. Understanding the biological basis of this phenomenon is expected to inspire new strategies to curb malaria incidence in countries where vector control via insecticides is unpractical, or inefficient because insecticide resistance genes have spread across mosquito populations. Several aspects of mosquito biology that condition the success of the parasite in colonizing its vector begin to be understood at the molecular level, and a wealth of recently published data highlights the multifaceted nature of the mosquito response against parasite invasion. In this brief review, we attempt to provide an integrated view of the challenges faced by the parasite to successfully invade its mosquito host, and discuss the possible intervention strategies that could exploit this knowledge for the fight against human malaria.     

Immune responses in Anopheles gambiae

Transmission of human malaria requires a successful development of Plasmodium parasites in anopheline mosquitoes. Insects have developed efficient immune responses to oppose microbial and eukaryotic invaders. The completion of the sequencing of the Anopheles genome provides a wealth of information on putative immune genes that are homologous to components of the Drosophila and mammalian immune systems. In this review, we will summarize our present knowledge of immune responses in the mosquito Anopheles gambiae and attempt a comparative analysis of insect immune systems.     

Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.     

Epidemiological consequences of a newly discovered cryptic subgroup of Anopheles gambiae

A cryptic subgroup of Anopheles gambiae sensu stricto mosquitoes was recently discovered in West Africa. This 'GOUNDRY' subgroup has increased susceptibility to Plasmodium falciparum, the most deadly form of malaria. Unusual for this major malaria vector, GOUNDRY mosquitoes also seem to bite exclusively outdoors. A mathematical model is developed to assess the epidemiological implications of current vector control tools, bednets and indoor residual spray, preferentially suppressing the more typical indoor biting mosquitoes. It is demonstrated that even if the GOUNDRY mosquitoes have a decreased preference for human blood, vector controls which select for increased GOUNDRY abundance relative to their indoor biting counterparts risks intensifying malaria transmission. Given the widely observed phenomenon of outdoor biting by major malaria vectors, this behaviour should not be ignored in future modelling efforts and warrants serious consideration in control programme strategy.     

Harnessing immune responses against Plasmodium for rational vaccine design

In recent years, groundbreaking advances have been made in understanding the biology of and immune mechanisms against the Plasmodium spp. parasite, the causative agent of malaria. Novel features of the Plasmodium life cycle have been unravelled and immune mechanisms, which take place during both infection and immunization, have been dissected. We have undoubtedly enhanced our knowledge, but the question now is how to use this information to manipulate immune responses against Plasmodium and to develop an efficacious malaria vaccine. In this review, we discuss the latest developments in the field and speculate on how immune responses against Plasmodium could be harnessed for rational vaccine design and application.     

Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest

Malaria parasites use vertebrate hosts for asexual multiplication and Culicidae mosquitoes for sexual and asexual development, yet the literature on avian malaria remains biased towards examining the asexual stages of the life cycle in birds. To fully understand parasite evolution and mechanism of malaria transmission, knowledge of all three components of the vector-host-parasite system is essential. Little is known about avian parasite-vector associations in African rainforests where numerous species of birds are infected with avian haemosporidians of the genera Plasmodium and Haemoproteus. Here we applied high resolution melt qPCR-based techniques and nested PCR to examine the occurrence and diversity of mitochondrial cytochrome b gene sequences of haemosporidian parasites in wild-caught mosquitoes sampled across 12 sites in Cameroon. In all, 3134 mosquitoes representing 27 species were screened. Mosquitoes belonging to four genera (Aedes, Coquillettidia, Culex and Mansonia) were infected with twenty-two parasite lineages (18 Plasmodium spp. and 4 Haemoproteus spp.). Presence of Plasmodium sporozoites in salivary glands of Coquillettidia aurites further established these mosquitoes as likely vectors. Occurrence of parasite lineages differed significantly among genera, as well as their probability of being infected with malaria across species and sites. Approximately one-third of these lineages were previously detected in other avian host species from the region, indicating that vertebrate host sharing is a common feature and that avian Plasmodium spp. vector breadth does not always accompany vertebrate-host breadth. This study suggests extensive invertebrate host shifts in mosquito-parasite interactions and that avian Plasmodium species are most likely not tightly coevolved with vector species.     

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.     

Plasmodium berghei: inhibition of the sporogonous cycle by alpha-difluoromethylornithine

alpha-Difluoromethylornithine (DFMO), an enzyme inhibitor of ornithine decarboxylase, inhibits the sporogonous cycle of the malaria parasite Plasmodium berghei in the mosquito vector Anopheles stephensi. DFMO was administered to the mosquitoes dissolved either in the sugar solution at their disposal in the cages or through blood meals taken from treated mice. The mice subsequently bitten by mosquitoes treated with DFMO by both routes of administration did not contract malaria.     

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.     

Midgut specific immune response of vector mosquito Anopheles stephensi to malaria parasite Plasmodium

Innate immune-related polypeptides expression in midgut in the ageing vector mosquito A. stephensi following infection by malaria parasite, Plasmodium yoelii yoelii has been studied. Twenty polypeptides were induced by an infected blood meal during various stages of adult life. A 24 kDa polypeptide was induced generally in most of the stages. Maximum parasite induced polypeptides i.e. 22, 33, 111, 122, 127, 140, 143 and 146 kDa were found in 5 days of post blood feeding (PBF) which coincides with the presence of oocysts on the midgut. However, in addition, three polypeptides in 11 days PBF and 8 polypeptides in 20 days PBF were also induced due to parasite infection in aged mosquitoes. Quantitatively, the amount of soluble proteins in the midgut in oocyst-sporozoite-positive mosquitoes was always less as compared to their normal counterparts. The parasite evidently elicits defined immune responses by inducing specific polypeptides in the midgut of the mosquito.     

Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae

Endosymbiotic Wolbachia bacteria are potent modulators of pathogen infection and transmission in multiple naturally and artificially infected insect species, including important vectors of human pathogens. Anopheles mosquitoes are naturally uninfected with Wolbachia, and stable artificial infections have not yet succeeded in this genus. Recent techniques have enabled establishment of somatic Wolbachia infections in Anopheles. Here, we characterize somatic infections of two diverse Wolbachia strains (wMelPop and wAlbB) in Anopheles gambiae, the major vector of human malaria. After infection, wMelPop disseminates widely in the mosquito, infecting the fat body, head, sensory organs and other tissues but is notably absent from the midgut and ovaries. Wolbachia initially induces the mosquito immune system, coincident with initial clearing of the infection, but then suppresses expression of immune genes, coincident with Wolbachia replication in the mosquito. Both wMelPop and wAlbB significantly inhibit Plasmodium falciparum oocyst levels in the mosquito midgut. Although not virulent in non-bloodfed mosquitoes, wMelPop exhibits a novel phenotype and is extremely virulent for approximately 12-24 hours post-bloodmeal, after which surviving mosquitoes exhibit similar mortality trajectories to control mosquitoes. The data suggest that if stable transinfections act in a similar manner to somatic infections, Wolbachia could potentially be used as part of a strategy to control the Anopheles mosquitoes that transmit malaria.     

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.     

Genetic engineering of malaria parasite resistance in vector mosquitoes

Malaria is the most significant vector‐borne disease and mostly affects people living in the lesser developed countries of tropical and sub‐tropical regions. Climate changes, rapid global transportation, immigration and invasion of exotic mosquito vectors bring the threat of introduction of the disease to developed nations. Sustainability of malaria control requires the discovery of therapeutic and prophylactic drugs, development of effective vaccines and control of vector mosquitoes. Drug development and vaccine research have been pursued aggressively over the past 20 years, and progress in novel approaches to vector control is now evident. Our long‐term objective is the production and utilization of strains of vector mosquitoes that are genetically refractory to the transmission of malaria parasites. These insects will be used to test the hypothesis that an increase in the frequency of a gene or allele that confers decreased vector competence to a population of mosquitoes will result in a reduction in the incidence and prevalence of malaria. Completed studies make it possible to develop strains of Anopheles mosquitoes expressing specific effector molecules that interfere completely with the transmission of the most lethal human malaria parasite, Plasmodium falciparum. Data are reviewed here that support the use of single‐chain monoclonal antibodies (scFv) that disable parasites in the midgut and hemolymph of transgenic mosquitoes.     

Genetic and environmental determinants of malaria parasite virulence in mosquitoes

Models of malaria epidemiology and evolution are frequently based on the assumption that vector-parasitic associations are benign. Implicit in this assumption is the supposition that all Plasmodium parasites have an equal and neutral effect on vector survival, and thus that there is no parasite genetic variation for vector virulence. While some data support the assumption of avirulence, there has been no examination of the impact of parasite genetic diversity. We conducted a laboratory study with the rodent malaria parasite, Plasmodium chabaudi and the vector, Anopheles stephensi, to determine whether mosquito mortality varied with parasite genotype (CR and ER clones), infection diversity (single versus mixed genotype) and nutrient availability. Vector mortality varied significantly between parasite genotypes, but the rank order of virulence depended on environmental conditions. In standard conditions, mixed genotype infections were the most virulent but when glucose water was limited, mortality was highest in mosquitoes infected with CR. These genotype-by-environment interactions were repeatable across two experiments and could not be explained by variation in anaemia, gametocytaemia, blood meal size, mosquito body size, infection rate or oocyst burden. Variation in the genetic and environmental determinants of virulence may explain conflicting accounts of Plasmodium pathogenicity to mosquitoes in the malaria literature.     

Cross-species regulation of malaria parasitaemia in the human host

Longitudinal genetic analysis of the composition of malaria parasites infecting humans has demonstrated that individuals living in endemic areas are chronically infected with multiple genotypes and species of Plasmodium. The accumulation of infections is a consequence of superinfection from the bites of many infected anopheline mosquitoes. The clinical outcome of infection is determined by the host's ability to regulate the density of malaria parasites in the blood. Interestingly, most infections do not cause symptoms of malarial disease after a degree of immunity is acquired. Here, we review data from the first genetic study of the longitudinal dynamics of multiple Plasmodium species and genotypes in humans. The data show that the total parasite density of Plasmodium species oscillates around a threshold and that peaks of infection with each species do not coincide. We propose that malaria parasitaemia is controlled in a density-dependent manner in these semi-immune children. This implies that a cross-species mechanism of parasite regulation exists. A model of how multiple immune responses could act in concert to explain these within host dynamics is discussed in relation to known regulatory mechanisms.