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.     

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.     

Local adaptation and vector-mediated population structure in Plasmodium vivax malaria

Plasmodium vivax in southern Mexico exhibits different infectivities to 2 local mosquito vectors, Anopheles pseudopunctipennis and Anopheles albimanus. Previous work has tied these differences in mosquito infectivity to variation in the central repeat motif of the malaria parasite's circumsporozoite (csp) gene, but subsequent studies have questioned this view. Here we present evidence that P. vivax in southern Mexico comprised 3 genetic populations whose distributions largely mirror those of the 2 mosquito vectors. Additionally, laboratory colony feeding experiments indicate that parasite populations are most compatible with sympatric mosquito species. Our results suggest that reciprocal selection between malaria parasites and mosquito vectors has led to local adaptation of the parasite. Adaptation to local vectors may play an important role in generating population structure in Plasmodium. A better understanding of coevolutionary dynamics between sympatric mosquitoes and parasites will facilitate the identification of molecular mechanisms relevant to disease transmission in nature and provide crucial information for malaria control.     

The energetic budget of Anopheles stephensi infected with Plasmodium chabaudi: is energy depletion a mechanism for virulence?

Evidence continues to accumulate showing that the malaria parasites (Plasmodium spp.) reduce the survival and fecundity of their mosquito vectors (Anopheles spp.). Our ability to identify the possible epidemiological and evolutionary consequences of these parasite-induced fitness reductions has been hampered by a poor understanding of the physiological basis of these shifts. Here, we explore whether the reductions in fecundity and longevity are the result of a parasite-mediated depletion or reallocation of the energetic resources of the mosquito. Mosquitoes infected with Plasmodium chabaudi were expected to have less energetic resources than uninfected mosquitoes, and energy levels were predicted to be lowest in mosquitoes infected with the most virulent parasite genotypes. Not only was there no evidence of a parasite-mediated reduction in the overall energetic budget of mosquitoes, but Plasmodium was actually associated with increased levels of glucose, a key insect nutritional and energetic resource. The data strongly suggest the existence of an increase in sugar feeding in mosquitoes infected with Plasmodium. We suggest different adaptive explanations for an enhanced sugar uptake in infected mosquitoes and call for more studies to investigate the physiological role of glucose in the Plasmodium-mosquito interaction.     

Anatomical dissemination of circumsporozoite protein in wild Afrotropical Anopheles affects malaria sporozoite rate determination by ELISA

The head, thorax, wings, legs and abdomen of 320 wild-caught Anopheles gambiae Giles sensu lato and 115 An.funestus Giles were tested by an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum Welch to determine how anatomical dissemination of circumsporozoite (CS) protein could affect the estimation of malaria sporozoite rates by ELISA. Of fifty-three Anopheles with CS protein detected in any body part, positive reactions were observed for 58.5% of heads, 67.0% of thoraces, 39.6% of wings, 52.8% of legs and 60.4% of abdomens. Mean absorbance values (range 0-2.00) were highest in thorax samples (1.17), followed by heads (0.80), abdomens (0.67), wings (0.48) and legs (0.46). Circumsporozoite protein was present in the wings or legs, but not in the head or thorax, in 11.3% (6/53) of the infected Anopheles. The ELISA infection rate of 12.8% (41/320) for An.gambiae would have increased to 14.7% (47/320) by inclusion of six mosquitoes with CS protein in wings or legs alone. The slight overestimation of the proportion of infective mosquitoes due to disseminated CS protein would have little effect on estimates of relative infection rates by ELISA for field-collected Anopheles, with abdomens removed prior to testing. However, the widespread dissemination of CS protein indicates that sporozoite load estimates by ELISA, for mosquitoes without abdomens, may not provide adequate measurements of the numbers of sporozoites in the salivary glands. Operationally, careful processing of mosquito samples for the determination of infectivity rates by ELISA is necessary to prevent the mixing of wings or legs among samples representing individual mosquitoes.     

Anopheles gambiae innate immunity

The successful development of Plasmodium in Anopheles mosquitoes is governed by complex molecular and cellular interactions that we are just beginning to understand. Anopheles immune system has received particular attention as genetic evidence points clearly to its critical role in eliminating the majority of parasites invading the midgut epithelium. Several factors regulating Plasmodium development have been identified and tentatively assigned to the individual steps leading to mosquito immune reactions; non-self-recognition, signal modulation, signal transduction and effector mechanisms. Detailed knowledge of these steps and their underlying molecular mechanisms may offer novel perspectives to abort Plasmodium development in the vector. Here, we summarize our current knowledge of mosquito innate immunity highlighting both, recent advances and areas where additional research is required.     

Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon

A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.     

Malaria parasite growth is stimulated by mosquito probing

The ability of malaria parasites to respond positively to the presence of feeding mosquito vectors would clearly be advantageous to transmission. In this study, Anopheles stephensi mosquitoes probed mice infected with the rodent malaria parasite, Plasmodium chabaudi. Growth of asexual stages was accelerated and gametocytes appeared 1-2 days earlier than in controls. This first study, to our knowledge, of the effects of mosquitoes on 'in-host' growth and development of Plasmodium has profound implications for malaria epidemiology, suggesting that individuals exposed to high mosquito numbers can contribute disproportionately high numbers of parasites to the transmission pool.     

Investigations on anopheline mosquitoes close to the nest sites of chimpanzees subject to malaria infection in Ugandan Highlands

ABSTRACT: BACKGROUND: Malaria parasites (Plasmodium sp.), including new species, have recently been discovered as low grade mixed infections in three wild chimpanzees (Pan troglodytes schweinfurthii) sampled randomly in Kibale National Park, Uganda. This suggested a high prevalence of malaria infection in this community. The clinical course of malaria in chimpanzees and the species of the vectors that transmit their parasites are not known. The fact that these apes display a specific behaviour in which they consume plant parts of low nutritional value but that contain compounds with anti-malarial properties suggests that the apes' health might be affected by the parasite. The avoidance of the night-biting anopheline mosquitoes is another potential behavioural adaptation that would lead to a decrease in the number of infectious bites and consequently malaria. METHODS: Mosquitoes were collected over two years using suction-light traps and yeast-generated CO2 traps at the nesting and the feeding sites of two chimpanzee communities in Kibale National Park. The species of the female Anopheles caught were then determined and the presence of Plasmodium was sought in these insects by PCR amplification. RESULTS: The mosquito catches yielded a total of 309 female Anopheles specimens, the only known vectors of malaria parasites of mammalians. These specimens belonged to 10 species, of which Anopheles implexus, Anopheles vinckei and Anopheles demeilloni dominated. Sensitive DNA amplification techniques failed to detect any Plasmodium-positive Anopheles specimens. Humidity and trap height influenced the Anopheles capture success, and there was a negative correlation between nest numbers and mosquito abundance. The anopheline mosquitoes were also less diverse and numerous in sites where chimpanzees were nesting as compared to those where they were feeding. CONCLUSIONS: These observations suggest that the sites where chimpanzees build their nests every night might be selected, at least in part, in order to minimize contact with anopheline mosquitoes, which might lead to a reduced risk in acquiring malaria infections.     

Humoral response to the Anopheles gambiae salivary protein gSG6: a serological indicator of exposure to Afrotropical malaria vectors

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.     

Anopheline species and their Plasmodium infection status in Aligarh,India

Malaria is a global issue and India contributes substantially to global malaria incidence. Information related to malaria vectors is very limited in Aligarh. The environmental and climatological situations permit the continual breeding of vectors in permanent breeding sites. This study was designed with the aim to screen all the anophelines species and possible malaria vectors in three different localities of Aligarh. Anopheles mosquitoes were collected from three different localities (Fort, Jalali and Tappal) during peak malaria transmission season (July to November) by using mouth aspirator and CDC light traps. Enzyme-linked immunosorbent assay (ELISA) was done to detect Plasmodium falciparum, Plasmodium vivax-210 and P. vivax-247 circumsporozoite proteins (CSP) from the collected female species. A total of 794 female anopheline mosquitoes belonging to 7 species were collected by different methods. Circumsporozoite protein–enzyme-linked immunosorbent assay was performed with 780 anopheline mosquitoes out of which 13 mosquitoes were positive in CSP–ELISA. Thus, the overall infection rate was 1.66% (13/780). Four (0.51%) mosquitoes belonging to three species were positive for P. falciparum, 7 (0.89%) mosquitoes belonging to three species were positive for VK 210 and 2 (0.25%) mosquitoes belonging to Anopheles culicifacies and Anopheles stephensi species were positive for VK 247. No mixed infection was found in this study. According to species, the highest infection rate was observed in An. culicifacies (7/288, 2.43%) followed by An. stephensi (2.40%) and Anopheles annularis (1.98%). An. culicifacies and An. stephensi were previously incriminated as malaria vectors in Aligarh. There was, however, no previous report in favor of infections in An. annularis in Aligarh. The on-going Malaria Control Program in India needs up to date information on malaria vectors. A major challenge is the lack of knowledge about vectors and their role in malaria transmission. Findings of this study suggested that in the absence of major malaria vectors there is a possibility that other Anopheles species may have been playing a role in malaria transmission in Aligarh.     

The effect of Plasmodium yoelii nigeriensis infection on the feeding persistence of Anopheles stephensi Liston throughout the sporogonic cycle.

Vector-borne parasites such as malaria have been shown to modify the feeding behaviour of their invertebrate hosts so as to increase the probability of transmission. However, evolutionary consideration of developmental changes in malaria within Anopheles mosquitoes suggests that the nature of altered feeding by mosquitoes should differ depending on the developmental stage of the parasite. We present laboratory evidence that the feeding persistence of female Anopheles stephensi towards a human host is decreased in the presence of Plasmodium yoelii nigeriensis oocysts (which cannot be transmitted), but increased when the malaria has developed into transmissible sporozoites in the salivary glands. In ten-minute trials, 33% of uninfected mosquitoes gave up their feeding attempt before the test period had ended, 53% of those harbouring oocysts had given up, but only 20% of those infected with sporozoites gave up by this time. We conclude that changes in feeding behaviour of mosquitoes mediated by parasite infection are sensitive to the developmental stage of the parasite and that these changes have important implications for malaria epidemiology.     

Chesson strain Plasmodium vivax in Saimiri sciureus boliviensis monkeys

Nine Saimiri sciureus boliviensis monkeys were inoculated with sporozoites of Plasmodium vivax (Chesson strain) dissected from Anopheles stephensi mosquitoes infected by feeding on blood from infected chimpanzees. The animals were splenectomized 7 days after inoculation. Seven animals developed infections with prepatent periods ranging from 12 to 43 days (mean of 19.6 days). Parasitemias were low during the first 50 days. Maximum parasitemias in 5 animals in which the strain adapted ranged from 10,000 to 46,800 per mm3. Anopheles freeborni mosquitoes were infected by feeding on 4 of the monkeys.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Studies on a newly isolated strain of Plasmodium brasilianum in Aotus and Saimiri monkeys and different anophelines

A strain of Plasmodium brasilianum was isolated from an Aotus vociferans monkey from Peru. The parasite readily infected Aotus monkeys from Bolivia and Columbia and Saimiri sciureus monkeys from Peru and Bolivia. Highest level mosquito infections were obtained by feeding on the Saimiri monkeys. The most susceptible mosquito was Anopheles freeborni, followed by Anopheles dirus, Anopheles stephensi, Anopheles gambiae, Anopheles culicifacies, Anopheles maculatus and Anopheles albimanus. Anopheles quadrimaculatus were also susceptible to infection. Degenerating oocysts were observed in An. dirus mosquitoes infected with this parasite. Transmission via the bites of infected An. maculatus mosquitoes was obtained to 3 Bolivian Saimiri monkeys; prepatent periods were 27, 27, and 29 days.     

Differential expression of proteins in the midgut of Anopheles albimanus infected with Plasmodium berghei

The main vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito. The midgut of disease-transmitting mosquitoes carries out a variety of functions that are related to blood feeding. We analyzed the midgut of A. albimanus infected with Plasmodium berghei (resistant mosquito) using a proteomic approach to identify putative short peptides that are enriched in the midgut after blood feeding. Mosquito midguts were analyzed by two-dimensional electrophoresis to determine the changes in protein profiles. We identified 21 spot proteins that are differentially expressed in the blood of mosquitoes during the immune challenge. Molecular weight of the spots varied from 13 to 36 kDa, with a broad isoelectric point range of 3.92–8.90. We identified the differentially expressed proteins using mass spectrometry and constructed a proteomic data base of the A. albimanus midgut with diverse functions, some of them proteins with digestive and immunologic functions. Identification of these proteins may have important implications for understanding the blood meal digestion process, as well as developing novel vector control strategies and understanding parasite vector interactions.     

Malaria infection increases bird attractiveness to uninfected mosquitoes

The epidemiology of vector‐borne pathogens is largely determined by the host‐choice behaviour of their vectors. Here, we investigate whether a Plasmodium infection renders the host more attractive to host‐seeking mosquitoes. For this purpose, we work on a novel experimental system: the avian malaria parasite Plasmodium relictum, and its natural vector, the mosquito Culex pipiens. We provide uninfected mosquitoes with a choice between an uninfected bird and a bird undergoing either an acute or a chronic Plasmodium infection. Mosquito choice is assessed by microsatellite typing of the ingested blood. We show that chronically infected birds attract significantly more vectors than either uninfected or acutely infected birds. Our results suggest that malaria parasites manipulate the behaviour of uninfected vectors to increase their transmission. We discuss the underlying mechanisms driving this behavioural manipulation, as well as the broader implications of these effects for the epidemiology of malaria.     

The sporogonic cycle of Plasmodium reichenowi

Plasmodium reichenowi, a malarial parasite of the chimpanzee, was infective to Anopheles freeborni, Anopheles quadrimaculatus, Anopheles stephensi, Anopheles maculatus, Anopheles dirus, and Anopheles culicifacies mosquitoes. Anopheles gambiae and Anopheles albimanus were not infected. Mean oocyst diameters of P. reichenowi were smaller than those of the other chimpanzee parasite, Plasmodium schwetzi. Sporozoites were present in the salivary glands of An. freeborni at 15 days when held at 25 to 26 C.