No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Corrigendum to “The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum, and their transmission by Anopheles stephensi, Anopheles gambiae and Aedes aegypti” [International Journal for Parasitology 33 (2003) 933–943]
Abstract
Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.
... During the first half of sporogony, ingested gametocytes form gametes, and then ookinetes which invade the mosquito MG and develop into oocysts ( Figure 1A). This transition from gametocyte to oocysts results in high losses (Plasmodium bottleneck) [Alavi et al., 2003]; only 0.2% of ingested P. berghei gametocytes become viable ookinetes, and only 2-20% of them develop into mature oocysts [Alavi et al., 2003]. Similar losses have been documented through the macrogametocyte to oocyst stage in P. falciparum laboratory cultured gametocytes that were membrane fed to An. Gambiae . ...
... During the first half of sporogony, ingested gametocytes form gametes, and then ookinetes which invade the mosquito MG and develop into oocysts ( Figure 1A). This transition from gametocyte to oocysts results in high losses (Plasmodium bottleneck) [Alavi et al., 2003]; only 0.2% of ingested P. berghei gametocytes become viable ookinetes, and only 2-20% of them develop into mature oocysts [Alavi et al., 2003]. Similar losses have been documented through the macrogametocyte to oocyst stage in P. falciparum laboratory cultured gametocytes that were membrane fed to An. Gambiae . ...
In eco‐evolutionary studies of parasite‐host interactions, virulence is defined as a reduction in host fitness as a result of infection relative to an uninfected host. Pathogen virulence may either promote parasite transmission, when correlated with higher parasite replication rate or decrease the transmission rate if the pathogen quickly kills the host. This evolutionary mechanism, referred to as ‘trade‐off’ theory proposes that pathogen virulence evolves towards a level that most benefits the transmission. It has been generally predicted that pathogens evolve towards low virulence in their insect vectors, mainly due to the high dependence of parasite transmission on their vector‐survival. Therefore the degree of virulence which malaria parasites impose on mosquito vectors may depend on several external and internal factors. Here we review briefly (I) the role of mosquito in parasite development, with a particular focus on mosquito midgut as the battleground between Plasmodium and the mosquito host. We aim to point out (II) the histology of the mosquito midgut epithelium and its role in host defence against parasite's countermeasures in the three main battle sites, namely a‐ the lumen (microbiota and biochemical environment) b‐ the peritrophic membrane (physical barrier), and c‐ the tubular epithelium including the basal membrane (physical and biochemical barrier). Lastly, (III) we describe the impact which malaria parasite and its virulence factors have on mosquito fitness. This article is protected by copyright. All rights reserved
... Multiplication and transformation of the parasites in avian hosts are accountable for disease outcome and transmission to other mosquito vectors. Unlike P. falciparum whose vector is limited to Anopheles mosquitoes, P. gallinaceum is transmitted by wider genera and species of mosquitoes such as Aedes, Anopheles, and Culex (Alavi et al., 2003). However, the effects of parasite density in these competent vectors on oocyst formation, mortality rate of infected mosquitoes, and transmission rate to avian hosts remain unclear. ...
... All mosquitoes used in this study were 4-10 days of age. After the starvation from sucrose for 12 h, they were allowed to feed on blood of a gametocyte donor chicken for 20 min (Alavi et al., 2003). Gametocyte donor chickens were 7-day-old Isa Babcock that were intravenously inoculated with 10 6 infected red blood cells (iRBCs). ...
Avian malaria caused by Plasmodium gallinaceum is an important mosquito-borne disease. Eradication of this disease remains problematic since its competent vectors are diverse and widely distributed across the globe. Several mosquito species were implicated as competent vectors for this parasite. However, studies on vector competence for P. gallinaceum remain limited. In this study, vector competence in the two most predominant mosquito vectors in tropical countries, Aedes albopictus and Ae. aegypti, was compared. In order to determine their infection rates, Ae. albopictus (>F10), Ae. aegypti (>F10), and Ae. aegypti (<F10) mosquitoes were equally divided and allowed to feed on infected chickens with 15 different gametocyte levels (0.1–5.2%). On day five post blood feeding (PBF), 60 mosquitoes from each group were dissected, and the number of oocysts in midgut was examined. Infections with P. gallinaceum were observed in all groups, but the number of oocysts produced was significantly different. To evaluate the transmission ability of these mosquitoes after having been fed on different levels of gametocytes, the infected mosquitoes were allowed to feed on naïve chickens on day 10 PBF, and the chicken bloods were monitored for 21 days PBF. All groups of mosquitoes demonstrated a high degree of infection rates. The infection rates in chickens were 80–100% after having been bitten by infected Ae. albopictus ( > F10) and Ae. aegypti ( > F10) and 40–60% by infected Ae. aegypti (<F10). These findings demonstrated that Ae. albopictus (>F10) and Ae. aegypti (>F10) were highly competent vectors for P. gallinaceum infections. These mosquitoes play a crucial role in the transmission cycle of this parasite in nature.
... Phytoplasmas are transmitted by a narrow range of phloem-feeding insect species, mainly including leafhoppers, planthoppers, and psyllids, whereas their plant host range is usually broader [10]. Insect vector specificity plays a key role in the epidemiology of several vector-borne pathogens [11,12]. A class of membrane proteins in phytoplasmas have been identified as immunodominant membrane proteins (IDPs), which can directly affect vector insects and host plants and play a crucial role in plant and insect vector transmission [2,13]. ...
Phytoplasmas are uncultivable, phloem-limited, phytopathogenic bacteria that represent a major threat to agriculture worldwide. Phytoplasma membrane proteins are in direct contact with hosts and presumably play a crucial role in phytoplasma spread within the plant as well as by the insect vector. Three highly abundant types of immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). Although recent results indicate that Amp is involved in host specificity by interacting with host proteins such as actin, little is known about the pathogenicity of IDP in plants. In this study, we identified an antigenic membrane protein (Amp) of rice orange leaf phytoplasma (ROLP), which interacts with the actin of its vector. In addition, we generated Amp-transgenic lines of rice and expressed Amp in tobacco leaves by the potato virus X (PVX) expression system. Our results showed that the Amp of ROLP can induce the accumulation of ROLP and PVX in rice and tobacco plants, respectively. Although several studies have reported interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins, this example demonstrates that Amp protein can not only interact with the actin protein of its insect vector but can also directly inhibit host defense responses to promote the infection. The function of ROLP Amp provides new insights into the phytoplasma-host interaction.
... Our observation raises the possibility that there exists some invasion plasticity to cater for the host environment, such a phenomenon would be an important consideration in the development of transmission blocking vaccines. For example, P. berghei infections in An. stephensi are more intense (i.e. have more oocysts) than in An. coluzzii (Alavi et al., 2003). The differentially expressed genes identified here may be actively involved in modulating infection intensity or may be differentially expressed via selection on the parasite population. ...
The crossing of the mosquito midgut epithelium by the malaria parasite motile ookinete form represents the most extreme population bottleneck in the parasite life cycle and is a prime target for transmission blocking strategies. However, we have little understanding of the clonal variation that exists in a population of ookinetes in the vector, partially because the parasites are difficult to access and are found in low numbers. Within a vector, variation may result as a response to specific environmental cues or may exist independent of those cues as a potential bet-hedging strategy. Here we use single-cell RNA-seq to profile transcriptional variation in Plasmodium berghei ookinetes across different vector species, and between and within individual midguts. We then compare our results to low-input transcriptomes from individual Anopheles coluzzii midguts infected with the human malaria parasite Plasmodium falciparum. Although the vast majority of transcriptional changes in ookinetes are driven by development, we have identified candidate genes that may be responding to environmental cues or are clonally variant within a population. Our results illustrate the value of single-cell and low-input technologies in understanding clonal variation of parasite populations.
... Relatively little is known about the architecture and protein composition of the IMC in these mosquito infecting stages. However, freeze fracture SEM images of ookinetes from the poultry parasite P. gallinaceum provide a glimpse of what appears to be a largely distinct IMC morphology from other stages (Alavi et al., 2003). The micrographs show a single vesicle punctured by multiple large pores. ...
Apicomplexan parasites, such as human malaria parasites, have complex lifecycles encompassing multiple and diverse environmental niches. Invading, replicating, and escaping from different cell types, along with exploiting each intracellular niche, necessitate large and dynamic changes in parasite morphology and cellular architecture. The inner membrane complex (IMC) is a unique structural element that is intricately involved with these distinct morphological changes. The IMC is a double membrane organelle that forms de novo and is located beneath the plasma membrane of these single-celled organisms. In Plasmodium spp. parasites it has three major purposes: it confers stability and shape to the cell, functions as an important scaffolding compartment during the formation of daughter cells, and plays a major role in motility and invasion. Recent years have revealed greater insights into the architecture, protein composition and function of the IMC. Here, we discuss the multiple roles of the IMC in each parasite lifecycle stage as well as insights into its sub-compartmentalization, biogenesis, disassembly and regulation during stage conversion of P. falciparum.
... In fact, it could indicate an ongoing process of close contact between wild raptors and domestic birds. Plasmodium gallinaceum is known to be very effective at infecting its mosquito vector, facilitating transmission to the vertebrate host, and has been used as a model of human malaria (Paraense, 1945;Alavi et al., 2003). ...
The order Haemosporida is widely distributed parasitizing members of the Aves class. In birds of prey, infection with Plasmodium spp. parasites varies from an apathogenic form to a clinical syndrome. However, studies on Haemosporida in raptors from the neotropical region are scarce. The aim of this study was to investigate natural infection by Plasmodium spp., Haemoproteus spp. and Leucocytozoon spp. in free-ranging wild raptors from southern Brazil. For this, we sampled 206 individuals of 23 species: 94 live-trapped Southern Caracaras (Caracara plancus) and 112 raptors from other species that were brought to rehabilitation centers. The presence of infection was investigated using a nested-PCR for Haemosporida performed on blood samples. Overall, 56 out of 206 birds were positive for Plasmodium spp./Haemoproteus spp. Twenty-two percent (21/94) of the C. plancus samples were positive. Of the 112 wild raptors rescued, 31% (35/112) of those belonging to 15 other species tested positive. No sample was positive for Leucocytozoon spp. Herein, we demonstrated nine lineages of Haemosporidian parasites (eight Plasmodium sp. and one Haemoproteus sp.) in free-living species of Brazilian birds of prey, being six of them potential novel lineages. It suggests that information currently available on South-American haemosporidian from these birds greatly underestimate the potential lineage diversity in this region.
... These Plasmodium species have been used in different laboratories for several years to infect An. gambiae, Anopheles funestus, An. quadrimaculatus and An. stephensi, all of which are malaria vectors in Africa and Asia, mainly due to the vectors' high susceptibility to infection with various malaria parasite species and strains (Yoeli et al. 1964, Vaughan et al. 1991, Alavi et al. 2003, Akaki & Dvorak 2005, Frischknecht et al. 2006, Hume et al. 2007, Lo & Coetzee 2013, Xu et al. 2013). ...
... Inside the mosquito, parasites are attacked by an array of immune responses (1,2). Most parasite losses occur during the ookinete-tooocyst transition (3,4). Ookinete traversal of the mosquito midgut leads to activation of JNK (c-Jun N-terminal kinase) signaling, inducing apoptosis of the invaded cells. ...
After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 ( Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito–parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.
... Maintenance of insects in artificial breeding conditions favors accumulation of traits that favor survival in the new environment, resulting in a change in genetic make-up over generations [25]. Parasite-mosquito combinations and their susceptibility to malaria infection are regulated at multiple steps during the development of the parasites [26] and numerous factors may modulate this interaction. These factors range from mosquito genetics [27,28] and immune system [29] to parasite polymorphisms that allow evasion of the mosquito immune system [30]. ...
Background:
Mosquito-feeding assays that assess transmission of Plasmodium from man-to-mosquito typically use laboratory mosquito colonies. The microbiome and genetic background of local mosquitoes may be different and influence Plasmodium transmission efficiency. In order to interpret transmission studies to the local epidemiology, it is therefore crucial to understand the relationship between infectivity in laboratory-adapted and local mosquitoes.
Methods:
We assessed infectivity of Plasmodium vivax-infected patients from Adama, Ethiopia, using laboratory-adapted (colony) and wild-caught (wild) mosquitoes raised from larval collections in paired feeding experiments. Feeding assays used 4-6 day-old female Anopheles arabiensis mosquitoes after starvation for 12 h (colony) and 18 h (wild). Oocyst development was assessed microscopically 7 days post-feeding. Wild mosquitoes were identified morphologically and confirmed by genotyping. Asexual parasites and gametocytes were quantified in donor blood by microscopy.
Results:
In 36 paired experiments (25 P. vivax infections and 11 co-infections with P. falciparum), feeding efficiency was higher in colony (median: 62.5%; interquartile range, IQR: 47.0-79.0%) compared to wild mosquitoes (median: 27.8%; IQR: 17.0-38.0%; Z = 5.02; P < 0.001). Plasmodium vivax from infectious individuals (51.6%, 16/31) infected a median of 55.0% (IQR: 6.7-85.7%; range: 5.5-96.7%; n = 14) of the colony and 52.7% (IQR: 20.0-80.0%; range: 3.2-95.0%; n = 14) of the wild mosquitoes. A strong association (ρ(16) = 0.819; P < 0.001) was observed between the proportion of infected wild and colony mosquitoes. A positive association was detected between microscopically detected gametocytes and the proportion of infected colony (ρ(31) = 0.452; P = 0.011) and wild (ρ(31) = 0.386; P = 0.032) mosquitoes.
Conclusions:
Infectivity assessments with colony and wild mosquitoes yielded similar infection results. This finding supports the use of colony mosquitoes for assessments of the infectious reservoir for malaria in this setting whilst acknowledging the importance of mosquito factors influencing sporogonic development of Plasmodium parasites.
... This suggests that interactions between microbes and parasites reduce Plasmodium sp. transmission [25]. Decreasing the vector's transmission efficacy by modifying its microbiota is a very active and novel field of research [8,26]. ...
Plasmodium falciparum is transmitted by mosquitoes from the Anopheles gambiae sensu lato (s.l) species complex and is responsible for severe forms of malaria. The composition of the mosquitoes’ microbiota plays a role in P. falciparum transmission, so we studied midgut bacterial communities of An. gambiae s.l from Burkina Faso. DNA was extracted from 17 pools of midgut of mosquitoes from the Anopheles gambiae complex from six localities in three climatic areas, including cotton-growing and cotton-free localities to include potential differences in insecticide selection pressure. The v3–v4 region of the 16S rRNA gene was targeted and sequenced using Illumina Miseq (2 × 250 nt). Diversity analysis was performed using QIIME and R software programs. The major bacterial phylum was Proteobacteria (97.2%) in all samples. The most abundant genera were Enterobacter (32.8%) and Aeromonas (29.8%), followed by Pseudomonas (11.8%), Acinetobacter (5.9%) and Thorsellia (2.2%). No statistical difference in operational taxonomic units (OTUs) was found (Kruskal–Wallis FDR—p > 0.05) among the different areas, fields or localities. Richness and diversity indexes (observed OTUs, Chao1, Simpson and Shannon indexes) showed significant differences in the cotton-growing fields and in the agroclimatic zones, mainly in the Sudano-Sahelian area. OTUs from seven bacterial species that mediate refractoriness to Plasmodium infection in An. gambiae s.l were detected. The beta diversity analysis did not show any significant difference. Therefore, a same control strategy of using bacterial species refractoriness to Plasmodium to target mosquito midgut bacterial community and affect their fitness in malaria transmission may be valuable tool for future malaria control efforts in Burkina Faso.
... However, how the parasite may use these proteins to sense the environment and trigger the egress process is a subject that also needs evaluation. On top of that, the fact that some parasites share the same mode of egression but differ from others may be a clue that the host could have molecules influencing this process, as some parasite species can only complete development in specific mosquito species (Alavi et al., 2003). An exchange of the mosquito species and detection of changes in the egression pattern in a specific parasite could provide clues for better understanding of how mosquito factors contribute to this process. ...
Although much progress has been made in the fight against malaria, the number of people that contract this disease due to the bite of an Anopheline mosquito remains unacceptably high. A better understanding of the relationship between the Plasmodium parasite and its vector is of extreme importance and may allow for the development of new tools to fight the disease. In particular, new genome editing techniques may allow the modification or removal of factors critical for parasite traversal of the vector mosquito. In this review, we aim to highlight what is known about important molecules encoded by the mosquito or parasite involved in this close interaction, focused on a specific window of the parasite life cycle inside the mosquito: from egress and release of the sporozoites from the oocyst into the hemolymph, until salivary gland penetration. While sophisticated molecular tools have already helped to understand aspects of parasite-mosquito interactions, our understanding of this brief period is still limited. Here, we discuss the evidence surrounding essential parasite and mosquito factors relating to sporozoite invasion of the salivary glands, and emphasize the areas where the lack of information is limiting the advance of strategies to manipulate the mosquito in order to block the transmission of malaria parasites.
... Mainly, sporozoites should reach the salivary glands and mature completely [2]. The losses of malaria parasites occur on each phase of sporogonic development, especially before ookinete stage [57], thus our results together with Hellgren's [23] findings provide strong evidence that these two lineages likely belong to a single P. relictum species. These results are similar to those in the human malaria agent P. falciparum lines 3D7 and HB3 that differ in a single nucleotide base pair in the mitochondrial cyt b gene. ...
Over 50 avian Plasmodium species have been described. However, PCR-based information shows much broader diversity of genetic lineages in these parasites. This discrepancy indicates insufficient knowledge about taxonomic diversity and boundaries of a single species in avian Plasmodium species. In recent taxonomy, most of genetically closely related lineages that share the same morphology and development patterns are attributed to the same biological species, but there is no information if these lineages are able to cross. This information is crucial to understand if these lineages form single or multiple evolutionary units. Due to presence of sexual process and sporogonic development of Plasmodium parasites in mosquitoes, self and cross-fertilization can occur and be identified during the oocyst stage. We initiated in vivo hybridization experiments of two widespread Plasmodium relictum lineages (pSGS1 and pGRW11) in experimentally infected Culex pipiens pipiens form molestus mosquitoes. To study putative hybrid oocysts, we used a laser microdissection technique together with PCR-based analyses of mitochondrial and nuclear genes. We demonstrate that both pSGS1 and pGRW11 lineages develop in infected mosquitoes in parallel, but also form hybrid oocysts of these two lineages. Our results are in accord to a recent global phylogeographic study of P. relictum that suggested that cross-fertilization between pSGS1 and pGRW11 might occur. This information helps to understand population structure, gene flow and the evolutionary process of haemosporidian parasites.
... However, the susceptibility of C. pipiens was parasite species specific, suggesting that vector competence for a given Plasmodium spp. is fixed and thus hereditary (Huff, 1930). These early results have been confirmed subsequently across several mosquito species, which range widely in their ability to support development of distinct Plasmodium spp., depending on the vector and parasite species combination (Alavi et al., 2003;Huff, 1929Huff, , 1927Vaughan et al., 1994). Similar observations were made in field-derived populations of Anopheles gambiae and Plasmodium falciparum in different locations of human malaria transmission in sub-Saharan Africa (Lambrechts et al., 2005;Niaré et al., 2002). ...
Mosquito immunity to malaria infection has received much attention since it was proposed as a major determinant of vector competence nearly 90 years ago. Understanding the mosquito's immune system holds the promise of devising strategies that eliminate all parasites within the vector and thus prevent malaria transmission. Herein, we review the field of mosquito innate immunity as it pertains to malaria parasite killing, providing a historical perspective and summary of current knowledge. Mosquito immunity targets each life stage of the malaria parasite as it undergoes development within its mosquito vector. Gametes and ookinetes are killed within the midgut lumen by nitric oxide produced by the mosquito midgut. Ookinetes and developing oocysts are killed by a complex process that is initiated by ookinete migration through the midgut epithelium. This process, involving nitration and opsonization, leads to parasite killing by a mechanism that is not entirely understood. A similar process is likely responsible for the sporozoite losses that occur in the hemolymph. Mosquito immune reactions are tightly controlled by a large regulatory network of extra- and intracellular signaling cascades. Their combined action is crucial for mosquito fitness as these regulatory cascades balance the positive effects of pathogen elimination with the harmful side effects of tissue damage. Several proof of principle studies have now demonstrated that genetic manipulation of the mosquito's immune system can render susceptible mosquito species refractory to malaria parasite infection. This field is thus ready to devise a framework to evaluate these approaches in the ecological context of malaria transmission.
... Based on laboratory studies, it has been shown that Aedes albopictus is a competent vector for at least 22 arboviruses (Malcolm 2009). In addition, it is known that some mosquito species can carry more than one type of Plasmodium (Alavi et al. 2003;Perez-Tris and Bensch 2005). Avian malaria parasites are present in numerous mosquito species, for example, Culex, Aedes, Culiseta, Anopheles, Mansonia, Aedeomya, and Coquillettidia; the Bparasite-vector-vertebrate host^interactions remain understudied, as do reasons for the pathogenicity of different species and few experts have the knowledge required to identify avian Plasmodium spp. ...
It has been claimed that infectious agents transmitted by mosquitoes (Diptera: Culicidae) may have a greater connection to cancer then hitherto supposed and that the immune system struggles to recognize and fight some of these infectious agents. One of the claims made is that there is a connection between human malaria and brain cancers in the USA. However, the USA declared itself free of human malaria in the last century, yet cancer incidences remain high, suggesting any overall cancer connection is slight. Two fundamental questions arise from the possible mosquito-cancer connection. Firstly, if mosquitoes are able to vector some pathogens and parasites linked with cancer pathogenesis, why has the fact not been discovered decades ago? Secondly, if there is a connection (other than in relation to Burkett's lymphoma), what is its extent? The answers may well lie with the various types of malarias known to exist. The discovery in humans of the simian malaria, caused by Plasmodium knowlesi, suggests that other forms of simian or even avian malaria may be capable of survival in humans, albeit at low levels of parasitemia, and humans may be a dead-end host. Other carcinogenic infectious agents transmitted by mosquitoes may also go undetected because either no one is looking for them, or they are looking in wrong anatomical locations and/or with inadequate tools. Research on false negative test results with respect to many infectious agents is sadly lacking, so its extent is unknown. However, electronic and other media provide numerous instances of patients failing to be diagnosed for both human malaria and Lyme's disease, to take just two examples. This review suggests that to shed light on a potential mosquito-cancer connection, more research is required to establish whether other simian and avian forms of malaria play a part. If so, then they potentially provide unique markers for early cancer detection.
... After motile ookinetes are formed, they reach the inner membrane of the midgut and activate the mosquito's innate immune system, involving both cellular and humoral defense mechanisms (Dimopoulos, 2003). Huge losses of parasites are observed during formation of oocysts, even in susceptible mosquitoes (Alavi et al., 2003). Sporogonic development during the oocyst stage may be critical for successful sporogony. ...
Plasmodium elongatum causes severe avian malaria and is distributed worldwide. This parasite is of particular importance due to its ability to develop and cause lethal malaria not only in natural hosts, but also in non-adapted endemic birds such as the brown kiwi and different species of penguins. Information on vectors of this infection is available but is contradictory. PCR-based analysis indicated the possible existence of a cluster of closely related P. elongatum lineages which might differ in their ability to develop in certain mosquitoes and birds. This experimental study provides information about molecular and morphological characterisation of a virulent P. elongatum strain (lineage pERIRUB01) isolated from a naturally infected European robin, Erithacus rubecula. Phylogenetic analysis based on partial cytochrome b gene sequences showed that this parasite lineage is closely related to P. elongatum (lineage pGRW6). Blood stages of both parasite lineages are indistinguishable, indicating that they belong to the same species. Both pathogens develop in experimentally infected canaries, Serinus canaria, causing death of the hosts. In both these lineages, trophozoites and erythrocytic meronts develop in polychromatic erythrocytes and erythroblasts, gametocytes parasitize mature erythrocytes, exoerythrocytic stages develop in cells of the erythrocytic series in bone marrow and are occasionally reported in spleen and liver. Massive infestation of bone marrow cells is the main reason for bird mortality. We report here on syncytium-like remnants of tissue meronts, which slip out of the bone marrow into the peripheral circulation, providing evidence that the syncytia can be a template for PCR amplification. This finding contributes to better understanding positive PCR amplifications in birds when parasitemia is invisible and improved diagnostics of abortive haemosporidian infections. Sporogony of P. elongatum (pERIRUB01) completes the cycle and sporozoites develop in widespread Culex quinquefasciatus and Culex pipiens pipiens form molestus mosquitoes. This experimental study provides information on virulence and within species lineage diversity in a single pathogenic species of haemosporidian parasite.
... Upon Plasmodium infection, the female Anopheles mosquito mounts an effective immune response: it is estimated that only 1 in 50 ookinetes present in the mosquito midgut survive to form oocysts [51]. Four immune signalling pathways, including the nuclear factor (NF)-kappaB-dependent Toll (Toll), immune deficient (IMD), Jun-N-terminal kinase (JNK) and Janus kinase/signal transducers and activators of transcription (JAK/STAT), have been linked to killing the parasite in the mosquito [52][53][54][55][56][57][58]. ...
... Malaria parasite needs to go through its developmental stages inside the mosquito mid-gut to complete its life cycle. Parasite bears a very high cost for infecting the vector as significant losses occur during its development inside the mosquito [2]. Inside Anopheles mosquito mid-gut, malaria parasite shares microenvironment with gut resident bacteria which can be a factor to parasite loss. ...
Malaria control is getting tougher each day due to development of resistance to anti-malaria drugs in the parasite and resistance to insecticides in malaria vectors. Alternate strategies are thus required to break the complex malaria parasite interaction. Malaria vectors like other living beings carries diverse flora in their gut, which may inhibit the malaria parasite development inside the mosquito. We earlier identified a natural gut residing lactic acid bacteria Carnobacterium sp., isolated from lab-reared Anopheles stephensi mosquito. An. stephensi is known vector of malaria in Indian subcontinent. Thus, in the present manuscript efforts were made to understand the sporogonic development of Plasmodium vinckei petteri in presence of the bacterium. The bacterium was established in mosquito gut by feeding orally along with sugar meal to a group of gut sanitized mosquito. Groups of naïve, gut sanitized and Carnobacterium sp. fed mosquitoes were used to compare the difference in sporogony of P. v. petteri. This bacterium shows antagonistic effect on development of the malaria parasite.
... Importantly, this period is marked by a major drop of parasite numbers. The rate of parasite losses during the ookinete to oocyst transition in An. gambiae is shown to reach 50, 41 and 69 folds for P. berghei [1], P. yoelii [2,3] and P. falciparum [4], respectively. ...
Background:
Mosquito infection with malaria parasites depends on complex interactions between the mosquito immune response, the parasite developmental program and the midgut microbiota. Simultaneous monitoring of the parasite and bacterial dynamics is important when studying these interactions. PCR based methods of genomic DNA (gDNA) have been widely used, but their inability to discriminate between live and dead cells compromises their application. The alternative method of quantification of mRNA mainly reports on cell activity rather than density.
Method:
Quantitative real-time (qrt) PCR in combination with Propidium Monoazide (PMA) treatment (PMA-qrtPCR) has been previously used for selectively enumerating viable microbial cells. PMA penetrates damaged cell membranes and intercalates in the DNA inhibiting its PCR amplification. Here, we tested the potential of PMA-qrtPCR to discriminate between and quantify live and dead Plasmodium berghei malarial parasites and commensal bacteria in the midgut of Anopheles coluzzii Coetzee & Wilkerson 2013 (formerly An. gambiae M-form).
Results:
By combining microscopic observations with reverse transcriptase PCR (RT-PCR) we reveal that, in addition to gDNA, mRNA from dead parasites also persists inside the mosquito midgut, therefore its quantification cannot accurately reflect live-only parasites at the time of monitoring. In contrast, pre-treating the samples with PMA selectively inhibited qrtPCR amplification of parasite gDNA, with about 15 cycles (Ct-value) difference between PMA-treated and control samples. The limit of detection corresponds to 10 Plasmodium ookinetes. Finally, we show that the PMA-qrtPCR method can be used to quantify bacteria that are present in the mosquito midgut.
Conclusion:
The PMA-qrtPCR is a suitable method for quantification of viable parasites and bacteria in the midgut of Anopheles mosquitoes. The method will be valuable when studying the molecular interactions between the mosquito, the malaria parasite and midgut microbiota.
... and transmissionblocking targets at (1) fertilization of male and female gametes to form zygote (P230 and P48/45); (2) development of ookinetes, penetration of peritrophic matrix and midgut epithelium to form oocysts at the basal face of the midgut (P25, P28, Chitnase, WARP, CTRP, MAOP, SOAP, CelTos, APN1, CBP, Serpin) and (3) invasion of salivary glands by sporozoites (saglin). and finally oocysts (Alavi et al., 2003;Vaughan et al., 1992), indicating this as a biological bottleneck during parasite development (Sinden, 2010). The mosquito-stage transmission-blocking vaccines (TBVs), the focus of this chapter, are designed to induce antibody responses in vaccinees. ...
Despite decades of effort battling against malaria, the disease is still a major cause of morbidity and mortality. Transmission-blocking vaccines (TBVs) that target sexual stage parasite development could be an integral part of measures for malaria elimination. In the 1950s, Huff et al. first demonstrated the induction of transmission-blocking immunity in chickens by repeated immunizations with Plasmodium gallinaceum-infected red blood cells. Since then, significant progress has been made in identification of parasite antigens responsible for transmission-blocking activity. Recombinant technologies accelerated evaluation of these antigens as vaccine candidates, and it is possible to induce effective transmission-blocking immunity in humans both by natural infection and now by immunization with recombinant vaccines. This chapter reviews the efforts to produce TBVs, summarizes the current status and advances and discusses the remaining challenges and approaches.
Copyright © 2015 Elsevier Ltd. All rights reserved.
... First, the largely intracellular malaria parasite becomes 'naked' upon entering mosquito midgut and remains accessible to intervening agents for up to 24 h. Second, a significant reduction in number occurs during each developmental succession from gametocytes, gametes, zygotes, ookinetes and finally oocysts (Alavi et al., 2003;Vaughan et al., 1992), indicating this as a biological bottleneck during parasite development (Sinden, 2010). The mosquito-stage transmission-blocking vaccines (TBVs), the focus of this chapter, are designed to induce antibody responses in vaccinees. ...
Movement of malaria across international borders poses a major obstacle to achieving malaria elimination in the 34 countries that have committed to this goal. In border areas, malaria prevalence is often higher than in other areas due to lower access to health services, treatment-seeking behaviour of marginalized populations that typically inhabit border areas, difficulties in deploying prevention programmes to hard-to-reach communities, often in difficult terrain, and constant movement of people across porous national boundaries. Malaria elimination in border areas will be challenging and key to addressing the challenges is strengthening of surveillance activities for rapid identification of any importation or reintroduction of malaria. This could involve taking advantage of technological advances, such as spatial decision support systems, which can be deployed to assist programme managers to carry out preventive and reactive measures, and mobile phone technology, which can be used to capture the movement of people in the border areas and likely sources of malaria importation. Additionally, joint collaboration in the prevention and control of cross-border malaria by neighbouring countries, and reinforcement of early diagnosis and prompt treatment are ways forward in addressing the problem of cross-border malaria.
Copyright © 2015 Elsevier Ltd. All rights reserved.
... Wuchereria sp. and Dirofilaria immitis (Russel et al. 2005) and avian parasite Plasmodium gallinaceum (Alavi et al. 2003). They enter buildings to feed and prefer to lay their eggs in artificial containers (Christopher 1960). ...
A survey of the larval population of tree hole breeding Aedes mosquito was conducted from January to December, 2010 in four different areas of Dhaka University Campus and its adjacent Suhrawardi park. A total of 689 mosquito larvae was collected during the survey. Among them 396 (57.47%) were Aedes albopictus, 51 (7.02%) were Ae. aegypti and 242 (35.12%) other Aedes species, and the number of male and female among them was counted 347 (50.36%) and 342 (49.63%), respectively. Among the varieties of plant species in the study area only 27 species of trees were found with 56 holes wherein 34 holes of 17 species trees were used by mosquitoes for breeding ground. The highest number (160) of larvae was found in bamboo (Bambusa nutans) stumps hole and lowest (13) in Delonix regia hole. The highest number of larval population was collected in September and the larval population increased from March to September and then gradually decreased. No mosquito larva was found in the tree holes in January and February. The number of larvae collected from different survey areas also varied which were 309 (44.84%), 152 (22.06%), 145 (21.04%) and 83 (12.04%) in Suhrawardi park, and the Arts building, Curzon hall and Botanical garden of Dhaka University, respectively. DOI: http://dx.doi.org/10.3329/bjz.v40i1.12887 Bangladesh J. Zool. 40(1): 1-11, 2012
... These Plasmodium species have been used in different laboratories for several years to infect An. gambiae, Anopheles funestus, An. quadrimaculatus and An. stephensi, all of which are malaria vectors in Africa and Asia, mainly due to the vectors' high susceptibility to infection with various malaria parasite species and strains (Yoeli et al. 1964, Vaughan et al. 1991, Alavi et al. 2003, Akaki & Dvorak 2005, Frischknecht et al. 2006, Hume et al. 2007, Lo & Coetzee 2013, Xu et al. 2013). ...
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.
Mosquitoes are the deadliest animal in the word, transmitting a variety of insect-borne infectious diseases, such as malaria, dengue fever, yellow fever, and Zika, causing more deaths than any other vector-borne pathogen. Moreover, in the absence of effective drugs and vaccines to prevent and treat insect-borne diseases, mosquito control is particularly important as the primary measure. In recent decades, due to the gradual increase in mosquito resistance, increasing attention has fallen on the mechanisms and effects associated with pathogen infection. This review provides an overview of mosquito innate immune mechanisms in terms of physical and physiological barriers, pattern recognition receptors, signalling pathways, and cellular and humoral immunity, as well as the antipathogenic effects of mosquito symbiotic bacteria. This review contributes to an in-depth understanding of the interaction process between mosquitoes and pathogens and provides a theoretical basis for biological defence strategies against mosquito-borne infectious diseases.
Graphical Abstract
Leucine-rich repeat (LRR) proteins are commonly involved in innate immunity of animals and plants, including for pattern recognition of pathogen-derived elicitors. The Anopheles secreted LRR proteins APL1C and LRIM1 are required for malaria ookinete killing in conjunction with the complement-like TEP1 protein. However, the mechanism of parasite immune recognition by the mosquito remains unclear, although it is known that TEP1 lacks inherent binding specificity. Here, we find that APL1C and LRIM1 bind specifically to Plasmodium berghei ookinetes, even after depletion of TEP1 transcript and protein, consistent with a role for the LRR proteins in pathogen recognition. Moreover, APL1C does not bind to ookinetes of the human malaria parasite Plasmodium falciparum , and is not required for killing of this parasite, which correlates LRR binding specificity and immune protection. Most of the live P . berghei ookinetes that migrated into the extracellular space exposed to mosquito hemolymph, and almost all dead ookinetes, are bound by APL1C, thus associating LRR protein binding with parasite killing. We also find that APL1C binds to the surface of P . berghei sporozoites released from oocysts into the mosquito hemocoel and forms a potent barrier limiting salivary gland invasion and mosquito infectivity. Pathogen binding by APL1C provides the first functional explanation for the long-known requirement of APL1C for P . berghei ookinete killing in the mosquito midgut. We propose that secreted mosquito LRR proteins are required for pathogen discrimination and orientation of immune effector activity, potentially as functional counterparts of the immunoglobulin-based receptors used by vertebrates for antigen recognition.
Anopheles darlingi is the main malarial vector in the Brazilian Amazon region. An. nuneztovari s.l., An. triannulatus s.l., An. evansae, and An. benarrochi s.l. do not have a defined role as malarial vectors, although they have been found to be naturally infected with Plasmodium vivax, and some develop oocysts. In this study, we evaluated the importance of low numbers of oocysts in sporozoite salivary gland invasion and transmission. Field-collected mosquitoes were experimentally infected with P. vivax. The infection rates and oocyst and sporozoite infection intensities were evaluated and compared with those of An. aquasalis. We found the highest number of oocysts in An. darlingi (mean = 39.47) and the lowest in An. nuneztovari s.l. (mean = 2). The highest number of sporozoites was observed in An. darlingi (mean = 610) and lowest in An. benarrochi s.l. (mean = 30). Plasmodium vivax DNA was detected in the saliva of all mosquito species after a blood meal. Regardless of the number of oocysts, all species transmitted sporozoites during blood meals. Considering the abundance of these mosquitoes and transmission of sporozoites, it is logical to assume that An. nuneztovari s.l. and An. triannulatus s.l. are involved in the transmission of P. vivax.
Avian malaria (Plasmodium) and related genera (Haemoproteus and Leucocytozoon) are diverse and widespread parasites. Despite the extent of knowledge on avian haemosporidian parasites, information about domestic and wild bird's blood parasites is overall insufficient in Iran. Prevalence of the haemosporidian parasites' and phylogenetic relationship of lineages are studied by using molecular and morphological results of 152 examined hosts belonging to 17 species. Molecular analysis for haemosporidian detections demonstrated overall prevalence 22.36%. Inspected hosts mostly belonging to Common Pigeons (Columba livia) parasitized by Haemoproteus spp., and Hooded Crows (Corvus cornix) and Carrion Crow (C. corone) were identified as hosting Plasmodium spp. Detected lineages COLIV03, COQUI05, LINN01, ROFI04 and SGS01 are identified as new reports from Iran. We detected no evidence of Leucocytozoon lineages, while the high prevalence of H. columbae was found in Common Pigeons. Such investigation on avian blood parasites contributes to providing new information on the prevalence, epidemiology and geographical distribution of haemosporidian parasites circulating in domestic, pets and wild birds.
Over the last few decades, a substantial number of anti-malarial effector genes have been evaluated for their ability to block parasite infection in the mosquito vector. While many of these approaches have yielded significant effects on either parasite intensity or prevalence of infection, just a few have been able to completely block transmission. Additionally, many approaches, while effective against the parasite, also disrupt or alter important aspects of mosquito physiology, leading to corresponding changes in lifespan, reproduction, and immunity. As the most promising approaches move towards field-based evaluation, questions of effector gene robustness and durability move to the forefront. In this forum piece, we critically evaluate past effector gene approaches with an eye towards developing a deeper pipeline to augment the current best candidates.
Within-host processes (representing the entry, establishment, growth, and development of a parasite inside its host) may play a key role in parasite transmission but remain challenging to observe and quantify. We develop a general model for measuring host defenses and within-host disease dynamics. Our stochastic model breaks the infection process down into the stages of parasite exposure, entry, and establishment and provides associated probabilities for a host's ability to resist infections with barriers and clear internal infections. We tested our model on Daphnia dentifera and the parasitic fungus Metschnikowia bicuspidata and found that when faced with identical levels of parasite exposure, Daphnia patent (transmitting) infections depended on the strength of internal clearance. Applying a Gillespie algorithm to the model-estimated probabilities allowed us to visualize within-host dynamics, within which signatures of host defense could be clearly observed. We also found that early within-host stages were the most vulnerable to internal clearance, suggesting that hosts have a limited window during which recovery can occur. Our study demonstrates how pairing longitudinal infection data with a simple model can reveal new insight into within-host dynamics and mechanisms of host defense. Our model and methodological approach may be a powerful tool for exploring these properties in understudied host-parasite interactions.
Malaria is a life‐threatening disease caused by one of the five species of Plasmodium, among which Plasmodium falciparum cause the deadliest form of the disease. Plasmodium species are dependent on a vertebrate host and a blood‐sucking insect vector to complete their life cycle. Plasmodium chitinases belonging to the GH18 family are secreted inside the mosquito midgut, during the ookinete stage of the parasite. Chitinases mediate the penetration of parasite through the peritrophic membrane, facilitating access to the gut epithelial layer. In this review, we describe Plasmodium chitinases with special emphasis on chitinases from P. falciparum and P. vivax, the representative examples of the short and long forms of this protein. In addition to the chitinase domain, chitinases belonging to the long form contain a pro‐domain and chitin‐binding domain. Amino acid sequence alignment of long and short form chitinase domains reveals multiple positions containing variant residues. A subset of these positions was found to be conserved or invariant within long or short forms, indicating the role of these positions in attributing form‐specific activity. The reported differences in affinities to allosamidin for P. vivax and P. falciparum were predicted to be due to different residues at two amino acid positions, resulting in altered interactions with the inhibitor. Understanding the role of these amino acids in Plasmodium chitinases will help us elucidate the mechanism of catalysis and the mode of inhibition, which will be the key for identification of potent inhibitors or antibodies demonstrating transmission‐blocking activity. This article is protected by copyright. All rights reserved.
Infection with malarial parasites renders hosts more mosquito attractive than their uninfected, healthy, counterparts. One volatile organic compound, α-pinene, is associated with Plasmodium spp. infection in multiple studies and is a known mosquito attractant. However, how malarial infection results in elevated levels of host-associated α-pinene remains unclear. One study suggests that erythrocyte exposure to the malarial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), results in increased levels of α-pinene. Here, we establish that endogenous levels of α-pinene are present in human erythrocytes, that these levels vary widely by erythrocyte donor, and that α-pinene levels are not altered by HMBPP treatment.
Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and propose that the mosquito complement-like system contributes to the species-specific dynamics of Plasmodium invasion.
The mosquito’s immune blood cells, ‘hemocytes’ imparts a highly selective immune response against various micro‐organisms/pathogens. Among several immune effectors, FREPs (Fibrinogen related proteins) have been recognized as key modulator of cellular immune responses; however, their physiological relevance has not been investigated in detail. Our ongoing comparative RNA‐Seq analysis identified a total of 13 FREPs originating from naïve sugar‐fed, blood‐fed, bacterial challenged, and Plasmodium vivax‐infected hemocytes in the mosquito Anopheles stephensi hemocytes. Transcriptional profiling of the selected seven FREP transcripts showed distinct responses against different pathophysiological conditions, where an exclusive induction of FREP12 after 10 days of P. vivax infection, suggesting its possible role against free circulating sporozoites for future exploration. When challenged with live bacterial injection in the thorax, we observed a higher affinity of FREP13 and FREP65 towards gram‐negative and gram‐positive bacteria in the mosquito hemocytes, respectively. Furthermore, we observed increased bacterial survival and proliferation, which is likely compromised by the downregulation of TEP1, in FREP13 mRNA depleted mosquito hemolymph. On the contrary, post‐blood‐feeding, we also noticed a significant delay of 24h in the enrichment of gut endosymbionts in the FREP13 silenced mosquitoes. Taken together, we conclude that hemocyte‐specific FREP13 carries the unique ability of tissue‐specific regulation, having an antagonistic antibacterial role in the hemolymph, and an agonistic role against gut endosymbionts.
Advances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood-feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito-microbiome studies and highlights an understudied source of bioactive small molecules.
Avian Plasmodium is of special interest to health care scientists and veterinarians due to the potency of causing avian malaria in non-adapted birds and their evolutionary phylogenetic relationship with human malaria species. This article aimed to provide a comprehensive list of the common avian Plasmodium parasites in the birds and mosquitoes, to specify the common Plasmodium species and lineages in the selected regions of West of Asia, East of Europe, and North of Africa/Middle East, and to determine the contribution of generalist and host-specific Plasmodium species and lineages. The final list of published infected birds includes 146 species, among which Passer domesticus was the most prevalent in the studied areas. The species of Acrocephalus arundinaceus and Sylvia atricapilla were reported as common infected hosts in the examined regions of three continents. The highest numbers of common species of infected birds between continent pairs were from Asia and Europe, and no common record was found from Europe and Africa. The species of Milvus migrans and Upupa epops were recorded as common species from Asia and Africa. The lineage of GRW11 and species of P. relictum were the most prevalent parasites among all the infection records in birds. The most prevalent genus of vectors of avian malaria belonged to Culex and species of Cx. pipiens. The lineage SGS1 with the highest number of occurrence has been found in various vectors comprising Cx. pipiens, Cx. modestus, Cx. theileri, Cx. sasai, Cx. perexiguus, Lutzia vorax, and Culicoides alazanicus. A total of 31 Plasmodium species and 59 Plasmodium lineages were recorded from these regions. SGS1, GRW04, and GRW11, and P. relictum and P. vaughani are specified as common generalist avian malaria parasites from these three geographic areas. The presence of avian Plasmodium parasites in distant geographic areas and various hosts may be explained by the movement of the infected birds through the migration routes. Although most recorded lineages were from Asia, investigating the distribution of lineages in some of the countries has not been done. Thus, the most important outcome of this review is the determination of the distribution pattern of parasite and vector species that shed light on gaps requiring further studies on the monitoring of avian Plasmodium and common vectors extension. This task could be achieved through scientific field and laboratory networking, performing active surveillance and designing regional/continental control programs of birds' malaria and other zoonotic diseases.
In this paper, we developed a novel deterministic coupled model tying together the effects of within-host and population level dynamics on malaria transmission dynamics. We develop within-host and within-vector dynamic models, population level between-hosts models, and a nested coupled model combining these levels. The unique feature of this work is the way the coupling and feedback for the model use the various life stages of the malaria parasite both in the human host and the mosquito vector. Analysis of the coupled and the within-human host models indicate the existence of locally asymptotically stable infection- and parasite-free equilibria when the associated reproduction numbers are less than one. The population-level model, on the other hand, exhibits backward bifurcation, where the stable disease-free equilibrium co-exists with a stable endemic equilibrium. A global sensitivity analysis was carried out to measure the effects of the sensitivity and uncertainty in the various model parameters estimates. The results indicate that the most important parameters driving the pathogen level within an infected human are the production rate of the red blood cells from the bone marrow, the infection rate, the immunogenicity of the infected red blood cells, merozoites and gametocytes, and the immunosensitivity of the merozoites and gametocytes. The key parameters identified at the population level are the human recovery rate, the death rate of the mosquitoes, the recruitment rate of susceptible humans into the population, the mosquito biting rate, the transmission probabilities per contact in mosquitoes and in humans, and the parasite production and clearance rates in the mosquitoes. Defining the feedback functions as a linear function of the mosquito biting rate, numerical exploration of the coupled model reveals oscillations in the parasite populations within a human host in the presence of the host immune response. These oscillations dampen as the mosquito biting rate increases. We also observed that the oscillation and damping effect seen in the within-human host dynamics fed back into the population level dynamics; this in turn amplifies the oscillations in the parasite population within the mosquito-host.
Background
Plasmodium berghei is a rodent malaria parasite and has been very valuable means in the progress of our understanding of the essential molecular and cellular biology of the malaria parasites. Availability of hosts such as mice and vectors such as Anopheles stephensi has made this parasite a suitable system to study the parasite-host and vector-parasite relationships.
Methods
This study was performed at Medical Entomology and Parasitology laboratories of the School of Public Health, Tehran University of Medical Sciences, Iran in 2016. The investigation was carried out to describe life cycle and parameters influencing maintenance of the parasite within the mice or the mosquito.
Results
Results have revealed details and addressed some parameters and points influence maintenance of various life stages of the parasite including merozoites, macrogametocytes, ookinetes, oocysts and sporozoites in the laboratory model P. berghei–A. stephensi-BALB/c mouse. Injection of fresh infected blood results in higher gametocytemia in the animals. The more injected parasites result in earlier and higher parasitemia and exfelagellation centers in the mice blood. However, the highest number of infected mosquitoes and oocysts formation were observed when the parasitemia and exflagellation centers per microscopic field were 9% and 3.6 in the infected mice respectively. The infected mosquitoes should be maintained on 8% (w/v) fructose, 0.05% (w/v) PABA at 20±1 °C and 50%–80% relative humidity.
Conclusion
This study helps to understand the biology of vertebrate-parasite and mosquito-malaria interactions that may aid in the development of a new generation of drug/vaccine and vector-based measures for malaria control.
Each year malaria kills hundreds of thousands of people and infects hundreds of millions of people despite current control measures. An effective malaria vaccine will likely be necessary to aid in malaria eradication. Vaccination using whole sporozoites provides an increased repertoire of immunogens compared to subunit vaccines across at least two life cycle stages of the parasite, the extracellular sporozoite, and intracellular liver stage. Three potential whole sporozoite vaccine approaches are under development and include genetically attenuated parasites, radiation attenuated sporozoites, and wild-type sporozoites administered in combination with chemoprophylaxis. Pre-clinical and clinical studies have demonstrated whole sporozoite vaccine immunogenicity, including humoral and cellular immunity and a range of vaccine efficacy that depends on the pre-exposure of vaccinated individuals. While whole sporozoite vaccines can provide protection against malaria in some cases, more recent studies in malaria-endemic regions demonstrate the need for improvements. Moreover, challenges remain in manufacturing large quantities of sporozoites for vaccine commercialization. A promising solution to the whole sporozoite manufacturing challenge is in vitro culturing methodology, which has been described for several Plasmodium species, including the major disease-causing human malaria parasite, Plasmodium falciparum. Here, we review whole sporozoite vaccine immunogenicity and in vitro culturing platforms for sporozoite production.
This chapter examines the biology of the infectious gametocyte in the vertebrate host, of the sexual and sporogonic development in the mosquito vectors, and of the delivery of infectious sporozoites into the proboscis of the mosquito. The growing understanding of how the biology of the parasites and their hosts affect each of the component variables in this formula may facilitate the future design and implementation of new and effective transmission-blocking interventions that can be applied in the diverse endemic situations encountered worldwide. The chapter attempts to identify what are currently believed to be key aspects in these sciences in the hope that this knowledge may promote the development of rational and sustainable transmission-blocking interventions. Other analyses have described the comparative insensitivity of the membrane-feeding assay compared to the direct skin-feed assay, while simultaneously recognizing the eminent utility of the latter in that multiple, and comparative, observations can be made on gametocytes from a single source.
Background & objectives: Under certain ecological circumstances, pathogens are able to rapidly adapt to new
vectors. The great capacity of Plasmodium spp. to adapt to new anopheline mosquito vectors on different continents and the continuous ecological changes attributed to humans might promote their adaptation to culicine vectors, which are known to infect humans. Based on our current knowledge, it is difficult to predict whether such adaptations will occur. This study was aimed to determine the infection susceptibility of Anopheles sinensis, Culex tritaeniorhynchus and Cx. pipiens pallens to Plasmodium vivax in Shandong Province of China.
Methods: The susceptibility of the three predominant species of mosquitoes —An. sinensis, Cx.tritaeniorhynchus and Cx. pipiens pallens in Shandong Province was compared with a direct membrane feeding assay with 15 batches of Shandong strain mono-infected gametocyte-containing blood collected from Plasmodium vivax-infected patients. Infectivity was measured by dissecting the midguts and salivary glands of the mosquitoes. The presence of oocysts and sporozoites was determined by microscopy at 6 and 22 days post-blood feeding.
Results: From the 15 batches of mosquitoes that were fed infected blood, oocysts and sporozoites were detected only in 7th, 13th and 15th batches of infection for An. sinensis, and no oocysts or sporozoites were detected in Cx. tritaeniorhynchus or Cx. pipiens pallens. The positive rate of An. sinensis infection was 21.2, 13 and 36.3% in the three batches of mosquitoes, with an average infection rate of 23.5%.
Interpretation & conclusion: The susceptibility of An. sinensis to P. vivax was very high in Shandong Province.
Cx. tritaeniorhynchus and Cx. pipiens pallens failed to exhibit susceptibility to P. vivax.
Studies of the lowland avifauna in the Neotropical Region have shown a paucity of Leucocytozoon species. However, surveys conducted in the Colombian highlands revealed a great diversity of these parasites infecting resident birds. To further investigate the relationship between Leucocytozoon diversity, the potential vectors, and altitudinal distribution, birds from 41 families were sampled at low and high elevations in Colombia. Blood samples were screened by microscopy, and a fragment of cytochrome b was amplified from Leucocytozoon-positive samples. The complete mitochondrial genome was also obtained for each morphospecies of Leucocytozoon. Leucocytozoon species were detected in resident birds, with various degrees of host specificity, at elevations from 2,400 to 3,950 meters above sea level, where five new host-parasite associations were discovered. Phylogenetic analysis based on the cytochrome b fragment suggested that two nominal taxa, L. fringillinarum and L. majoris, are species complexes. Blood sources of Simuliidae revealed generalist-feeding habits that included avian and mammalian hosts. Molecular analysis of parasites in black flies indicated a close relationship with the parasites found in birds. Our investigation provides further evidence that the distribution and transmission of Leucocytozoon species in the Neotropics are influenced by elevation, with the highest prevalence between 2,400 and 3,200 m asl.
Mosquitoes are responsible for the transmission of diseases with a serious impact on global human health, such as malaria and dengue. All mosquito-transmitted pathogens complete part of their life cycle in the insect gut, where they are exposed to mosquito-encoded barriers and active factors that can limit their development. Here we present the current understanding of mosquito gut immunity against malaria parasites, filarial worms, and viruses such as dengue, Chikungunya, and West Nile. The most recently proposed immune mediators involved in intestinal defenses are discussed, as well as the synergies identified between the recognition of gut microbiota and the mounting of the immune response.
Microsporidia are obligate intracellular parasites characterized by a unique invasive mechanism, and, in some species, by a highly compact nuclear genome. To improve our knowledge of gene expression in these opportunistic pathogens, experiments on transcriptional regulation mechanisms were undertaken. In the microsporidia Encephalitozoon cuniculi (a mammalian parasite with a genome size of 2.9 Mb), we demonstrated a polycistronic gene organization. At the time of transcription, the transcripts are randomly processed leading to an mRNA population composed of monocistronic and polycistronic mRNA. To study the structure of microsporidian genomes and their adaptation capacities, the sequencing of Brachiola algerae genome (an insect and mammalian microsporidia) has been initiated in collaboration with Genoscope. Thirty chromosome bands from 160 to 2240 kb were identified, giving a haploid genome size estimated of 23 Mb. The study of the chromosomal distribution of the 16S rRNA unit showed the presence of 16S copy on at least 9 chromosome bands. The first analysis of this AT rich genome (more than 75%) revealed the presence of different kinds of transposable elements as well as genes encoding for enzymes involved in the RNA interference process. We further demonstrated that two Anopheles gambiae cellular lineages Sua4.0 (haemocytic cell) and Mos 55 (embryonic cells) can be infected by B. algerae providing a model to study in vitro the interaction between An. gambiae and its pathogens. The successful use of the RNA interference in these two types of cells will contribute to a better understanding of the adaptation mechanisms of pathogens like P. falciparum and B. algerae.
This study elucidated the influence of blood meal type on reproductive performance of Anopheles gambiae s.s. Starting from three days old, An. gambiae mosquitoes, from a laboratory colony maintained following1 standard anopheline rearing techniques, were offered seven different blood meal types, through an artificial membrane. Thereafter, the mosquitoes were monitored for pre-larval and immature stage reproductive attributes. The results indicated that except for Embryony Period (EP) and Pupal Stage Duration (PSD), all attributes investigated varied significantly (p<0.05) among blood meals. While Blood Feeding Rate (BFR) was significantly highest and least in groups of mosquitoes fed with human (Mean = 94.69±3.36%) and pig (Mean = 48.54±10.13%) blood meals, respectively; the shortest Pre-oviposition Period (POP) were recorded among mosquitoes given human and cattle blood meals (Mean = 3.96±0.88 and 4.30±1.25 days, respectively). Also, human blood meal was the most ideal for Oviposition Rate (OR) (Mean = 79.20±12.39%) while chicken and pig blood meals were the least favourable (Mean = 46.80±9.55 and 48.05±6.34%, respectively). Fecundity (F) had similar distribution pattern as BFR while Egg Hatching Rate (HER) was generally low (<60%), irrespective of blood meal type. While Total Immature Stage Duration (TISD) varied widely among blood meal types, Total Larval Stage Duration (TLSD) was only significantly (p<0.05) longest in mosquitoes fed with pig blood meal (12.33±1.63 days). The epidemiological implications of these results were highlighted and discussed.
evelopment of effective strategies to control malaria transmission is an important issue in malaria research. This study aimed to investigate refractoriness of laboratory and field strains of the malaria vector Culex quinquefasciatus to the avian malaria parasite Plasmodium gallinaceum, a model of the human malaria disease. Transmission potential was determined by feeding batches of Cx. quinquefasciatus on P. gallinaceum infected chickens with 10% and 30% parasitemia. The mosquitoes were examined for percent infectivity and numbers of oocysts. Our study showed that when the mosquitoes were fed on the infected chickens with high parasitemia, the laboratory isolate of Cx. quinquefasciatus was more susceptible to P. gallinaceum transmission and produced significantly higher oocyst numbers than the field isolate. There were, however, no differences in term of the transmission potential and infectivity of the malaria parasite when the mosquitoes were allowed to feed on the P. gallinaceum-infected chickens with low parasitemia. In conclusion, our study demonstrated the differences in refractoriness of the natural vector Cx. quinquefasciatus to the transmission of the avian malaria P. gallinaceum, implying the existence of the natural pathogen resistance mechanisms in the Culex mosquito.
Green fluorescent protein (GFP) is a well-established reporter protein for the examination of biological processes. This report describes a recombinant Plasmodium berghei, PbGFPCON, that constitutively expresses GFP in a growth responsive manner in its cytoplasm from a transgene that is integrated into the genome and controlled by the strong promoter from a P. berghei elongation factor-1α gene. All life cycle forms of PbGFPCON except for male gametes can be easily visualized by fluorescent microscopy. PbGFPCON showed similar growth characteristics to wild type P. berghei parasites throughout the whole life cycle and can therefore be used as a reference line for future investigations of parasite–host cell interactions. The principle of automated fluorescence-based counting and sorting of live parasites from host cell backgrounds and different parasite forms from complex mixtures such as asynchronous blood stages is established. PbGFPCON allows the visualization and investigation of live parasite stages that are difficult and labor-intensive to observe, such as the liver and mosquito stages. PbGFPCON can be employed to establish the phenotype of independent mutant parasites. With the recent development of a second, independent selectable marker in P. berghei, PbGFPCON is a useful tool to investigate the effect of further genetic modifications on host–parasite interactions.
Nearly 1200 species of Aedes have been described and given a formal Latin name. These species can be found in diverse habitats from Polynesian islands and African deserts to ice pools at the Arctic Circle. The larval nutrition and adult female host preference for blood-feeding is equally variable. Mating behaviours and preference for oviposition substrate are also distinctive for a given species. Considering first, the number of species and then, the diversity in habitat, nutrition and behaviour among these species, a single, simple rearing method cannot be devised that will be successful for all of them. In a recent manual of mosquito rearing, 59 species of Aedes are listed with methods for laboratory rearing (Gerberg et al., 1994). This listing includes species that are difficult or impossible to maintain in continuous colony. This is undoubtedly a very conservative estimate of the species that have been colonized but, even so, it indicates that far less than 10% of the described species have been successfully colonized.
The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding
the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to
invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading
the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions.
We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene,PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone
derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC50 of 7 μm and differentially inhibited two chromatographically separable P. gallinaceumookinete-produced chitinase activities with IC50 values of 7 and 12 μm, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that theP. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion.
The population dynamics of cultured Plasmodium falciparum parasites was examined during their sporogonic development in Anopheles gambiae mosquitoes. Estimates of absolute densities were determined for each life stage, and life tables were constructed for each of 38 experimental infections. Macrogametocyte and ookinete mortalities contributed equally to the overall mortality. On average, there was a 40-fold decrease in parasite numbers in the transition from the macrogametocyte to the ookinete stage, a 69-fold decrease in the transition from ookinete to oocyst stages, and a total net decrease in parasite numbers from macrogametocyte to oocyst stage of 2,754-fold (i.e., multiplicative). There was no relationship between macrogametocyte and ookinete densities due to the inherent variability in fertility among different gametocyte cultures. There was a curvilinear relationship (r2 = 0.66) between ookinete and oocyst densities. Above a threshold of about 30 ookinetes/mosquito, the oocyst yield per ookinete became increasingly greater with increasing ookinete density. There was a linear relationship (r2 = 0.73) between oocyst and sporozoite densities, with an average of 663 salivary gland sporozoites produced per oocyst. Sporozoite production per oocyst was not affected by oocyst density and virtually all oocyst infections resulted in sporozoite infections of the salivery glands. This quantitative study indicates that the sporogony of cultured P. falciparum in laboratory-infected A. gambiae is an inefficient process and that the ookinete is the key transitional stage affecting the probability of vector infectivity.
Plasmodium gallinaceum develops up to the ookinete stage in the non-compatible mosquito, Anopheles stephensi, this development occurring over the same time period and with the same success as in the compatible vector, Aedes aegypti. The slower digestive rate in An. stephensi, smaller blood-meal and lower enzyme activity than in Aedes, and an acceleration of the formation of the peritrophic membrane (PM) do not inhibit the development of the parasite. However, the ookinetes are not able to penetrate the midgut epithelium. The role of blood, vector and parasite in establishing a parasite infection in the mosquito are all considered. In particular, the role of vector-parasite recognition mechanisms in determining vector-parasite specificity are discussed.
Sporogonic development of Plasmodium yoelii yoelii 17XNL was examined in 5 species of Anopheles mosquitoes; A. albimanus, A. dirus, A. freeborni, A. gambiae, and A. stephensi. The kinetics of ookinete formation differed among species. In A. freeborni, A. gambiae, and A. stephensi, mature ookinetes formed synchronously at 8 hr, then quickly subsided. In A. albimanus and A. dirus, ookinete formation was more protracted, and ookinete densities peaked from 12 to 24 hr. Losses in parasite abundance during the conversion of ookinetes to oocysts were similar between A. dirus and A. gambiae (55- and 41-fold losses, respectively) but were an order of magnitude less in A. stephensi (1.3-fold loss). Ookinete conversion to oocysts in A. albimanus was nil. Melanotic encapsulation of oocysts occurred in 25-30% of infected A. gambiae and A. dirus. Melanized parasites in A. gambiae at days 7-10 were small (10 microns diameter) and retort-shaped, whereas melanized parasites in A. dirus were generally as large as normal oocysts (60 microns) and many were incompletely melanized. Melanotic encapsulation did not occur in A. stephensi, A. freeborni, or A. albimanus. On day 16, sporozoites were present in the salivary glands of A. freeborni, A. gambiae, and A. stephensi, but only half of mosquitoes were mature oocysts also had gland infections. When present in the glands, sporozoites were successfully transmitted to mice via mosquito bite. Parasite populations were not normally distributed in any mosquito species but were adequately described by a negative binomial type of distribution.
Wild-caught Anopheles gambiae s. l. and An. funestus were dissected and their midguts were examined for the presence of Plasmodium falciparum oocyst infections. The mean intensity of infection and the prevalence of infected mosquitoes were determined for each sample, with one sample representing the mosquitoes caught in a single house at any given time. The patterns of infection were investigated using the relationships between prevalence, intensity, and variance within samples, and were found to be consistent with laboratory infections. The overall distribution of oocysts is characterized by a mixture of negative binomial distributions with means determined by the infectiousness of the human hosts, and a constant degree of aggregation (k = 0.0767) presumably determined by the development of oocysts within mosquitoes. The prevalence/intensity relationship was treated as a bivariate distribution to ascertain the effect of sample size on the accuracy of estimation, and to allow inference of intensity from prevalence. In mathematical models fitted to the collected data, sample size affected directly the minimum possible prevalence of infection, and the accuracy of both mean and prevalence estimations. Based on minimum possible positive prevalence rates, data from samples of less than 20-25 mosquitoes would provide unacceptable errors in prevalence estimations. However, natural oocyst rates are consistently higher than the minimum prevalence, and it is suggested that any interpretations from samples of less than approximately 40 mosquitoes must be treated with some caution. Such variation in natural samples means that prediction of intensity of infection from prevalence (or vice versa) is extremely inaccurate.
Developmentally arrested malarial gametocytes undergo gamete formation in the mosquito midgut immediately after ingestion of the infected bloodmeal. In the rodent malaria parasite Plasmodium berghei male gametogenesis (exflagellation) can be induced in vitro by a temperature decrease (from 39 degrees C in the vertebrate host to 20 degrees C) and a concomitant pH increase (from 7.3 in mouse blood to 8.0). We report the presence of additional Gametocyte Activating Factor(s) (GAF) present in Anopheles stephensi tissue extracts, which induce both male and female gametogenesis at the otherwise nonpermissive pH of 7.3 in vitro but are unable to overcome the low temperature requirement. All constituent cellular events of microgametogeneis studied here are induced by the same triggers in vitro. A temperature decrease is also required for exflagellation in the mosquito midgut. The possible role of GAF as a second obligatory natural trigger of gametogenesis is discussed.
We have discovered that the mosquito Anopheles stephensi, a natural vector of human malaria, limits parasite development with inducible synthesis of nitric oxide (NO). Elevated expression of A. stephensi NO synthase (NOS), which is highly homologous to characterized NOS genes, was detected in the midgut and carcass soon after invasion of the midgut by Plasmodium. Early induction is likely primed by bacterial growth in the blood meal. Later increases in A. stephensi NOS expression and enzyme activity occurred at the beginning of sporozoite release. Circulating levels of nitrite/nitrate, end-products of NO synthesis, were significantly higher in Plasmodium-infected mosquitoes. Dietary provision of the NOS substrate L-arginine reduced Plasmodium infections in A. stephensi. In contrast, dietary provision of a NOS inhibitor significantly increased parasite numbers in infected mosquitoes, confirming that A. stephensi limits Plasmodium development with NO.
The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions. We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that the P. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion.
Malaria is a devastating public health menace, killing over one million people every year and infecting about half a billion.
Here it is shown that the protozoan Plasmodium gallinaceum, a close relative of the human malaria parasite Plasmodium falciparum, can develop in the fruit fly Drosophila melanogaster. Plasmodium gallinaceum ookinetes injected into the fly developed into sporozoites infectious to the vertebrate host with similar kinetics as seen
in the mosquito host Aedes aegypti. In the fly, a component of the insect's innate immune system, the macrophage, can destroy Plasmodia. These experiments suggest that Drosophila can be used as a surrogate mosquito for defining the genetic pathways involved in both vector competence and part of the parasite
sexual cycle.
The purpose of this introduction is to describe in outline only the salient aspects of the biology of Plasmodium and the mosquito vector that are required to understand the rationale of the protocols given. For a more detailed analysis the reader is directed to the comprehensive works on the subject published by Shute and Maryon (1966), Vanderberg and Gwadz (1980), Sinden (1984) and Carter and Graves (1988). An in-depth review of the complex factors regulating the transmission of Plasmodium to the mosquito (Sinden et al., 1996) may also be useful.
This study investigated the successive losses in the parasite densities of #Plasmodium falciparum$ stages during the early sporogony in laboratory-reared #Anopheles gambiae$ infected by membrane feeding with blood from naturally infected gametocyte carriers (> 50 gametocytes/mm3). The developmental stages of #P. falciparum$ in the mosquito were studied from zygote to oocyst, by immunofluorescent method using monoclonal antibodies against the Pfs25 protein present on the surface of newly formed gametes. This method allows for assessment of the various sporogonic stages before, during and after passage of the midgut wall. Parasite densities were determined within the entire blood meal at 3 h (zygotes and macrogametes) and 24 h (ookinetes) post-infection. At 48 h after the mosquito blood meal, midguts were checked for the presence of early oocysts. For the mid-size oocysts count, classic microscopy examination was used at day 7 post-infection. The parasite efficacy was estimated by following successive losses in parasite densities between different early stages of the sporogonic cycle in #A. gambiae$. Thirty-seven experimental infections were realized with high gametocyte densities, ranging from 64 to 2392 gametocytes/mm3. All gametocyte carriers showed infection with round forms 100% ; ookinetes were found in 91.9%. The prevalences of infections with oocysts were 48.6% at day 2 (young oocyst) and 37.8% at day 7 (mid-size oocyst). The mean densities per mosquito for each parasite stage were 12.6 round forms, 5.5 ookinetes, 1.8 young oocyst and 2 mid-size oocysts. Significant correlations were found between two consecutive parasite stages (round forms/ookinetes, ookinetes/young oocysts, young oocysts/mid-size oocysts) and between round forms and mid-size oocysts. The mean parasite density significantly decreased between round forms and ookinetes (41.6%) and between ookinetes and young oocysts (61.4%)... (D'après résumé d'auteur
Gametogenesis of Plasmodium in vitro can be induced by the combined stimulus of a 5 °C fall in temperature and the presence of xanthurenic acid (XA). In-vitro experiments showed that P. gallinaceum (EC50=80 nM) is much more sensitive to XA than P. berghei (9 μM), P. yoelii (8 μM), and P. falciparum (2 μM). However, in the mosquito vector, we do not know whether the temperature shift and XA are the only gametocyte-activating factors (GAF), nor do we know with certainty the true source(s) of XA in the mosquito blood meal. Previous studies indicate that XA is the only source of GAF in the mosquito. By defining, and then contrasting, the ability of an XA-deficient mutant of Aedes aegypti, with the wild-type mosquito to support exflagellation and ookinete formation in vivo, we determined the roles of parasite-, mosquito- and host blood-derived GAF in the regulation of gametogenesis of P. gallinaceum. Removal of both host and vector sources of GAF totally inhibited both exflagellation and ookinete production, whilst the lack of either single source resulted in only a partial reduction of exflagellation and ookinete formation in the mosquito gut. Both sources can be effectively replaced/substituted by synthetic XA. This suggests (1) both mosquito- and vertebrate-derived factors act as GAF in the mosquito gut in vivo; (2) the parasite itself is unable to produce any significant GAF activity. Studies are underway to determine whether vertebrate-derived GAF is XA. These data may form the basis of further studies of the development of new methods of interrupting malarial transmission.
In this paper, Peter Billingsley and Robert Sinden examine the specificity of interactions between mosquito vectors and malaria parasites as areas for the design of rational intervention strategies.
Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were homogenized and microvillar membranes prepared by calcium precipitation and differential centrifugation. Oligosaccharides present on the microvillar glycoproteins were identified by lectin blotting before and after in vitro and in situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins expressed a structurally restricted range of terminal sugars and oligosaccharide linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid N-linked oligosaccharides, some with α1–6 linked fucose as a core residue. Complex-type N-linked oligosaccharides on eight glycoproteins all possessed terminal N-acetylglucosamine, and α- and β-linked N-acetylgalactosamine. Eight glycoproteins expressed O-linked oligosaccharides all containing N-acetylgalactosamine with or without further substitutions of fucose and/or galactose. Galactoseβ1–3/4/6N-acetylglucosamine-, sialic acidα2–3/6galactose-, fucoseα1–2galactose- and galactoseα1–3galactose- were not detected. Terminal α-linked N-acetylgalactosamine residues on N-linked oligosaccharides are described for the first time in insects. The nature and function of these midgut glycoproteins have yet to be identified, but the oligosaccharide side chains are candidate receptors for ookinete binding and candidate targets for transmission blocking strategies
Malaria is transmitted from vertebrate host to mosquito vector by mature sexual blood-living stages called gametocytes. Within seconds of ingestion into the mosquito bloodmeal, gametocytes undergo gametogenesis. Induction requires the simultaneous exposure to at least two stimuli in vitro: a drop in bloodmeal temperature to 5 degrees C below that of the vertebrate host, and a rise in pH from 7.4 to 8.0-8.2. In vivo the mosquito bloodmeal has a pH of between 7.5 and 7.6. It is thought that in vivo the second inducer is an unknown mosquito-derived gametocyte-activating factor. Here we show that this factor is xanthurenic acid. We also show that low concentrations of xanthurenic acid can act together with pH to induce gametogenesis in vitro. Structurally related compounds are at least ninefold less effective at inducing gametogenesis in vitro. In Drosophila mutants with lesions in the kynurenine pathway of tryptophan metabolism (of which xanthurenic acid is a side product), no alternative active compound was detected in crude insect homogenates. These data could form the basis of the rational development of new methods of interrupting the transmission of malaria using drugs or new refractory mosquito genotypes to block parasite gametogenesis.
Blood digestion of Aedes aegypti and development of Plasmodium gallinaceum were shifted against each other by giving the mosquitoes two consecutive blood meals. In this way the parasites were exposed to an environment where blood digestion was more advanced than in single blood meals. This procedure had an inhibiting effect on oocyst production when the two blood meals overlapped; an enhancing effect when they were well separated. The results are explained by the action of trypsin-like proteases on the parasites and indicate that plasmodia 0-10 h after blood meal are more sensitive to the enzymes than later stages of the parasites.
After exposure to extracts from blood fed A. aegupti cultured ookinetes of P. gallinaceum were damaged to various, defined extents. Immature ookinetes were found to be more sensitive to damage than mature ones. The damage was dependent on the digestion time after which the Aedes extracts had been prepared and could be correlated with the proteolytic activity in the extracts. Control experiments demonstrated that the factors responsible for damage were neither present in unfed mosquitoes nor in blood alone and that the damage was not a result of osmotic stress. After the treatment of the Aedes extracts with lima bean trypsin inhibitor the ookinete damage was much less, indicating that the Aedes trypsin was the major agent of damage. These results were supported by experiments in which the tryptic activity of the extracts was eliminated by thermal denaturation. It is concluded that in the mosquito midgut most of the ookinetes are damaged by digestive enzymes and that this is one factor leading to the discrepancy between the number of ingested macrogametocytes and the number of oocysts which is usually found in nature. It seems that the only ookinetes which have a chance of surviving are those which develop in the centre of the blood clot, away from the site of enzyme action.
Phagocytosis of the extracellular gametocytes of Plasmodium falciparum has been demonstrated in vitro, and in vivo in the bloodmeal within the mosquito midgut. Up to 84% of the gametocytes were ingested within two hours in vitro, compared to 7% in the same period in the mosquito gut.
Secretion and luminal formation of the peritrophic membrane (PM) were induced in female Anopheles stephensi and Aedes aegypti by feeding the mosquitoes on a warmed suspension of latex particles in Ringer's solution. The PM in A. stephensi was produced from apical secretion vesicles stored in the midgut epithelial cells and secreted into the lumen during feeding. In A. aegypti, the PM was formed de novo. When the latex feeding was followed 24 hr later by a meal of lyophilized pig blood, the 2 mosquito species exhibited very different modifications to their PM structure; in A. stephensi no PM was formed around the blood meal, whereas de novo synthesis of the PM in A. aegypti continued during the blood meal, with the resulting PM greatly thickened compared to the normal feeding. This artificial induction of PM formation was used as the basis to study the role of the PM in blood meal digestion and in infectivity of mosquitoes by the appropriate species of Plasmodium. The feeding of a latex suspension alone had no stimulatory effect on the 2 major midgut proteases, trypsin and aminopeptidase, in either species. After a blood meal alone, proteases rose to maximum activity at 30 hr and 24 hr after feeding in A. stephensi and A. aegypti, respectively. After double feeding, protease activities in both species were almost identical to those in blood-fed mosquitoes. Neither the absence of a PM (in A. stephensi) nor the presence of a thickened PM (in A. aegypti), therefore, has any effect on the ability of mosquitoes to digest a blood meal. Malaria infectivity, measured by oocyst counts, also was compared after normal and double feeding using infective blood meals. Infectivity of A. stephensi by Plasmodium berghei was unaffected by the presence or absence of the PM. The thickened PM produced by double feeding in A. aegypti caused a reduction of midgut infectivity by Plasmodium gallinaceum. These results suggest that the PM may act as a partial, but not an absolute, barrier to invasion of the midgut by the ookinete.
Malaria parasites (ookinetes) appear to digest the peritrophic membrane in the mosquito midgut during penetration. Previous studies demonstrated that lectins specific for N-acetylglucosamine bind to the peritrophic membrane and proposed that the membrane contains chitin [Rudin, W. & Hecker, H. (1989) Parasitol. Res. 75, 268-279]. In the present study, we show that the peritrophic membrane is digested by Serratia marcescens chitinase (EC 3.2.1.14), leading to the release of N-acetylglucosamine and fragmentation of the membrane. We also report the presence of a malaria parasite chitinase that digests 4-methylumbelliferyl chitotriose. The enzyme is not detectable until 15 hr after zygote formation, the time required for maturation of the parasite from a zygote to an ookinete, the invasive form of the parasite. At 20 hr, the enzyme begins to appear in the culture supernatant. The chitinase extracted from the parasite and found in the culture supernatant consists of a major band and two minor bands of activity on native polyacrylamide gel electrophoresis. The presence of chitin in the peritrophic membrane, the disruption of the peritrophic membrane during invasion, and the presence of chitinase in ookinetes suggest that the chitinase in ookinetes is used in the penetration of the peritrophic membrane.
Blood digestion was studied in strains of Anopheles stephensi which had been genetically selected for either refractoriness or susceptibility to infection by Plasmodium falciparum. Females of the refractory Pb3-9a strain ingested more blood than selected (Sda-500) and unselected (Punjab) susceptible females and began to degrade the haemoglobin soon after feeding. In susceptible females, haemoglobin degradation started only after a significant post-feeding lag period. Total protein content of the midgut after the bloodmeal was correspondingly higher for refractory than for susceptible females, but absolute and relative rates of protein degradation were not significantly different between the different mosquito strains. Bloodmeal induction of midgut trypsin activity and the maximal trypsin activity were the same for the different strains. The residual aminopeptidase activity and its relative post-feeding activity (enzyme units per midgut) were significantly higher in refractory females. However, when converting to specific aminopeptidase activity, no differences between strains were evident. The results indicate that both the early initiation of haemoglobin degradation and higher aminopeptidase activity in the Pb3-9a refractory strain are important in the limitation of parasite development within the mosquito midgut, whereas trypsin plays no role in this process.
The presence and distribution of binding sites for eight different lectins, Con A, DBA, HPL, LFA, RCA I, SBA, UEA I, and WGA, were compared in the midguts of Plasmodium gallinaceum-infected Aedes aegypti and Plasmodium berghei-infected Anopheles stephensi. Lectins with high specificity for N-acetyl-D-glucosamine (GlcNAc) exhibited high binding preference for the peritrophic membrane and microvillar glycocalyx of Ae. aegypti; the same structures were preferentially labeled by N-Acetyl-D-galactosamine (GalNAc)-specific lectins in An. stephensi. No differences could be observed in the lectin-binding patterns of the intercellular spaces or cellular organelles and structures. The Plasmodium ookinete surface did not react with any of the lectins tested. It appears that sugars are involved in vector recognition by the parasite and that the peritrophic membrane and/or glycocalyx may be crucial structures for the penetration of the gut epithelium by the ookinete.
Magainins and cecropins are families of peptides with broad antimicrobial and antiparasitic activities derived respectively from the skin of frogs or from giant silk moths. In insects, cecropins function as part of an inducible immune system against a number of bacterial infections. When injected into anopheline mosquitoes previously infected with a variety of Plasmodium species, both magainins and cecropins disrupt sporogonic development by aborting the normal development of oocysts; sporozoites are not formed and the vector cannot transmit the parasite to another host. It may be possible to induce effective transmission-blocking immunity in the mosquito vector by the introduction and expression of genes coding for magainins, cecropins, or similarly acting parasiticidal peptides into the mosquito genome.
We determined the time and site of secretion of the precursors of the peritrophic membrane (PM) in Aedes aegypti and when the structure is assembled. The fine structure of the developing membrane of blood-feed females was described, and the pattern of secretion of injected tritiated glucosamine analyzed autoradiographically. Immediately following blood feeding, ingested red cells rapidly become compressed, such that the surrounding plasma is extruded to the margin of the midgut contents. Thereby, ingested fluids form a narrow margin separating the blood mass from the midgut epithelium. By electron microscopy, the PM first becomes evident at about 4 to 8 h after blood is ingested, and the membrane attains mature texture by 12 h. The compacted mass of ingested erythrocytes seems to serve as a template for the forming structure. In contrast, tritiated glucosamine, injected into freshly engorged mosquitoes, begins to concentrate on the midgut microvilli by 2 h after feeding. By 8 h the label assumes the layered appearance that characterizes the fine structure of the mature membrane. In contrast to the prevailing concept that the PM of mosquitoes first assumes texture anteriorly immediately after blood is ingested, we find that this potential barrier to pathogen development forms no earlier than 4 h after feeding and that it is formed from precursors secreted along the entire length of the epithelium overlying the food mass.
The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.
Oocysts of Plasmodium knowlesi developed normally on the gut of the mosquito Anopheles freeborni, rupturing and releasing sporozoites at 10-14 days post-infection. Subsequently, however, sporozoites were never found in this mosquito's salivary glands. Heterologous transplants of whole salivary glands from uninfected An. freeborni and An. dirus, a completely susceptible mosquito, into the abdomens of insects heavily infected with mature oocysts were done. Sporozoites failed to infect An. freeborni glands implanted in An. dirus but did enter An. dirus glands implanted in An. freeborni. These experiments suggest that P. knowlesi sporozoites are unable to recognize An. freeborni glands.
An experimental study of the mechanisms and patterns of resistance to Plasmodium berghei in different mosquito species revealed a diversity of factors which prevent or inhibit sporogonic development in its different phases and in different sites in the mosquito vector. The experiments showed that Culex salinarius was a totally resistant species in which exflagellation and ookinete formation are prevented. In Aedes aegypti, ookinetes in small or moderate numbers are formed but penetration of the mosquito midgut wall is blocked and oocysts are not formed. In the three experimental vectors, Anopheles quadrimaculatus, Anopheles aztecus, and Anopheles stephensi grades of enhanced susceptibility are recognized. They are expressed in lesser numbers of abnormal and degenerative oocysts, in higher numbers of sporozoites in the salivary gland, and greater viability and invasiveness of these sporozoites. In Anopheles dureni, the natural vector of rodent malaria, one observes both in nature and under experimental conditions the highest adaptation and most pronounced grade of susceptibility to P. berghei.
The extent and rate of development of Plasmodium gallinaceum Brumpt in the hemocoel of the refractory mosquito Culex pipiens pipiens L. and in that of the susceptible mosquito Aedes aegypti (L.) have been determined. Infected blood, with a high gametocyte count, was injected into the hemocoel of the mosquitoes and the development of the parasites in both hosts was compared with each other and with the natural exogenous cycle. Serial sections of the infected mosquitoes were used in these experiments. It was concluded that (1) the parasites develop at the same rate in the hemocoel of the injected susceptible mosquito as on the stomach wall of the naturally infected host; (2) development of the parasites in the hemocoels of both susceptible and refractory mosquitoes was parallel for the early stages until the 3rd day; (3) oocysts in the refractory host deteriorated and receded from the host tissues after the 4th day; (4) adequate metabolites were available to support development in the resistant host for the first 3 days but undetermined toxic factors were probably responsible for the resultant deterioration and death of the parasites.
A quantitative study was made of naturally acquired malaria infections among over 37,000 Anopheles gambiae and Anopheles funestus, collected from huts in a hyperendemic area in north-east Tanzania.
The numbers of sporozoites estimated to be present in the salivary glands commonly lay between 2,000 and 4,000. The medians and means were higher, with those in A. gambiae exceeding those in A. funestus. The difference is attributed mainly to the circumstance that almost all the heaviest infections encountered, in both guts and glands, occurred in A. gambiae.
Over 98% of the oöcysts were identified as those of P. falciparum; the remaining 4 infections, considered to be due to P. malariae, were all in A. funestus.
Formation and solubility of the peritrophic membranes (pm) in the midgut (stomach) of female Anopheles stephensi depended on salt concentration in the gut lumen. High calcium drastically reduced the solubility of the pm in vitro. Thin-layer chromatography revealed the presence of N-acetylgalactosamine and galactose in the pm. In contrast to Aedes aegypti no N-acetylglucosamine was present indicating significant differences between Anopheles and Aedes mosquitoes with respect to pm formation and composition. Experimental distension of the stomach epithelium did not trigger secretion of proteases, but membrane-bound granules were released from the stomach cells, and pm were formed. When females were fed blood 18 hr after an enema with salt solution, when practically no granules were present in the gut cells, protease activity was increased, and blood was digested normally, but no pm were formed. A functional correlation of the membrane-bound granules and pm formation is postulated. alpha-Amanitin prevented normal digestion, protease activity was only insignificantly increased, and no pm formed after blood intake.
Previous in vivo studies have implicated trypsin-like proteinases in the destruction of ookinetes of Plasmodium gallinaceum in the gut of the susceptible mosquito Aedes aegypti (Gass 1977). An in vitro study has shown that the ookinetes are destroyed by crude extracts of blood-fed A. aegypti and that this destruction is largely inhibited by an inhibitor of mosquito proteinases, lima bean trypsin inhibitor (Gass and Yeates 1979). In the present study ookinetes of P. gallinaceum have been incubated with highly purified preparations of the three principal trypsin-like proteinases of A. aegypti. It is shown that all three enzymes destroy the ookinetes and that the purified preparations are about as active as crude mosquito extracts.
Transmission of malaria parasites occurs by relatively few species of mosquitoes. One proposed mechanism of refractoriness is an inability of certain Plasmodium spp. to cross the peritrophic matrix (PM) in the midgut of an incompatible mosquito. We have tested this hypothesis by studying sporogonic development of Plasmodium gallinaceum in susceptible (Aedes aegypti and Anopheles gambiae G3) and refractory (Anopheles stephensi) mosquito species in the presence and absence of the PM. In the presence of the PM the number of oocytes that developed in A. gambiae G3 was about 20% of that in A. aegypti, whereas no oocysts developed in A. stephensi. To disrupt PM formation we added, to an infectious bloodmeal, either exogenous fungal chitinase or polyoxin D, the latter being a potent inhibitor of chitin synthase. The absence of the PM did not increase the susceptibility of A. aegypti and A. gambiae nor did it make A. stephensi susceptible to P. gallinaceum infection. The data indicate that the PM is not the primary determinant of P. gallinaceum compatibility in these mosquitoes and suggest that determinant(s) of refractoriness occurs after the parasite crosses the mosquito PM.
The effect of a synthetic cecropin-like peptide, Shiva-3, on in vitro ookinete development and on the early sporogonic stages of Plasmodium berghei in the midgut of Anopheles albimanus was investigated. Peptide concentrations of 75 and 100 microM were effective (P < 0.05) in reducing ookinete production and the number of infected mosquitoes in almost all experiments. These peptide concentrations in the midgut were not toxic for the survival of the mosquitoes. Complete inhibition was obtained if 100 microM Shiva-3 was applied in the first 8 hr of parasite development. The deleterious effect of the peptide on the parasite was effective after exposure for as short as 50 sec and the permanence of free peptide in the mosquito midgut was estimated to be of a minimum of 5 min. These observations indicate the possibility of using Shiva-like peptide genes to engineering malaria-resistant vectors as an alternative in malaria control strategies.
We have identified a mechanism for refractoriness to a bird malaria, Plasmodium gallinaceum, in the African vector of human malaria, Anopheles gambiae. Oocysts fail to develop in the refractory mosquitoes as a result of ookinete death which occurs within 27 hr of midgut invasion. Ultrastructural studies showed that parasite death occurs while the ookinete lies free in the midgut epithelial cell cytosol, usually surrounded by an organelle-free zone that consists of finely fibrillar material. The mechanism of parasite killing does not involve a previously described refractory mechanism of parasite encapsulation. We selected genetic lines which are refractory and susceptible to midgut infection. Genetic crossing of the lines suggests that the refractory trait is inherited as a single dominant genetic locus. Other loci probably influence oocyst number in susceptible mosquitoes. Intracellular ookinete killing appears to involve a previously unrecognized host defense mechanism against malaria parasites that involves direct destruction of the invading organism.
Sporogonic development of cultured Plasmodium falciparum was compared in six species of Anopheles mosquitoes. A reference species, A. gambiae, was selected as the standard for comparison. Estimates of absolute densities were determined for each lifestage. From these data, four aspects of parasite population dynamics were analyzed quantitatively: 1) successive losses in abundance as parasites developed from gametocyte to ookinete to oocyst stages, 2) oocyst production of sporozoites, 3) correlation between various lifestage parameters, and 4) parasite distribution. Parasite populations in A. gambiae incurred a 316-fold loss in abundance during the transition from macrogametocyte to ookinete stage, a 100-fold loss from ookinete to oocyst stage, yielding a total loss of approximately 31,600-fold (i.e., losses are multiplicative). Comparative susceptibilities in order were A. freeborni > A. gambiae, A. arabiensis, A. dirus > A. stephensi, A. albimanus. The key transition(s) determining overall susceptibility differed among species. Despite species differences in oocyst densities and infection rates, salivary gland sporozoite production per oocyst (approximately 640) was the same among species. The most consistent association among lifestage parameters was a positive correlation between densities and infection rates of homologous lifestages. A curvilinear relationship between ookinete and oocyst densities in A. gambiae indicated a threshold density was required for ookinete conversion to oocysts (approximately 30 ookinetes per mosquito). The same relationship in A. freeborni was linear, with no distinct threshold. Ookinete and oocyst populations were negative binomially distributed in all species. Indices of heterogeneity in mosquito susceptibility to infection indicated that gene frequencies determining susceptibility fluctuated with time in all species, except A. freeborni where susceptibility remained homogenous throughout the study. This approach provides a framework for identifying mechanisms of susceptibility and evaluating Plasmodium sporogonic development in naturally occurring vector species in nature.
Oocyst prevalence and intensity have been recorded in 349 laboratory infections of Anopheles stephensi with Plasmodium berghei. Intensity and prevalence of infection are shown to be predictably related. The structure and heterogeneity in the infections has been analysed with the objective of describing the biological mechanisms by which the observed negative binomial oocyst distributions are generated. The analysis has revealed that the most likely processes lie within the population dynamic events of malaria within the mosquito, namely gametogenesis, fertilization and mortality. The distribution is similar in all Plasmodium-mosquito combinations examined so far, whether they are of laboratory (P. gallinaceum in Aedes aegypti) or field (P. vivax in An. albimanus and P. falciparum in An. gambiae s.l. and An. funestus) origin. Further we conclude that there is competition between parasites in the vector. Oocyst frequency distribution analysis shows that under natural conditions of transmission intensity, and even under the best laboratory conditions, significant numbers (> 10%) of fully susceptible mosquitoes will not be infected under conditions where the mean infection is as high as 250 oocysts. Failure to infect is not therefore an absolute indicator of refractoriness. In assessing transmission data it is shown that sample sizes should not be less than 50, and ideally 100 mosquitoes, if reliable data are to be obtained. In field it is suggested that difficulties in determining the low natural intensity of oocyst infections indicate that prevalence estimates are a useful and accessible parameter to measure.(ABSTRACT TRUNCATED AT 250 WORDS)
It has been possible to infect mosquitoes routinely with cultured gametocytes of Plasmodium falciparum since 1980. This has enabled the development of a reliable bio-assay for potential transmission-blocking vaccines and research on the role of specific antibodies from the host on the parasitic stages in the mosquito midgut. After some development and fine-tuning of the assay, it became apparent that the immune responses of the human host, as well as factors from the parasite and the mosquito, determined the final outcome of the mosquito infection. The age of the mosquito, crowding of parasites inside the peritrophic membrane and the quantity and particularly the quality of the gametocytes ingested all influence the chance of successful transmission. Cytokines and/or other mediators of inflammation from the human host can also reduce transmission, probably by promoting phagocytosis of the freshly emerged gametes by leucocytes in the bloodmeal.
We examined several insect antimicrobial peptides to study their effect on Plasmodium gallinaceum zygotes, ookinetes, oocysts, and sporozoites. Only two insect defensins-Aeschna cyanea (dragon fly) and Phormia terranovae (flesh fly)-had a profound toxic effect on the oocysts in Aedes aegypti and on isolated sporozoites. The defensins affected the oocysts in a time-dependent manner. Injecting the peptide into the hemolymph 1 or 2 days after an infectious blood meal had no significant effect on prevalence of infection or relative oocyst density per mosquito. When injected 3 days after parasite ingestion, the relative oocyst density was significantly reduced. Injection on day 4 or later damaged the developing oocysts, although the oocysts density per mosquito was not significantly different when examined on day 8. The oocysts were swollen or had extensive internal vacuolization. The peptides had no detectable effect on the early stages of the parasite: the zygotes and ookinetes tested in vitro. Both the defensins were highly toxic to isolated sporozoites in vitro as indicated by disruption of the membrane permeability barrier, a change in morphology, and loss of motility. In contrast to the toxicity of cecropin and magainin for mosquitoes, defensin, at concentrations that kill parasites, is not toxic to mosquitoes, suggesting that defensin should be studied further as a potential molecule to block sporogonic development of Plasmodium.
During the course of its development in the mosquito and transmission to a new vertebrate host, the malaria parasite must interact with the mosquito midgut and invade the gut epithelium. To investigate how the parasite recognizes the midgut before invasion, we have developed an in vitro adhesion assay based on combining fluorescently labelled ookinetes with isolated midgut epithelia from blood-fed mosquitoes. Using this assay, we found that Plasmodium gallinaceum ookinetes readily adhered to midguts of Aedes aegypti, mimicking the natural recognition of the epithelium by the parasite. This interaction is specific: the ookinetes preferentially adhered to the lumen (microvillar) side of the gut epithelium and did not bind to other mosquito tissues. Conversely, the binding was not due to a non-specific adhesive property of the midguts, because a variety of other cell types, including untransformed P. gallinaceum zygotes or macrogametes, did not show similar binding to the midguts. High concentrations of glycosylated (fetuin, orosomucoid, ovalbumin) or non-glycosylated (bovine serum albumin) proteins, added as non-specific competitors, failed to compete with the ookinetes in binding assays. We also found that the adhesion of ookinetes to the midgut surface is necessary for sporogonic development of the parasite in the mosquito. Antibodies and other reagents that blocked adhesion in vitro also reduced oocyst formation when these reagents were combined with mature ookinetes and fed to mosquitoes. Chemical modification of the midguts with sodium periodate at pH 5.5 destroyed adhesion, indicating that the ookinete binds to a carbohydrate ligand on the surface of the midgut. The ligand is sensitive to periodate concentrations of less than 1 mmol l-1, suggesting that it may contain sialic-acid-like sugars. Furthermore, free N-acetylneuraminic acid competed with the ookinetes in binding aasays, while other monosaccharides had no effect. However, in agreement with the current belief that adult insects do not contain sialic acids, we were unable to detect any sialic acids in mosquito midguts using the most sensitive HPLC-based fluorometric assay currently available. We postulate that a specific carbohydrate group is used by the ookinete to recognize the midgut epithelium and to attach to its surface. This is the first receptor-ligand interaction demonstrated for the ookinete stage of a malaria parasite. Further characterization of the midgut ligand and its parasite counterpart may lead to novel strategies of blocking oocyst development in the mosquito.
An invertebrate intestinal mucin gene, AgMuc1, was isolated from the malaria vector mosquito Anopheles gambiae. The predicted 122-residue protein consists of a central core of seven repeating TTTTVAP motifs flanked by hydrophobic N- and C-terminal domains. This structure is similar to that of mucins that coat the protozoan parasite Trypanosoma cruzi. Northern blot analysis indicated that the gene is expressed exclusively in the midgut of adult mosquitoes. A length polymorphism and in situ hybridization were used to genetically and cytogenetically map AgMuc1 to division 7A of the right arm of the second chromosome. The subcellular localization of the encoded protein in tissue culture cells was examined by using a baculovirus vector to express AgMuc1 protein tagged with the green fluorescent protein (GFP). The results indicated that this protein is found at the cell surface and that both hydrophobic domains are required for cell surface targeting. We propose that AgMuc1 is an abundant mucin-like protein that lines the surface of the midgut microvilli, potentially protecting the intestinal epithelium from the proteinase-rich environment of the gut lumen. An intriguing possibility is that, as an abundant surface protein, AgMuc1 may also interact with the malaria parasite during its invasion of the mosquito midgut.
Mosquitoes transmit malaria, but only a few species permit the complete development and transmission of the parasite. Also, only a fraction of the ingested parasites develop in the vector. The attrition occurs in different compartments during the parasite's complex developmental scheme in the insect. A number of factors, both physical and biochemical, that affect the development have been proposed or demonstrated. Each of these factors is located within a specific space in the insect. We have divided this space into six compartments, which are distinct in their biochemical and biophysical nature: Endoperitrophic space, Peritrophic matrix, Ectopretrophic space, Midgut epithelium, Haemocoel and Salivary gland. Because factors that influence a particular stage of parasite development share the same microenvironment within these compartments, they must be considered collectively to exploit them for designing effective transmission blocking strategies. In this article we discuss these factors according to their spatial location in the mosquito.
Transmission blocking vaccines (TBVs) against malaria are intended to induce immunity against the stages of the parasites which infect mosquitoes so that TBV-immunised individuals cannot transmit malaria. As malarial infections are transmitted mainly within a few hundreds of meters from an infectious human source, TBVs used within in a community would protect the immediate neighbourhood of the vaccinated individuals. TBVs against the two major species of human malaria, Plasmodium falciparum and P. vivax, are under development. Candidate TBV constructs for both Plasmodium species have been successfully tested in animal systems and testing is in progress with clinical grade material in humans.
We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.