Existing Infection Facilitates Establishment and Density of Malaria Parasites in Their Mosquito Vector

Very little is known about how vector-borne pathogens interact within their vector and how this impacts transmission. Here we show that mosquitoes can accumulate mixed strain malaria infections after feeding on multiple hosts. We found that parasites have a greater chance of establishing and reach higher densities if another strain is already present in a mosquito. Mixed infections contained more parasites but these larger populations did not have a detectable impact on vector survival. Together these results suggest that mosquitoes taking multiple infective bites may disproportionally contribute to malaria transmission. This will increase rates of mixed infections in vertebrate hosts, with implications for the evolution of parasite virulence and the spread of drug-resistant strains. Moreover, control measures that reduce parasite prevalence in vertebrate hosts will reduce the likelihood of mosquitoes taking multiple infective feeds, and thus disproportionally reduce transmission. More generally, our study shows that the types of strain interactions detected in vertebrate hosts cannot necessarily be extrapolated to vectors.     

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

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

Mosquito mortality and the evolution of malaria virulence

Abstract Several laboratory studies of malaria parasites (Plasmodium sp.) and some field observations suggest that parasite virulence, defined as the harm a parasite causes to its vertebrate host, is positively correlated with transmission. Given this advantage, what limits the continual evolution of higher parasite virulence? One possibility is that while more virulent strains are more infectious, they are also more lethal to mosquitoes. In this study, we tested whether the virulence of the rodent malaria parasite P. chabaudi in the laboratory mouse was correlated with the fitness of mosquitoes it subsequently infected. Mice were infected with one of seven genetically distinct clones of P. chabaudi that differ in virulence. Weight loss and anemia in infected mice were monitored for 16–17 days before Anopheles stephensi mosquitoes were allowed to take a blood meal from them. Infection virulence in mice was positively correlated with transmission to mosquitoes (infection rate) and weakly associated with parasite burden (number of oocysts). Mosquito survival fell with increasing oocyst burden, but there was no overall statistically significant relationship between virulence in mice and mosquito mortality. Thus, there was no evidence that more virulent strains are more lethal to mosquitoes. Both vector survival and fecundity depended on parasite clone, and contrary to expectations, mosquitoes fed on infections more virulent to mice were more fecund. The strong parasite genetic effects associated with both fecundity and survival suggests that vector fitness could be an important selective agent shaping malaria population genetics and the evolution of phenotypes such as virulence in the vector.     

Avian Plasmodium lineages found in spot surveys of mosquitoes from 2007 to 2010 at Sakata wetland,Japan: do dominant lineages persist for multiple years?

The ecology and geographical distribution of disease vectors are major determinants of spatial and temporal variations in the transmission dynamics of vector‐borne pathogens. However, there are limited studies on the ecology of vectors that contribute to the natural transmission of most vector‐borne pathogens. Avian Plasmodium parasites are multihost mosquito‐borne pathogens transmitted by multiple mosquito species, which might regulate the diversity and persistence of these parasites. From 2007 to 2010, we conducted entomological surveys at Sakata wetland in central Japan, to investigate temporal variation in mosquito occurrence and prevalence of avian Plasmodium lineages in the mosquito populations. A polymerase chain reaction (PCR)‐based method was used to detect Plasmodium parasites and identify the blood sources of mosquitoes. Culex inatomii and C. pipiens pallens represented 60.0% and 34.8% of 11 mosquito species collected, respectively. Our results showed that the two dominant mosquito species most likely serve as principal vectors of avian Plasmodium parasites during June, which coincides with the breeding season of bird species nesting in the wetland reed beds. Fourteen animal species were identified as blood sources of mosquitoes, with the oriental reed warbler (Acrocephalus orientalis) being the commonest blood source. Although there was significant temporal variation in the occurrence of mosquitoes and prevalence of Plasmodium lineages in the mosquitoes, the dominant Plasmodium lineages shared by the two dominant mosquito species were consistently found at the same time during transmission seasons. Because vector competence cannot be confirmed solely by PCR approaches, experimental demonstration is required to provide definitive evidence of transmission suggested in this study.     

Can Wolbachia be used to control malaria?

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

Malaria infection increases bird attractiveness to uninfected mosquitoes

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

Male size does not affect mating success (of Anopheles gambiae in São Tomé)

Abstract For malaria control, the utility of transgenic vector Anopheles mosquitoes (Diptera: Culicidae) refractory to Plasmodium transmission, will depend on their interbreeding with the wild vector population. In many species, larger males are more successful in obtaining mates. In São Tomé island, we determined that size did not affect mating success of male Anopheles gambiae Giles sensu stricto, the main malaria vector in tropical Africa. Also we showed that larval intraspecific competition is probably insignificant in this population of An. gambiae. Thus, the potential success of transgenic An. gambiae is unlikely to be affected by size selection under field conditions.     

Apparent vector‐mediated parent‐to‐offspring transmission in an avian malaria‐like parasite

Parasite transmission strategies strongly impact host–parasite co‐evolution and virulence. However, studies of vector‐borne parasites such as avian malaria have neglected the potential effects of host relatedness on the exchange of parasites. To test whether extended parental care in the presence of vectors increases the probability of transmission from parents to offspring, we used high‐throughput sequencing to develop microsatellites for malaria‐like Leucocytozoon parasites of a wild raptor population. We show that host siblings carry genetically more similar parasites than unrelated chicks both within and across years. Moreover, chicks of mothers of the same plumage morph carried more similar parasites than nestlings whose mothers were of different morphs, consistent with matrilineal transmission of morph‐specific parasite strains. Ours is the first evidence of an association between host relatedness and parasite genetic similarity, consistent with vector‐mediated parent‐to‐offspring transmission. The conditions for such ‘quasi‐vertical’ transmission may be common and could suppress the evolution of pathogen virulence.     

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

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

Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii.     

Attraction of mosquitoes to primate odours and implications for zoonotic Plasmodium transmission

Vector-borne diseases often originate from wildlife and can spill over into the human population. One of the most important determinants of vector-borne disease transmission is the host preference of mosquitoes. Mosquitoes with a specialised host preference are guided by body odours to find their hosts in addition to carbon dioxide. Little is known about the role of mosquito host preference in the spillover of pathogenic agents from humans towards animals and vice versa. In the Republic of Congo, the attraction of mosquitoes to primate host odours was determined, as well as their possible role as malaria vectors, using odour-baited traps mimicking the potential hosts of mosquitoes. Most of the mosquito species caught showed a generalistic host preference. Anopheles obscurus was the most abundant Anopheles mosquito, with a generalistic host preference observed from the olfactory response and the detection of various Plasmodium parasites. Interestingly, Culex decens showed a much higher attraction towards chimpanzee odours than to human or cow odours. Human Plasmodium parasites were observed in both human and chimpanzee blood, although not in the Anopheles mosquitoes that were collected. Understanding the role of mosquito host preference for cross-species parasite transmission provides information that will help to determine the risk of spillover of vector-borne diseases.     

Dipsticks for rapid detection of Plasmodium in vectoring Anopheles mosquitoes

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Fine‐scale distribution modeling of avian malaria vectors in north‐central Kansas

Infectious diseases increasingly play a role in the decline of wildlife populations. Vector‐borne diseases, in particular, have been implicated in mass mortality events and localized population declines are threatening some species with extinction. Transmission patterns for vector‐borne diseases are influenced by the spatial distribution of vectors and are therefore not uniform across the landscape. Avian malaria is a globally distributed vector‐borne disease that has been shown to affect endemic bird populations of North America. We evaluated shared habitat use between avian malaria vectors, mosquitoes in the genus Culex and a native grassland bird, the Greater Prairie‐Chicken (Tympanuchus cupido), by (1) modeling the distribution of Culex spp. occurrence across the Smoky Hills of north‐central Kansas using detection data and habitat variables, (2) assessing the occurrence of these vectors at nests of female Greater Prairie‐Chickens, and (3) evaluating if shared habitat use between vectors and hosts is correlated with malarial infection status of the Greater Prairie‐Chicken. Our results indicate that Culex occurrence increased at nest locations compared to other available but unoccupied grassland habitats; however the shared habitat use between vectors and hosts did not result in an increased prevalence of malarial parasites in Greater Prairie‐Chickens that occupied habitats with high vector occurrence. We developed a predictive map to illustrate the associations between Culex occurrence and infection status with malarial parasites in an obligate grassland bird that may be used to guide management decisions to limit the spread of vector‐borne diseases.     

Temperature-Dependent Pre-Bloodmeal Period and Temperature-Driven Asynchrony between Parasite Development and Mosquito Biting Rate Reduce Malaria Transmission Intensity

A mosquito needs to bite at least twice for malaria transmission to occur: once to acquire parasites and, after these parasites complete their development in their mosquito host, once to transmit the parasites to the next vertebrate host. Here we investigate the relationship between temperature, parasite development, and biting frequency in a mosquito-rodent malaria model system. We show that the pre-bloodmeal period (the time lag between mosquito emergence and first bloodmeal) increases at lower temperatures. In addition, parasite development time and feeding exhibit different thermal sensitivities such that mosquitoes might not be ready to feed at the point at which the parasite is ready to be transmitted. Exploring these effects using a simple theoretical model of human malaria shows that delays in infection and transmission can reduce the vectorial capacity of malaria mosquitoes by 20 to over 60%, depending on temperature. These delays have important implications for disease epidemiology and control, and should be considered in future transmission models.     

New repellent effective against African malaria mosquito Anopheles gambiae: implications for vector control

Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is a vector for Plasmodium, the causative agent of malaria. Current control strategies to reduce the impact of malaria focus on reducing the frequency of mosquito attacks on humans, thereby decreasing Plasmodium transmission. A need for new repellents effective against Anopheles mosquitoes has arisen because of changes in vector behaviour as a result of control strategies and concern over the health impacts of current repellents. The response of A. gambiae to potential repellents was investigated through an electroantennogram screen and the most promising of these candidates (1‐allyloxy‐4‐propoxybenzene, 3c {3,6}) chosen for behavioural testing. An assay to evaluate the blood‐host seeking behaviour of A. gambiae towards a simulated host protected with this repellent was then performed. The compound 3c {3,6} was shown to be an effective repellent, causing mosquitoes to reduce their contact with a simulated blood‐host and probe less at the host odour. Thus, 3c {3,6} may be an effective repellent for the control of A. gambiae.     

Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito—parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission.     

Insecticide exposure impacts vector–parasite interactions in insecticide-resistant malaria vectors

Currently, there is a strong trend towards increasing insecticide-based vector control coverage in malaria endemic countries. The ecological consequence of insecticide applications has been mainly studied regarding the selection of resistance mechanisms; however, little is known about their impact on vector competence in mosquitoes responsible for malaria transmission. As they have limited toxicity to mosquitoes owing to the selection of resistance mechanisms, insecticides may also interact with pathogens developing in mosquitoes. In this study, we explored the impact of insecticide exposure on Plasmodium falciparum development in insecticide-resistant colonies of Anopheles gambiae s.s., homozygous for the ace-1 G119S mutation (Acerkis) or the kdr L1014F mutation (Kdrkis). Exposure to bendiocarb insecticide reduced the prevalence and intensity of P. falciparum oocysts developing in the infected midgut of the Acerkis strain, whereas exposure to dichlorodiphenyltrichloroethane reduced only the prevalence of P. falciparum infection in the Kdrkis strain. Thus, insecticide resistance leads to a selective pressure of insecticides on Plasmodium parasites, providing, to our knowledge, the first evidence of genotype by environment interactions on vector competence in a natural Anopheles–Plasmodium combination. Insecticide applications would affect the transmission of malaria in spite of resistance and would reduce to some degree the impact of insecticide resistance on malaria control interventions.     

Malaria's Missing Number: Calculating the Human Component of R0 by a Within-Host Mechanistic Model of Plasmodium falciparum Infection and Transmission

Human infection by malarial parasites of the genus Plasmodium begins with the bite of an infected Anopheles mosquito. Current estimates place malaria mortality at over 650,000 individuals each year, mostly in African children. Efforts to reduce disease burden can benefit from the development of mathematical models of disease transmission. To date, however, comprehensive modeling of the parameters defining human infectivity to mosquitoes has remained elusive. Here, we describe a mechanistic within-host model of Plasmodium falciparum infection in humans and pathogen transmission to the mosquito vector. Our model incorporates the entire parasite lifecycle, including the intra-erythrocytic asexual forms responsible for disease, the onset of symptoms, the development and maturation of intra-erythrocytic gametocytes that are transmissible to Anopheles mosquitoes, and human-to-mosquito infectivity. These model components were parameterized from malaria therapy data and other studies to simulate individual infections, and the ensemble of outputs was found to reproduce the full range of patient responses to infection. Using this model, we assessed human infectivity over the course of untreated infections and examined the effects in relation to transmission intensity, expressed by the basic reproduction number R₀ (defined as the number of secondary cases produced by a single typical infection in a completely susceptible population). Our studies predict that net human-to-mosquito infectivity from a single non-immune individual is on average equal to 32 fully infectious days. This estimate of mean infectivity is equivalent to calculating the human component of malarial R₀. We also predict that mean daily infectivity exceeds five percent for approximately 138 days. The mechanistic framework described herein, made available as stand-alone software, will enable investigators to conduct detailed studies into theories of malaria control, including the effects of drug treatment and drug resistance on transmission.     

Novel high‐throughput screens of Anopheles gambiae odorant receptors reveal candidate behaviour‐modifying chemicals for mosquitoes

Despite many decades of multilateral global efforts, a significant portion of the world population continues to be plagued with one or more mosquito‐vectored diseases. These include malaria and filariasis, as well as numerous arboviral‐associated illnesses, such as dengue and yellow fevers. The dynamics of disease transmission by mosquitoes is complex, and involves both vector competence and vectorial capacity. One area of intensive effort is the study of chemosensory‐driven behaviours in the malaria vector mosquito Anopheles gambiae Giles, the modulation of which is likely to provide opportunities for disease reduction. In this context, recent studies characterize a large divergent family of An. gambiae odorant receptors (AgORs) that play critical roles in olfactory signal transduction. This work facilitates high‐throughput, cell‐based calcium mobilization screens of AgOR‐expressing human embryonic kidney cells identifying a large number of conventional AgOR ligands, as well as the first nonconventional Orco (olfactory receptor co‐receptor) family agonist. As such, ligand‐mediated modulation serves as a proof‐of‐concept demonstration that AgORs represent viable targets for high‐throughput screening and for the eventual development of behaviour‐modifying olfactory compounds. Such attractants or repellents could foster malaria reduction programmes.     

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

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