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.     

‘Manipulation’ without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites

Previous studies have suggested that Plasmodium parasites can manipulate mosquito feeding behaviours such as probing, persistence and engorgement rate in order to enhance transmission success. Here, we broaden analysis of this ‘manipulation phenotype’ to consider proximate foraging behaviours, including responsiveness to host odours and host location. Using Anopheles stephensi and Plasmodium yoelii as a model system, we demonstrate that mosquitoes with early stage infections (i.e. non-infectious oocysts) exhibit reduced attraction to a human host, whereas those with late-stage infections (i.e. infectious sporozoites) exhibit increased attraction. These stage-specific changes in behaviour were paralleled by changes in the responsiveness of mosquito odourant receptors, providing a possible neurophysiological mechanism for the responses. However, we also found that both the behavioural and neurophysiological changes could be generated by immune challenge with heat-killed Escherichia coli and were thus not tied explicitly to the presence of malaria parasites. Our results support the hypothesis that the feeding behaviour of female mosquitoes is altered by Plasmodium, but question the extent to which this is owing to active manipulation by malaria parasites of host behaviour.     

Malaria Vector Surveillance in Ganghwa-do, a Malaria-Endemic Area in the Republic of Korea

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.     

A weather-driven model of malaria transmission

The classic formulae in malaria epidemiology are reviewed that relate entomological parameters to malaria transmission, including mosquito survivorship and age-at-infection, the stability index (S), the human blood index (HBI), proportion of infected mosquitoes, the sporozoite rate, the entomological inoculation rate (EIR), vectorial capacity (C) and the basic reproductive number (R ₀). The synthesis emphasizes the relationships among classic formulae and reformulates a simple dynamic model for the proportion of infected humans. The classic formulae are related to formulae from cyclical feeding models, and some inconsistencies are noted. The classic formulae are used to to illustrate how malaria control reduces malaria transmission and show that increased mosquito mortality has an effect even larger than was proposed by Macdonald in the 1950's.     

Mosquito-Bacteria Symbiosis: The Case of Anopheles gambiae and Asaia

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.     

Evidence for regular ongoing introductions of mosquito disease vectors into the Galápagos Islands

Wildlife on isolated oceanic islands is highly susceptible to the introduction of pathogens. The recent establishment in the Galápagos Islands of the mosquito Culex quinquefasciatus, a vector for diseases such as avian malaria and West Nile fever, is considered a serious risk factor for the archipelago''s endemic fauna. Here we present evidence from the monitoring of aeroplanes and genetic analysis that C. quinquefasciatus is regularly introduced via aircraft into the Galápagos Archipelago. Genetic population structure and admixture analysis demonstrates that these mosquitoes breed with, and integrate successfully into, already-established populations of C. quinquefasciatus in the Galápagos, and that there is ongoing movement of mosquitoes between islands. Tourist cruise boats and inter-island boat services are the most likely mechanism for transporting Culex mosquitoes between islands. Such anthropogenic mosquito movements increase the risk of the introduction of mosquito-borne diseases novel to Galápagos and their subsequent widespread dissemination across the archipelago. Failure to implement and maintain measures to prevent the human-assisted transport of mosquitoes to and among the islands could have catastrophic consequences for the endemic wildlife of Galápagos.     

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs^1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential^6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km² vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol^9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction⁹.Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality¹¹, a PCR for identification of Anopheles gambiae sibling species¹⁰ and a nested PCR for typing of Plasmodium falciparum infection¹². Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min'' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.     

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.     

A mathematical model for the dynamics of malaria in mosquitoes feeding on a heterogeneous host population

We describe and develop a difference equation model for the dynamics of malaria in a mosquito population feeding on, infecting and getting infected from a heterogeneous population of hosts. Using the force of infection from different classes of humans to mosquitoes as parameters, we evaluate a number of entomological parameters, indicating malaria transmission levels, which can be compared to field data. By assigning different types of vector control interventions to different classes of humans and by evaluating the corresponding levels of malaria transmission, we can compare the effectiveness of these interventions. We show a numerical example of the effects of increasing coverage of insecticide-treated bed nets in a human population where the predominant malaria vector is Anopheles gambiae.     

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.     

A mathematical model for zoonotic transmission of malaria in the Atlantic Forest: Exploring the effects of variations in vector abundance and acrodendrophily

Transmission foci of autochthonous malaria caused by Plasmodium vivax-like parasites have frequently been reported in the Atlantic Forest in Southeastern and Southern Brazil. Evidence suggests that malaria is a zoonosis in these areas as human infections by simian Plasmodium species have been detected, and the main vector of malaria in the Atlantic Forest, Anopheles (Kerteszia) cruzii, can blood feed on human and simian hosts. In view of the lack of models that seek to predict the dynamics of zoonotic transmission in this part of the Atlantic Forest, the present study proposes a new deterministic mathematical model that includes a transmission compartment for non-human primates and parameters that take into account vector displacement between the upper and lower forest strata. The effects of variations in the abundance and acrodendrophily of An. cruzii on the prevalence of infected humans in the study area and the basic reproduction number (R₀) for malaria were analyzed. The model parameters are based on the literature and fitting of the empirical data. Simulations performed with the model indicate that (1) an increase in the abundance of the vector in relation to the total number of blood-seeking mosquitoes leads to an asymptotic increase in both the proportion of infected individuals at steady state and R₀; (2) the proportion of infected humans at steady state is higher when displacement of the vector mosquito between the forest strata increases; and (3) in most scenarios, Plasmodium transmission cannot be sustained only between mosquitoes and humans, which implies that non-human primates play an important role in maintaining the transmission cycle. The proposed model contributes to a better understanding of the dynamics of malaria transmission in the Atlantic Forest.     

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Sulfadoxine-pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.     

Malaria vectors of Papua New Guinea

Understanding malaria transmission in Papua New Guinea (PNG) requires exact knowledge of which Anopheles species are transmitting malaria and is complicated by the cryptic species status of many of these mosquitoes. To identify the malaria vectors in PNG we studied Anopheles specimens from 232 collection localities around human habitation throughout PNG (using CO₂ baited light traps and human bait collections). A total of 22,970 mosquitoes were individually assessed using a Plasmodium sporozoite enzyme-linked immunosorbent assay to identify Plasmodiumfalciparum, Plasmodiumvivax and Plasmodiummalariae circumsporozoite proteins. All mosquitoes were identified to species by morphology and/or PCR. Based on distribution, abundance and their ability to develop sporozoites, we identified five species as major vectors of malaria in PNG. These included: Anophelesfarauti, Anopheleshinesorum (incriminated here, to our knowledge, for the first time), Anophelesfarauti 4, Anopheleskoliensis and Anophelespunctulatus. Anopheleslongirostris and Anophelesbancroftii were also incriminated in this study. Surprisingly, An. longirostris showed a high incidence of infections in some areas. A newly identified taxon within the Punctulatus Group, tentatively called An. farauti 8, was also found positive for circumsporozoite protein. These latter three species, together with Anopheleskarwari and Anophelessubpictus, incriminated in other studies, appear to be only minor vectors, while Anophelesfarauti 6 appears to be the major vector in the highland river valleys (>1500 m above sea level). The nine remaining Anopheles species found in PNG have been little studied and their bionomics are unknown; most appear to be uncommon with limited distribution and their possible role in malaria transmission has yet to be determined.     

Variation in relapse frequency and the transmission potential of Plasmodium vivax malaria

There is substantial variation in the relapse frequency of Plasmodium vivax malaria, with fast-relapsing strains in tropical areas, and slow-relapsing strains in temperate areas with seasonal transmission. We hypothesize that much of the phenotypic diversity in P. vivax relapses arises from selection of relapse frequency to optimize transmission potential in a given environment, in a process similar to the virulence trade-off hypothesis. We develop mathematical models of P. vivax transmission and calculate the basic reproduction number R₀ to investigate how transmission potential varies with relapse frequency and seasonality. In tropical zones with year-round transmission, transmission potential is optimized at intermediate relapse frequencies of two to three months: slower-relapsing strains increase the opportunity for onward transmission to mosquitoes, but also increase the risk of being outcompeted by faster-relapsing strains. Seasonality is an important driver of relapse frequency for temperate strains, with the time to first relapse predicted to be six to nine months, coinciding with the duration between seasonal transmission peaks. We predict that there is a threshold degree of seasonality, below which fast-relapsing tropical strains are selected for, and above which slow-relapsing temperate strains dominate, providing an explanation for the observed global distribution of relapse phenotypes.     

Last-come,best served? Mosquito biting order and Plasmodium transmission

A pervasive characteristic of parasite infections is their tendency to be overdispersed. Understanding the mechanisms underlying this overdispersed distribution is of key importance as it may impact the transmission dynamics of the pathogen. Although multiple factors ranging from environmental stochasticity to inter-individual heterogeneity may explain parasite overdispersion, parasite infection is also overdispersed in an inbred host population maintained under laboratory conditions, suggesting that other mechanisms are at play. Here, we show that the aggregated distribution of malaria parasites within mosquito vectors is partially explained by a temporal heterogeneity in parasite infectivity triggered by the bites of mosquitoes. Parasite transmission tripled between the mosquito''s first and last blood feed in a period of only 3 h. Surprisingly, the increase in transmission is not associated with an increase in parasite investment in production of the transmissible stage. Overall, we highlight that Plasmodium is capable of responding to the bites of mosquitoes to increase its own transmission at a much faster pace than initially thought and that this is partly responsible for overdispersed distribution of infection. We discuss the underlying mechanisms as well as the broader implications of this plastic response for the epidemiology of malaria.     

Local prevalence and transmission of avian malaria in the Alakai Plateau of Kauai,Hawaii, U.S.A.

Avian malaria is among the most important threats to native Hawaiian forest birds. It is caused by the parasite Plasmodium relictum and is transmitted by the introduced mosquito vector Culex quinquefasciatus. Temperature increases and precipitation declines due to climate change over the last decade may be responsible for the observed recent expansion in the range and prevalence of avian malaria on the Alakai Plateau, Kauai Island. To examine the hypothesis that conditions are now favorable for transmission of malaria on the Plateau, mosquitoes were sampled with CO₂ and Reiter oviposition traps at three sites (Kawaikoi, Halepa'akai, and Koke'e) on several occasions between October, 2013 and April, 2014. P. relictum infection was assessed by PCR or dissection under a microscope. We also surveyed mosquito larvae along Halepa'akai and Kawaikoi streams. We observed that Cx. quinquefasciatus is well established on the Alakai Plateau, as mosquitoes were caught on all field trips, except in April at Halepa'akai, and larvae were found throughout the year. We observed differences in adult abundance among sites and microhabitats (stream vs ridge lines).     

Evaluation of Antimalarial Activity and Toxicity of a New Primaquine Prodrug

Plasmodium vivax is the most prevalent of the five species causing malaria in humans. The current available treatment for P. vivax malaria is limited and unsatisfactory due to at least two drawbacks: the undesirable side effects of primaquine (PQ) and drug resistance to chloroquine. Phenylalanine-alanine-PQ (Phe-Ala-PQ) is a PQ prodrug with a more favorable pharmacokinetic profile compared to PQ. The toxicity of this prodrug was evaluated in in vitro assays using a human hepatoma cell line (HepG2), a monkey kidney cell line (BGM), and human red blood cells deficient in the enzyme glucose-6-phosphate-dehydrogenase (G6PD). In addition, in vivo toxicity assays were performed with rats that received multiple doses of Phe-Ala-PQ to evaluate biochemical, hematological, and histopathological parameters. The activity was assessed by the inhibition of the sporogonic cycle using a chicken malaria parasite. Phe-Ala-PQ blocked malaria transmission in Aedes mosquitoes. When compared with PQ, it was less cytotoxic to BGM and HepG2 cells and caused less hemolysis of G6PD-deficient red blood cells at similar concentrations. The prodrug caused less alteration in the biochemical parameters than did PQ. Histopathological analysis of the liver and kidney did show differences between the control and Phe-Ala-PQ-treated groups, but they were not statistically significant. Taken together, the results highlight the prodrug as a novel lead compound candidate for the treatment of P. vivax malaria and as a blocker of malaria transmission.     

Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.     

Anopheline species and their Plasmodium infection status in Aligarh,India

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