Recognition of Anopheles mid-gut molecules interacting with the malaria parasite is important as they can potentially be targeted to interrupt the life cycle of the parasite in the mosquito's body. The mid-gut of mosquitoes is covered with the glycocalyx, which is composed of various glycoproteins. Pieces of evidence show that mucin proteins are one of the most frequent ingredients of the glycocalyx. In the present study, we isolated and identified the sequence of mucin from the mid-gut of Anopheles stephensi, Liston and Kazerun strains. Anopheles stephensi mucin (AsMuc) has two central core repeats with the consensus sequence TTTTVAP flanked with a hydrophobic N-terminus and a C-terminus which it seems are both necessary for cell surface expression. To show if this molecule is expressed on the surface of the cell, we cloned AsMuc in a baculovirus vector and tracked the expression of the protein in Sf9 insect cells. Immune assays showed the surface localization of the recombinant mucin. AsMuc expression on the surface of the cell suggests it could be a potential ligand for Plasmodium spp. attachment to the Anopheles mid-gut.

A total of 39 strains of Anopheles stephensi, an important urban malaria vector, were collected from various parts of India and maintained in the insectary for this study. Based on the egg-float ridge number, 19 strains were classified into ecological variants and 32 strains were exposed to chlorpyrifos and propoxur to investigate their resistance status. Filter paper containing freshly laid eggs was taken, the ridge numbers on the floats were counted under the microscope, and strains were classified into ecological variants. Of the 19 strains, 18 were of ‘type form’, with ridge numbers ranging from 15 to 21. The Papareddipalya (PRP) strain belonged to the ‘intermediate form’, with 14 to 17 ridge numbers. Larval bioassays were carried out according to the procedure of the WHO. For chlorpyrifos, the lowest LC50 value was 0.00107 mg/l (Padmanabhanagar strain) and the highest value was 0.0403 mg/l (GOA-A strain). Furthermore, the lowest LC90 value was 0.00368 mg/l (Delhi strain) and the highest was 0.1746 mg/l (GOA-A strain). For propoxur, the lowest LC50 value was 0.00029 mg/l (Goraguntepalya strain) and the highest value was 0.0037 mg/l (JP Nagar strain). Moreover, the lowest LC90 value was 0.00094 mg/l (Goraguntepalya strain) and the highest value was 0.0115 mg/l (JP Nagar strain). The tolerance values ranged from 1.26 to 37.68 for chlorpyrifos and from 1.34 to 12.77 for propoxur. All the type forms were from urban and semi-urban locations, and the intermediate strain was from a semi-urban location. The bioassay results indicated that the strains of An. stephensi were more susceptible to propoxur than to chlorpyrifos.

Anopheles stephensi – Liston (Culicidae: Diptera) is an important urban malarial vector in the Indian sub-continent, accounting for about 15% of the total annual malaria incidence. Chemical control represents a key strategy in the management of this insect vector. However, owing to erratic and continuous application of insecticides, resistance has become a common phenomenon among them and their control has become an uphill task. The genetics of alphamethrin, a synthetic pyrethroid resistance was studied to determine its mode of inheritance. The late third instar larvae were selectively inbred for 27 and ten generations to synthesize homozygous resistant (R) and susceptible (S) stocks, respectively, to the diagnostic dose of 0.12 mg l−1. The log-dosage probit mortality relationships and degree of dominance (D) were calculated. Resistance was observed in both sexes, the dosage-mortality (d-m) line of F1 was towards the resistant parent and the ‘D’ value was found to be 0.8 indicating alphamethrin resistant (amr) gene to be autosomal and incompletely dominant. The d-m lines of F2/backcross exhibited a clear plateau of mortality across a range of doses indicating monogenic resistance. The null hypothesis for monogenic resistance was tested from mortality data of backcross progeny compared with theoretical expectations using the χ2 test and was found to be non-significant. Understanding genetics of insecticide resistance is significant in prediction and management of resistant insects. The amr genes can be used as genetic marker in A. stephensi, which can be used in several applications in conducting basic and applied genetic research.

Midgut proteases contribute to the success or failure of Plasmodium infection of the mosquito. This paper examines the reciprocal effect of Plasmodium yoelii nigeriensis on midgut trypsin, chymotrypsin, aminopeptidase and carboxypeptidase in the mosquito Anopheles stephensi. The total protein ingested and the rate of protein digestion were unaffected by the parasite, but more protein was ingested at the first than the second bloodmeal. All peptidases were unaffected by the presence of the parasite during the first gonotrophic cycle, when ookinetes were penetrating the midgut. In the second gonotrophic cycle, trypsin and chymotrypsin were unaffected by growing oocysts, but aminopeptidase activity was reduced in the midguts of infected mosquitoes. Chymotrypsin activity was depressed and aminopeptidase activity elevated during the second gonotrophic cycle. Plasmodium infection has a negligible effect on bloodmeal digestion and does not limit the availability of the protein for egg production. The significance of changes in aminopeptidase activity when oocysts are present is discussed.

Anopheles stephensi mosquitoes showed a reduction in fecundity over 3 successive gonotrophic cycles, after becoming infected with Plasmodium yoelii nigeriensis. This effect could be observed at high oocyst burdens (> 75) or at low oocyst burdens (mean of 4·36). Mean bloodmeal size of the infected mosquitoes was significantly reduced only when feeding upon a mouse with a high gametocytaemia and the conversion of the bloodmeal into eggs by the infected mosquitoes was disrupted. Patterns of infected mosquito mortality, over the 3 gonotrophic cycles, varied with severity of infection. Although in 1 case increased mortality and decreased bloodmeal size may have affected fecundity, this could not have accounted for all of the observed fecundity reduction. We propose that other, unknown parasite related factors, are involved.

The effect of temperature on early Plasmodium falciparum development was examined in Anopheles stephensi. The rates of both ookinete development and bloodmeal digestion were lengthened as temperatures decreased from 27 to 21 °. However, low temperatures (21–27 °) did not significantly influence infection rates or densities of either ookinetes or oocysts. In contrast, high temperatures (30 and 32 °) significantly impacted parasite densities and infection rates by interfering with developmental processes occurring between parasite fertilization and ookinete formation, especially during zygote and early ookinete maturation. This study demonstrates clearly that temperature affects the sporogonic development of P. falciparum in anophelines by altering the kinetics of ookinete maturation. These studies not only confirm the ookinete as the key development stage affecting the probability of vector infectivity, they provide new insights into the epidemiology of P. falciparum infections.

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.

Subtherapeutic doses of chloroquine have been reported to enhance infectivity of drug-resistant Plasmodium species to their vectors. In this investigation, Plasmodium yoelii nigeriensis N67 strain showed enhanced infectivity to mosquitoes when stimulated by chloroquine. Both sensitive and resistant clones were derived from the N67 strain by dilution, showing that this strain is polymorphic for the resistant trait. A resistant subline was derived by selection under drug pressure from a chloroquine-sensitive clone, but neither the sensitive nor the resistant clones or sublines showed enhancement of infectivity in the presence of chloroquine. This suggests that the enhancement of infectivity shown by the N67 strain is a response to chloroquine stimulation shown only by certain of the genotypes within it, and that this response to chloroquine is not a trait causally connected with the genes coding for chloroquine resistance.

This study investigated the significance of serum complement on transmission-reducing activity (TRA) of field sera from 24 infected Plasmodium falciparum gametocyte carriers (from Cameroon) against cultured NF54 P. falciparum. Laboratory-reared Anopheles stephensi were given infectious blood meals prepared either with sera from naïve Dutch donor (AB type) or pair-matched field serum samples, both with and without active complement. TRA of serum factors and host complement on mosquito infection rate and oocyst intensity were divided into the various components involved in the early stages of sporogony. The majority (>80%) of sera tested showed positive antibody titres to Pfs230, the relevant complement-dependent target of transmission-reducing mechanisms. Regardless of the presence of active complement, bloodmeals with field sera exhibited significantly lower infection rates and oocyst intensity than the control group. Serological reactivity in Capture-ELISA against Pfs230 was significantly correlated with the reduction of parasite infectivity. Contrary to our expectation, the presence of active complement in the mosquito bloodmeal did not increase parasite losses and therefore the magnitude of transmission reduction by individual immune sera. Our findings on P. falciparum are consistent with previous studies on animal hosts of Plasmodium, indicating that early P. falciparum sporogonic stages may be insensitive to the antibody-dependent pathways of complement in human serum.

Host responses to the transmittable stages of the malaria parasite may reduce transmission effectively. Transmission-reducing activity (TRA) of human serum can be determined as a percentage, using the Standard Membrane Feeding Assay (SMFA). This laboratory assay was evaluated using the results of 121 experiments with malaria-endemic sera among which many repeated measurements were obtained. The assay consists of the feeding of Anopheles stephensi mosquitoes with cultured Plasmodium falciparum gametocytes, mixed with human red blood cells, and control and experimental sera. The TRA of individual sera was determined by the comparison of oocyst densities between these sera. Bootstrap data on oocyst densities in individual mosquitoes in control feeds were used to construct confidence limits for TRA percentages of serum feeds. Low (<20%) and high TRA (>90%) values for individual sera were usually reproduced in a second experiment, whereas this was more difficult for values between 20% and 90%. The observed variability of TRA values is explained in part by the variability in oocyst density per mosquito. Oocyst densities in control feeds varied more between experiments than within experiments and showed a slight decline over the 3 years of experiments. Reproducibility of TRA of field sera was low (20%) between experiments, but much higher (61%) within experiments. A minimum of 35 oocysts per mosquito in control feeds gave optimal reproducibility (44%) between experiments. We recommend that (1) sera are compared within an experiment, or (2) assays are only analysed where controls have at least 35 oocysts per mosquito. The SMFA is under the recommended conditions appropriate for the study of factors that may influence TRA, e.g. transmission blocking vaccines.

Plasmodium falciparum gametocytes grown in vitro were fed through membrane feeders to laboratory-reared Anopheles stephensi mosquitoes. Intact midguts, including entire bloodmeal contents, were removed between 24 and 48 h post-bloodfeeding. Giemsa-stained histological sections were prepared from the midguts and examined by light microscopy. Contrary to previous reports, ookinetes were clearly visible within midgut epithelial cells, demonstrating intracellular migration across the midgut wall. Ookinetes entered epithelial cells through the lateral apical membrane at sites where 3 adjacent cells converged. There was no evidence for the existence of a morphologically distinct group of epithelial cells preferentially invaded by ookinetes. However, ookinete penetration was associated with significant morphological changes to invaded cells, including differential staining, condensation and fragmentation of the nucleus, vacuolization, loss of microvilli and various degrees of extrusion into the midgut lumen. Epithelial cells completely separated from the midgut wall were found within the midgut lumen. These cells were associated with invading parasites suggesting that ookinete penetration resulted in complete ejection of invaded cells from the midgut wall. Small clusters of morphologically altered midgut cells and invading parasites spanning the membranes of adjacent abnormal epithelial cells were observed, consistent with intracellular movement of ookinetes between neighbouring midgut cells. Extruded epithelial cells were also observed rarely in uninfected midguts. Epithelial cell extrusion, therefore, may be a general mechanism of tissue repair through which damaged cells are removed from the midgut wall rather than a parasite-specific response. These observations demonstrate that human malaria parasite infection of mosquitoes is consistent with, and provides further support for, the Time Bomb model of ookinete invasion of the mosquito midgut epithelium previously proposed for rodent malaria parasites.

Two studies were carried out to determine the effect of the rodent malaria Plasmodium yoelii nigeriensis on the blood feeding success of Anopheles stephensi. Initially, pairs of mice with similar packed cell volume (PCV) (measured by haematocrit) were selected. Following infection of one of the pair its PCV gradually fell. At various times post-infection, a comparison was made of the bloodmeal size (haemoglobin content) of mosquitoes feeding on these mice. The bloodmeal sizes increased with parasite-induced fall in PCV down to a haematocrit of 43–44%, which occurred approximately 48 h post-infection. Bloodmeals were significantly reduced, however, when mosquitoes fed on mice with higher parasitaemias and a haematocrit of 15–35%. Thus, at early stages of infection, mosquitoes ingested a bloodmeal significantly greater than did the mosquitoes feeding on the control mice. However, mosquitoes were not able to compensate for severe infection-associated anaemia. To compensate for variation due to innate differences in the mice, a second experiment was performed. Mosquitoes were fed on the same mice before (control) and after infection. Again, the bloodmeal size increased with decreasing PCV down to haematocrits of 42–45%, but declined thereafter. In this host–parasite–vector system, haematocrits that maximized erythrocyte intake were produced when gametocytes, capable of exflagellation, were present.

Malarial gametocytes circulate in the peripheral blood of the vertebrate host as developmentally arrested intra-erythrocytic cells, which only resume development into gametes when ingested into the bloodmeal of the female mosquito vector. The ensuing development encompasses sexual reproduction and mediates parasite transmission to the insect. In vitro the induction of gametogenesis requires a drop in temperature and either a pH increase from physiological blood pH (ca pH 7·4) to about pH 8·0, or the presence of a gametocyte-activating factor recently identified as xanthurenic acid (XA). However, it is unclear whether either the pH increase or XA act as natural triggers in the mosquito bloodmeal. We here use pH-sensitive microelectrodes to determine bloodmeal pH in intact mosquitoes. Measurements taken in the first 30 min after ingestion, when malarial gametogenesis is induced in vivo, revealed small pH increases from 7·40 (mouse blood) to 7·52 in Aedes aegypti and to 7·58 in Anophěles stephensi. However, bloodmeal pH was clearly suboptimal if compared to values required to induce gametogenesis in vitro. Xanthurenic acid is shown to extend the pH-range of exflagellation in vitro in a dose-dependent manner to values that we have observed in the bloodmeal, suggesting that in vivo malarial gametogenesis could be further regulated by both these factors.

Changes in the pattern of haemolymph polypeptides were studied to map the innate immune response of the malaria vector Anopheles stephensi (Diptera: Culicidae) to infection by the parasite Plasmodium yoelii yoelii. In addition, changes in the ontogenetic pattern due to glucose feeding, age, sex and gonotrophic cycle-related proteins were analysed. Six polypeptides were induced and 22 polypeptides disappeared due to glucose feeding during various stages of the mosquito's adult life. Five polypeptide patterns (91, 100, 108, 133 & 145 KDa) were found exclusively in female haemolymph. The changes in these polypeptide patterns have been correlated with sexual dimorphism in their feeding behaviour.

In total, 18 polypeptides were induced in the haemolymph of parasitised mosquitoes during different stages of development. Most of the polypeptides were induced in the early stages of infection, i.e. immediately after a bloodmeal. One polypeptide (25 KDa) was induced in all the stages. The expression of two polypeptides (32.5 and 70 KDa) on day 9 and one polypeptide (42 KDa) on day 15 was also enhanced following parasitism. The different induced proteins may help mosquitoes of different ages cope with parasite infection. The parasite evidently elicits immune responses in multiple tissues of the mosquito, including two epithelia which the parasite must traverse to complete its development. The mechanism of these responses and their significance in malaria transmission are discussed.

The effect of hexaflumuron, a chitin synthesis inhibitor, on the mating success of three species of vector mosquitoes, namely Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi, was investigated. The three species were exposed to concentrations giving 5% and 50% inhibition of emergence (EI5 and EI50, respectively) at the fourth instar and to 0.005 and 0.05 mg/1 hexaflumuron at the pupal stage. Emergent adults were allowed to mate with each other and with their untreated counterparts. Reduction in mating (evaluated as % insemination) was highest in Ae. aegypti exposed at the larval stage. Mosquitoes exposed at the pupal stage to 0.05 mg/1 hexaflumuron showed higher reduction in mating than those exposed to 0.005 mg/1. The effect of hexaflumuron appeared to be cumulative, resulting in a 78–80% reduction in % insemination when treated males were mated with treated females, compared with untreated males mated with untreated females.