Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae)

Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.     

Midgut antibodies reduce the reproductive capacity of Anopheles stephensi (Diptera: Culicidae)

Rabbits immunized with polypeptides of midgut of glucose fed A. stephensi resulted in high titer of antibodies (10(4)-10(6)) as detected by ELISA. Effect of antisera on fecundity, hatchability and engorgement was investigated. Fecundity was reduced drastically (62.4%). Eight polypeptides were recognized by the antisera raised against midgut tissues viz., 92, 85, 55, 52, 45, 38, 29 and 13 kDa. Cross reactivity of these antibodies with different tissues of A. stephensi as well as different species of Anopheles was also analyzed. The results indicated that anti-mosquito midgut antibodies had the potential to disrupt the reproductive physiology of mosquitoes in view of the present study, there is a need for further investigation with target antigens.     

Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: culicidae)

The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P. vivax was studied by feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies, reported to exhibit differential vectorial capacity. The mosquitoes that ingested anti-midgut antibodies along with infectious parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.     

Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles Stephensi mosquitoes

Abstract. Anopheles stephensi mosquitoes which had fed upon mice infected with Plasmodium yoelii nigeriensis malaria parasites produced significantly fewer eggs than mosquitoes fed on an uninfected mouse. Fecundity reduction was more pronounced when the bloodmeal contained malaria gametocytes and the mosquitoes developed oocysts. Egg production and haematin excretion were correlated for uninfected bloodfed mosquitoes; the presence of P.y. nigeriensis in the blood affected this relationship. Reduced fecundity was associated with a significant reduction of bloodmeal size (measured by haematin excretion) in mosquitoes which ingested gametocytaemic blood. The bloodmeal size in mosquitoes fed on parasitaemic blood without gametocytes was not significantly reduced. The use of haematin assays for determination of bloodmeal size in mosquitoes is discussed.     

Genetic and environmental determinants of malaria parasite virulence in mosquitoes

Models of malaria epidemiology and evolution are frequently based on the assumption that vector-parasitic associations are benign. Implicit in this assumption is the supposition that all Plasmodium parasites have an equal and neutral effect on vector survival, and thus that there is no parasite genetic variation for vector virulence. While some data support the assumption of avirulence, there has been no examination of the impact of parasite genetic diversity. We conducted a laboratory study with the rodent malaria parasite, Plasmodium chabaudi and the vector, Anopheles stephensi, to determine whether mosquito mortality varied with parasite genotype (CR and ER clones), infection diversity (single versus mixed genotype) and nutrient availability. Vector mortality varied significantly between parasite genotypes, but the rank order of virulence depended on environmental conditions. In standard conditions, mixed genotype infections were the most virulent but when glucose water was limited, mortality was highest in mosquitoes infected with CR. These genotype-by-environment interactions were repeatable across two experiments and could not be explained by variation in anaemia, gametocytaemia, blood meal size, mosquito body size, infection rate or oocyst burden. Variation in the genetic and environmental determinants of virulence may explain conflicting accounts of Plasmodium pathogenicity to mosquitoes in the malaria literature.     

Ovary specific immune response during Plasmodium yoelii yoelii infection in malaria vector Anopheles stephensi (Diptera:Insecta)

Innate immune related polypeptides expression during three gonotrophic cycles in the ovaries of major disease vector mosquito A. stephensi has been analyzed following infection by malaria parasite, P. yoelii yoelii. Seventeen polypeptides were induced in the ovaries of various stages due to parasitic infection. Most of proteins were induced systemically during early stages of infection suggesting the possibility of immune related signalling process. The reduction in the quantity of protein contents in infected mosquitoes has been ascribed to the repression of seven polypeptides and in turn correlated with the fecundity reduction. The mechanism of these responses and their significance for malaria transmission and fecundity reduction are discussed.     

Plasmodium falciparum: ingested anti-sporozoite antibodies affect sporogony in Anopheles stephensi mosquitoes

In endemic areas, malaria-infected mosquitoes may feed upon humans who possess antibodies against malaria sporozoites. Therefore, we examined the effect that ingested anti-sporozoite antibodies have upon Plasmodium falciparum sporogony within Anopheles stephensi mosquitoes. Anti-sporozoite antibodies (IgG) traversed the midgut into the hemocoel within 3 hr following ingestion and, depending upon the titer, persisted for 6-24 hr. When fed to infected A. stephensi at 12 days postinfection (p.i.), anti-sporozoite antibodies bound to sporozoites in the hemocoel, but not to sporozoites residing in the salivary glands of the same mosquitoes. Anti-sporozoite antibodies also bound to developing oocysts when fed to infected A. stephensi at 5 days p.i. Oocysts in mosquitoes that had been fed anti-sporozoite antibodies on Day 5 p.i. produced significantly more sporozoites than did oocysts in nonimmune-fed (Day 5 p.i.) mosquitoes. In addition, the sporozoites from Day 5 immune-fed mosquitoes were significantly more infective to cultured human hepatoma cells than were sporozoites from nonimmune-fed controls. Use of hetereologous immune feedings at Day 5 p.i. did not result in an enhanced production of sporozoites, suggesting that enhancement is related to the specificity of the antibody and is not merely a nutritional effect.     

Effects of in vivo exposure to DEET on blood feeding behavior and fecundity in Anopheles quadrimaculatus (Diptera: Culicidae)

This study determined the effects of contact with DEET on guinea pig skin on mortality, probing time, blood feeding rate, engorgement time, and fecundity responses in female Anopheles quadrimaculatus Say. Exposure, in this manner, to 10% DEET (in ethanol) for 5 min, resulted in 98% mortality in mosquitoes after 24h. The median probing time (PT(50)) required by females, when exposed to 0.1%, 1.0%, and 10% DEET, was significantly (P<0.0001) longer (12.5, 12.1, and 19.1s, respectively) than the 6.8s required by females to probe ethanol-treated skin (control). Similarly, mean blood feeding rates in populations of females exposed to 1.0% DEET for < or = 5 min (14.4%) was 6x lower (P<0.001) (85.5%) than in females exposed to ethanol-treated skin, whereas the mean engorgement time on skin treated with 1.0% DEET (66.3s) was significantly shorter (P<0.0001) than for females feeding on the control guinea pigs (105.9s). The mean number of mature o?cytes per female (fecundity) in treatment (1.0% DEET) and control mosquitoes was not significantly different. The responses to DEET observed in this study suggest that repeated exposure of female A. quadrimaculatus populations to this repellent, in laboratory bioassays, could result in confounding of toxicant and repellent effects and inaccurate estimates of DEET repellency.     

Plasmodium berghei: inhibition of the sporogonous cycle by alpha-difluoromethylornithine

alpha-Difluoromethylornithine (DFMO), an enzyme inhibitor of ornithine decarboxylase, inhibits the sporogonous cycle of the malaria parasite Plasmodium berghei in the mosquito vector Anopheles stephensi. DFMO was administered to the mosquitoes dissolved either in the sugar solution at their disposal in the cages or through blood meals taken from treated mice. The mice subsequently bitten by mosquitoes treated with DFMO by both routes of administration did not contract malaria.     

Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1’s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.     

A rodent malaria, Plasmodium berghei, is experimentally transmitted to mice by merely probing of infective mosquito, Anopheles stephensi

We found that infection of a rodent malaria, Plasmodium berghei, occurred when the sporozoites were injected into the skin, the muscle, the peritoneal cavity and the tail end. Mice, which were injected with sporozoites in the tail end and had the site cut 5 min later, did not develop malaria. We also found that mice developed malaria when malaria infective mosquitoes, Anopheles stephensi, were forced not to take blood but only to probe into the skin. Moreover, the mice probed by the infective mosquitoes were protected from malaria infection if the site was treated with Kyu (heat treatment) after the mosquitoes had probed. These findings indicate that malaria infection occurs not only by blood feeding of the infective mosquito but also by probing of the mosquito. Sporozoites injected into the skin remain at the injected site for at least 5 min, then migrate to the blood vessels and invade into the blood stream. At present, the mechanism is not clear, although we propose here the existence of the skin stage of malaria parasites before the liver stage and the blood stage.     

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 3. Carbohydrates.

Determinations were made of carbohydrates in hemolymph collected from adult female mosquitoes (Anopheles stephensi). First the hemolymph was fractionated by extraction and precipitation procedures, after which qualitative and quantitative determinations of carbohydrates were made by thin layer chromatography. The most abundant sugars found in the hemolymph were glucose and trehalose, though maltose, glucuronic acid, and inositol could be found after the mosquitoes took blood meals. After the mosquitoes ingested a noninfected blood meal, their hemolymph sugar levels rose almost 4-fold. There was less of an increase following a blood meal infected with the rodent malaria parasite, Plasmodium berghei. Depletion of sugars in the hemolymph of infected mosquitoes may result from direct utilization of sugar by the malaria parasite developing within the mosquito.     

Infection of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundity

The entomopathogenic fungus Metarhizium anisopliae is being considered as a biocontrol agent against adult African malaria vectors. In addition to causing significant mortality, this pathogen is known to cause reductions in feeding and fecundity in a range of insects. In the present study we investigated whether infection with M. anisopliae affected blood feeding and fecundity of adult female malaria vectors Anopheles gambiae Giles sensu stricto. Mosquitoes were contaminated with either a low or a moderately high dose of oil-formulated conidia of M. anisopliae, and offered a single human blood meal 48, 72, or 96 h later to assess feeding propensity and individual blood meal size. In a second experiment, individual fungus-infected females were offered a blood meal every third day (to a total of 8 gonotrophic cycles), and allowed to oviposit after each cycle in order to quantify feeding propensity and fecundity. Infected females took smaller blood meals and displayed reduced feeding propensity. It was found that mosquitoes, inoculated with a moderately high dose of fungal conidia, exhibited reduced appetite related to increasing fungal growth. Of the fungus-infected females, the proportion of mosquitoes taking the second blood meal was reduced with 51%. This was further reduced to 35.3% by the 4th blood meal. During 8 feeding opportunities, the average number of blood meals taken by uninfected females was 4.39, against 3.40 (low dose), and 2.07 (high dose) blood meals for the fungus-infected females. Moreover, infected females produced fewer eggs per gonotrophic cycle and had a lower life-time fecundity. Epidemiological models show that both blood feeding and fecundity are among the most important factors affecting the likelihood of a mosquito transmitting malaria, which suggests that this fungus may have potential as biocontrol agent for vector-borne disease control.     

Alterations in polypeptides pattern in malaria vector Anopheles stephensi, fed upon immunized blood causing fecundity reduction

Changes in polypeptides pattern of haemolymph, midgut, ovary and salivary glands of female mosquito A. stephensi were studied when fed upon anti-mosquito haemolymph antibodies. The expression of almost all polypeptides was reduced in haemolymph and ovary of the immune fed mosquitoes as compared to control. However, there was no significant difference in case of midgut and salivary glands. Seven polypeptides 100, 90, 84, 80, 62, 19 and 12.5 kDa were absent in haemolymph and five 92, 90, 80, 60 and 55 kDa were absent in ovaries. Changes in the polypeptide pattern have been correlated with the fecundity reduction due to immunized blood feeding.     

Studies on effects of Pelargonium citrosa leaf extracts on malarial vector,Anopheles stephensi Liston

Methanol extracts of Pelargonium citrosa leaf were tested for their biological, larvicidal, pupicidal, adulticidal, antiovipositional activity, repellency and biting deterrency against Anopheles stephensi. Larval mortality was dose dependent with the highest dose of 4% plant extract evoking 98% mortality. The extracts affected pupicidal and adulticidal activity and significantly decreased fecundity and longevity of A. stephensi. The larval, pupal and adult development were completely inhibited by the treatment. At 4% the extracts evoked strong repellent action. They also interfered with oviposition, egg hatchability, and exhibited a growth inhibiting effect against larvae and good repellency against adults of A. stephensi. The leaf extract treatment significantly enhanced biting deterrency. As naturally occurring insecticides, these plant derived materials could be useful as an alternative for synthetic insecticides controlling field populations of mosquitoes.     

The effects of blood feeding and exogenous supply of tryptophan on the quantities of xanthurenic acid in the salivary glands of Anopheles stephensi (Diptera: Culicidae)

Xanthurenic acid (XA), produced as a byproduct during the biosynthesis of insect eye pigment (ommochromes), is a strong inducer of Plasmodium gametogenesis at very low concentrations. In previous studies, it was shown that XA is present in Anopheles stephensi (Diptera: Culicidae) mosquito salivary glands and that during blood feeding the mosquitoes ingested their own saliva into the midgut. Considering these two facts together, it is therefore likely that XA is discharged with saliva during blood feeding and is swallowed into the midgut where it exerts its effect on Plasmodium gametocytes. However, the quantities of XA in the salivary glands and midgut are unknown. In this study, we used high performance liquid chromatography with electrochemical detection to detect and quantify XA in the salivary glands and midgut. Based on the results of this study, we found 0.28+/-0.05 ng of XA in the salivary glands of the mosquitoes, accounting for 10% of the total XA content in the mosquito whole body. The amounts of XA in the salivary glands reduced to 0.13+/-0.06 ng after mosquitoes ingested a blood meal. Approximately 0.05+/-0.01 ng of XA was detected in the midgut of nonblood fed An. stephensi mosquitoes. By adding synthetic tryptophan as a source of XA into larval rearing water (2 mM) or in sugar meals (10 mM), we evaluated whether XA levels in the mosquito (salivary glands, midgut, and whole body) were boosted and the subsequent effect on infectivity of Plasmodium berghei in the treated mosquito groups. A female specific increase in XA content was observed in the whole body and in the midgut of mosquito groups where tryptophan was added either in the larval water or sugar meals. However, XA in the salivary glands was not affected by tryptophan addition to larval water, and surprisingly it reduced when tryptophan was added to sugar meals. The P. berghei oocyst loads in the mosquito midguts were lower in mosquitoes fed tryptophan treated sugar meals than in mosquitoes reared on tryptophan treated larval water. Our results suggest that mosquito nutrition may have a significant impact on whole body and midgut XA levels in mosquitoes. We discuss the observed parasite infectivity results in relation to XA's relationship with malaria parasite development in mosquitoes.     

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.     

Uptake and persistence of ingested antibody in the mosquito Anopheles stephensi

A sensitive ELISA was developed to monitor the persistence of a specific antibody, rabbit anti-BSA, in the bloodmeal, haemolymph and tissues of the mosquito Anopheles stephensi Liston. Different concentrations of anti-BSA were fed to female mosquitoes in sheep blood, via a membrane-feeder, and it was found that antibody persisted in the gut as the bloodmeal was digested: concentrations present at 24 h were directly related to those fed. Homogenates of mosquito bodies, from which the intact guts had been removed, were always antibody-positive up to 9 days post-feeding, indicating that undigested antibody had passed through the gut wall into the haemocoele. Haemolymph was extracted from mosquitoes at different times post-feeding, using a microcapillary and manipulator, and antibody was detected in several of the assays. The level of antibodies in the haemolymph 24 h post-feeding was less than half of the level in mosquito heads, indicating removal of antibodies from the haemolymph, perhaps by binding onto haemocoelic tissues. The relevance of these results to the ingestion, survival and fate of antibody against malaria sporozoites is discussed.     

Fitness of anopheline mosquitoes expressing transgenes that inhibit Plasmodium development

One potential strategy for the control of malaria and other vector-borne diseases is the introduction into wild vector populations of genetic constructs that reduce vectorial capacity. An important caveat of this approach is that the genetic construct should have minimal fitness cost to the transformed vector. Previously, we produced transgenic Anopheles stephensi expressing either of two effector genes, a tetramer of the SM1 dodecapeptide or the phospholipase A2 gene (PLA2) from honeybee venom. Mosquitoes carrying either of these transgenes were impaired for Plasmodium berghei transmission. We have investigated the role of two effector genes for malaria parasite blockage in terms of the fitness imposed to the mosquito vector that expresses either molecule. By measuring mosquito survival, fecundity, fertility, and by running population cage experiments, we found that mosquitoes transformed with the SM1 construct showed no significant reduction in these fitness parameters relative to nontransgenic controls. The PLA2 transgenics, however, had reduced fitness that seemed to be independent of the insertion site of the transgene. We conclude that the fitness load imposed by refractory gene(s)-expressing mosquitoes depends on the effect of the transgenic protein produced in that mosquito. These results have important implications for implementation of malaria control via genetic modification of mosquitoes.