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.     

Antibodies to Anopheles midgut reduce vector competence for Plasmodium vivax malaria

Anopheles tessellatus mosquitoes ingested Plasmodium vivax gametocytes in human erythrocytes suspended in rabbit sera with and without anti-mosquito midgut antibodies. When the mosquito bloodmeal contained anti-midgut antibodies, fewer oocysts of P. vivax developed on the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. Complement inactivated serum also reduced the infection rate and load. A second bloodmeal containing anti-midgut antibodies, given 48 or 72 h later, did not enhance the transmission-blocking effect. IgG purified from antimidgut sera was shown to mediate the transmission-blocking effect.     

Effect of anti-mosquito antibodies on the infectivity of the rodent malaria parasite Plasmodium berghei to Anopheles farauti

The effect of mouse anti-mosquito antibodies, present in the bloodmeal, on the infectivity of Plasmodium berghei Vincke to Anopheles farauti Laveran was investigated. Significantly fewer oocysts developed in mosquitoes feeding on mice immunized with sugar-fed mosquito midgut antigens than in mosquitoes feeding on control mice. Mosquitoes feeding on mice immunized with the midgut antigens derived from sugar-fed mosquitoes also showed reduced mortality and had lower infection rates than those fed on unimmunized mice. Blood-fed midgut antigen was less effective in producing these effects than sugar-fed midgut antigen.     

Fecundity of Anopheles tessellatus reduced by the ingestion of murine anti-mosquito antibodies

Abstract. High titres of antibodies to antigens derived from head/thorax, midgut or abdomen of Anopheles tessellatus were produced in inbred mice. These antibodies, when ingested in a bloodmeal, reduced the fecundity of An.tessellatus by up to 29% in different experiments. It is postulated that antibodies directed against antigens shared between the head/thorax, abdomen and midgut tissues are involved in the reduction of fecundity.     

Effect of mating period and time‐of‐day for bloodmeal on rearing of Asian tiger mosquito (Aedes albopictus) in laboratory conditions

Colonization and maintenance of mosquitoes in the laboratory is required to study physiology, ecology, and behavior of mosquitoes and interactions between mosquito and pathogens. Artificial blood feeding systems have been widely used to maintain the laboratory colony of Aedes albopictus. In this study, we investigated the effects of mating period (1, 3, 6, and 10 days) and time‐of‐day for bloodmeal (08:00, 13:00, and 18:00) in the use of an artificial feeding system on blood‐feeding rate, female fecundity, egg hatching rate, and developmental time of the Asian tiger mosquito, A. albopictus. Younger females mated for three or fewer days reproduced more eggs compared to those of oldest females mated for ten days. Similar to the result for eggs laid, the mean egg‐hatching rate was significantly higher from the offspring of younger females than from those of older females. However, mating period and time‐of‐day for bloodmeal had no effect on the blood feeding rate and developmental time. Taken together, we suggest that three‐day mating with bloodmeal at 18:00 is optimal for maintaining colonies of A. albopictus in laboratory conditions.     

Effect of anti-fat body antibodies on reproductive capacity of mosquito Anopheles stephensi and transmission blocking of Plasmodium vivax

Effect of anti-mosquito-fat body antibodies on the development of the malaria parasite, Plasmodium vivax has been studied by feeding Anopheles stephensi mosquitoes with infected blood supplemented with serum from immunized rabbits. Immunogenic polypeptides were identified by western blot. Mosquitoes that ingested anti-fat body antibodies along with infectious blood meal had significantly fewer oocysts than the mosquitoes in the control group. Effect of anti-mosquito fat body antibodies on fecundity, hatchability, mortality and engorgement of mosquitoes has also been reported. A significant reduction in fecundity and hatchability was observed, however, effect on mortality and engorgement was variable and statistically insignificant. Results indicated that fat body antibodies have the potential to disrupt reproductive physiology of malaria vector An. stephensi.     

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.     

Human immune responses that reduce the transmission of Plasmodium falciparum in African populations

Malaria-infected individuals can develop antibodies which reduce the infectiousness of Plasmodium gametocytes to biting Anopheles mosquitoes. When ingested in a bloodmeal together with gametocytes, these antibodies reduce or prevent subsequent parasite maturation in the insect host. This transmission-blocking immunity is usually measured in human sera by testing its effect on the infectivity of gametocytes grown in vitro. Here we evaluate evidence of transmission-blocking immunity in eight studies conducted in three African countries. Plasmodium falciparum gametocytes isolated from each individual were fed to mosquitoes in both autologous plasma collected with the parasites, and permissive serum from non-exposed donors. Evidence of transmission reducing effects of autologous plasma was found in all countries. Experiments involving 116 Gambian children (aged 0.5-15 years) were combined to determine which factors were associated with transmission reducing immune responses. The chances of infecting at least one mosquito and the average proportion of infected mosquitoes were negatively associated with recent exposure to gametocytes and sampling late in the season. These results suggest that effective malaria transmission-reducing antibodies do not commonly circulate in African children, and that recent gametocyte carriage is required to initiate and/or boost such responses.     

Using bacteria to express and display anti-Plasmodium molecules in the mosquito midgut

Bacteria capable of colonizing mosquito midguts are attractive vehicles for delivering anti-malaria molecules. We genetically engineered Escherichia coli to display two anti-Plasmodium effector molecules, SM1 and phospholipase-A(2), on their outer membrane. Both molecules significantly inhibited Plasmodium berghei development when engineered bacteria were fed to mosquitoes 24h prior to an infective bloodmeal (SM1=41%, PLA2=23%). Furthermore, prevalence and numbers of engineered bacteria increased dramatically following a bloodmeal. However, E. coli survived poorly in mosquitoes. Therefore, Enterobacter agglomerans was isolated from mosquitoes and selected for midgut survival by multiple passages through mosquitoes. After four passages, E. agglomerans survivorship increased from 2 days to 2 weeks. Since E. agglomerans is non-pathogenic and widespread, it is an excellent candidate for paratransgenic control strategies.     

Comparative fecundity and associated factors for two sibling species of the Anopheles gambiaecomplex occurring sympatrically in The Gambia

Abstract. For two sibling species of mosquitoes belonging to the Anopheles gambiae complex of malaria vectors, the effects of body size (wing length) and bloodmeal size (haematin excretion) on fecundity of wild females were investigated in The Gambia, West Africa. Freshly blood-fed individuals from sympatric populations of An.arabiensis and An.gambiae sensu stricto were sampled by collection at 07.00–09.00 hours from within bednets during July/August 1993, at the beginning of the rainy season. The possible confounding effect of infection with Plasmodium parasites was removed by eliminating infected mosquitoes from the study samples. An.arabiensis females comprised 75% of the An.gambiae sensu law population and were significantly larger (greater mean wing length) than those of An.gambiae s.s. mosquitoes. Mean egg production per female (for the subsequent gonotrophic cycle, excluding pre-gravids) for the two species was not significantly different, though the relationship between wing length and egg production showed An.gambiae s.s. to be more fecund than the An.arabiensis of the same size. Pre-gravid An.gambiae s.s. had consumed significandy smaller bloodmeals than gravid females but the mean wing length of these two gonotrophic categories was not significantly different. In contrast, An.arabiensis pre-gravids were smaller and had consumed smaller bloodmeals than the gravids.     

Effect of adult nutrition on the melanization immune response of the malaria vector Anopheles stephensi

Two dietary resources - blood and sugar - were assessed for effects on the melanization immune response of the mosquito Anopheles stephensi Liston (Diptera: Culicidae) towards inoculated Sephadex beads (negatively charged C-25). This melanization is conferred by genetic factors capable of making the mosquito refractory to malaria parasites. If An. stephensi females had obtained a bloodmeal one day before inoculation with a bead, the efficacy of their immune response increased with the concentration of sugar ingested. At the highest sugar concentration (6%) tested, 38% of the mosquitoes completely melanized their bead, whereas at the lowest sugar concentration (2%), none of the mosquitoes were able to melanize their bead completely. Among mosquitoes not having a bloodmeal, the immuno-competence was low (c. 9% of the mosquitoes completely melanized their bead) and independent of sugar concentration. The observed interaction between these two resources indicates that both resources are required for the Anopheles female to develop an effective melanization immune response.     

Molecular methods for arthropod bloodmeal identification and applications to ecological and vector-borne disease studies

DNA-based methods have greatly enhanced the sensitivity and specificity of hematophagous arthropod bloodmeal identification. A variety of methods have been applied to study the blood-feeding behaviour of mosquitoes, ticks, black flies and other blood-feeding arthropods as it relates to host-parasite interactions and pathogen transmission. Overviews of the molecular techniques used for bloodmeal identification, their advantages, disadvantages and applications are presented for DNA sequencing, group-specific polymerase chain reaction primers, restriction fragment length polymorphism, real-time polymerase chain reaction, heteroduplex analysis, reverse line-blot hybridization and DNA profiling. Technical challenges to bloodmeal identification including digestion and analysis of mixed bloodmeals are discussed. Analysis of bloodmeal identification results remains a challenge to the field, particularly with regard to incorporation of vertebrate census and ecology data. Future research directions for molecular analysis of arthropod bloodmeals are proposed.     

Evaluation of procedures to determine absolute density of Plasmodium vivax ookinetes

The ookinete is the key determinant of infection within the mosquito vector, yet there are few population studies of ookinetes in nature. This investigation compared different techniques used to estimate ookinete densities in mosquitoes. Laboratory-reared Anopheles dirus mosquitoes were fed on gametocytemic blood drawn from 7 Plasmodium vivax patients at a malaria clinic in Mae Sot, Thailand. At 20-26 hr, bloodmeals were excised. Three techniques were evaluated, i.e., hemacytometer counts under phase-contrast microscope, Giemsa staining of bloodmeal smears, and immunofluorescent staining with a monoclonal antibody specific against the 25-kDa antigen expressed on the surface of P. vivax zygotes and ookinetes. Additional mosquitoes were dissected at day 10 for oocysts. The hemacytometer method was the simplest and quickest method but lacked precision at low ookinete densities. Immunofluorescent staining was the most sensitive, accurate, and the only method that enabled unequivocal detection of zygotes. Bloodmeals contained a mixture of zygotes, retorts, and mature ookinetes, indicating that postzygotic development of P. vivax in A. dirus was asynchronous. The conversion efficiency of zygotes/ookinetes to oocysts varied among patients and was independent of zygote-ookinete density, suggesting that variations in host blood composition, e.g., antibodies, drugs, etc., may influence the success of zygote-ookinete development.     

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.     

Stable isotope analysis can potentially identify completely-digested bloodmeals in mosquitoes

Background

Vertebrate bloodfeeding is a critical component of a mosquito''s ability to transmit pathogens that cause diseases such as malaria, dengue fever and viral encephalitis. Due to degradation by the digestive process, current methods to identify mosquito bloodmeal sources are only useful for approximately 36 hours post-feeding. A critical need exists for technologies to extend this window and gain a more complete picture of mosquito feeding behavior for epidemiological studies. Stable isotopes are useful for investigating organism feeding behavior because the isotopic ratio of an organism''s tissues reflects that of the material it ingests.

Methodology/Principal Findings

Proof-of-principle data indicates that after bloodfeeding, Aedes albopictus mosquitoes acquire diagnostic Carbon and Nitrogen stable isotope profiles from their vertebrate hosts that can be accurately identified one week post-feeding, approximately 4 days after the entire bloodmeal has been digested. Total C/N ratio served as a biomarker marker for bloodfeeding (P<0.02), while δN was the most informative variable which could distinguish between unfed, chicken-fed and human-fed mosquitoes (P<0.01). By plotting C/N vs. δN, all feeding treatments could be identified in a double-blind analysis.

Conclusions/Significance

These proof-of-principle experiments indicate that analysis of stable isotopes can be used to distinguish bloodfed from unfed mosquitoes, and also distinguish between different vertebrate bloodmeal sources even after all blood has been digested. The development of stable isotope-based assays for mosquito bloodmeal identification may be a powerful tool to investigate mosquito feeding ecology and the dynamics of vector-borne pathogens.     

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.     

Diversion of Anopheles gambiae from children to other hosts following exposure to permethrin-treated bednets

Permethrin-treated bednets reduce mortality and morbidity from malaria in Gambian children. However, it is not certain how this effect is achieved, as neither mosquito numbers nor the human blood index of indoor-resting female Anopheles gambiae Giles sensu lato (Diptera: Culicidae) mosquitoes have been reduced when treated bednets were introduced into a community. One possibility is that insecticide-treated bednets divert mosquitoes from children to adults. To investigate this hypothesis, a cross-over trial with insecticide-treated bednets was undertaken in two small Gambian villages. To differentiate mosquitoes that had fed on children from those that had fed on adults, all children in the study villages were immunized with rabies vaccine before the trial. Using the detection of rabies antibody in a bloodmeal as an indicator that a mosquito had bitten a child, it was found that the percentage of blood-fed mosquitoes caught indoors that had bitten a child fell significantly from 30.8% to 9.2% and from 28.0% to 6.9% in each village after insecticide-treated bednets were introduced. To investigate the possibility that some diversion to animals had occurred, a PCR analysis for human beta-globin DNA was undertaken on selected samples. The results of this investigation were confusing, as some rabies-antibody positive bloodmeals were negative for human DNA. This may have been due to cross-reacting antibodies in animal sera and/or DNA degradation by digestion in the mosquito. Although good evidence for diversion of mosquitoes away from children was obtained, it remains uncertain whether diversion was mainly to adult humans, to animals or to both.     

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.     

Plasmodium infection decreases fecundity and increases survival of mosquitoes

Long-lived mosquitoes maximize the chances of Plasmodium transmission. Yet, in spite of decades of research, the effect of Plasmodium parasites on mosquito longevity remains highly controversial. On the one hand, many studies report shorter lifespans in infected mosquitoes. On the other hand, parallel (but separate) studies show that Plasmodium reduces fecundity and imply that this is an adaptive strategy of the parasite aimed at redirecting resources towards longevity. No study till date has, however, investigated fecundity and longevity in the same individuals to see whether this prediction holds. In this study, we follow for both fecundity and longevity in Plasmodium-infected and uninfected mosquitoes using a novel, albeit natural, experimental system. We also explore whether the genetic variations that arise through the evolution of insecticide resistance modulate the effect of Plasmodium on these two life-history traits. We show that (i) a reduction in fecundity in Plasmodium-infected mosquitoes is accompanied by an increase in longevity; (ii) this increase in longevity arises through a trade-off between reproduction and survival; and (iii) in insecticide-resistant mosquitoes, the slope of this trade-off is steeper when the mosquito is infected by Plasmodium (cost of insecticide resistance).