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 specific immune response of vector mosquito Anopheles stephensi to malaria parasite Plasmodium

Innate immune-related polypeptides expression in midgut in the ageing vector mosquito A. stephensi following infection by malaria parasite, Plasmodium yoelii yoelii has been studied. Twenty polypeptides were induced by an infected blood meal during various stages of adult life. A 24 kDa polypeptide was induced generally in most of the stages. Maximum parasite induced polypeptides i.e. 22, 33, 111, 122, 127, 140, 143 and 146 kDa were found in 5 days of post blood feeding (PBF) which coincides with the presence of oocysts on the midgut. However, in addition, three polypeptides in 11 days PBF and 8 polypeptides in 20 days PBF were also induced due to parasite infection in aged mosquitoes. Quantitatively, the amount of soluble proteins in the midgut in oocyst-sporozoite-positive mosquitoes was always less as compared to their normal counterparts. The parasite evidently elicits defined immune responses by inducing specific polypeptides in the midgut of the mosquito.     

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.     

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.     

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.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Monoclonal antibodies AC-43 and AC-29 disrupt <Emphasis Type="Italic">Plasmodium vivax</Emphasis> development in the Indian malaria vector <Emphasis Type="Italic">Anopheles culicifacies</Emphasis> (Diptera: culicidae)

A repertoire of monoclonal antibodies (mAbs) was generated against the midgut proteins of Anopheles culicifacies mosquitoes. The mAbs AC-43 and AC-29 significantly inhibited Plasmodium vivax development inside the mosquito midgut. The number of oocysts that developed was reduced by 78.6% when mosquitoes ingested a combination of these two mAbs along with the blood meal. AC-43 mAb binds to the epitope common in 97, 80 and 43 kDa polypeptides from the midgut protein extract, as indicated by western blot analysis. Similarly, the mAb AC-29 recognized 52, 44, 40 and 29 kDa polypeptides. These female midgut-specific polypeptides are shared between An. culicifacies and An. stephensi, two major vectors of malaria in India. Deglycosylation assays revealed that O-linked carbohydrates are the major components in epitopes corresponding to AC-43 and AC-29. Gold particle labelling revealed that both these mAbs preferentially bind to glycoproteins at the apical microvilli and the microvillus-associated network present inside transverse sections of the gut epithelium. These regions are particularly known to have receptors for ookinetes, which enable them to cross this epithelial barrier and provide them with certain necessary chemicals or components for further development into oocysts. Therefore, these glycoproteins appear to be potential candidates for a vector-directed transmission-blocking vaccine (TBV).     

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.     

Lactate dehydrogenase allozyme differentiation of species in the Anopheles culicifacies complex

Abstract Genetically controlled enzyme variation exists within and between four sibling species of the Anopheles culicifacies complex of malaria vectors in India. A study on electrophoretic variation of nine enzymes in An.culicifacies sibling species revealed that the lactate dehydrogenase ( Ldh ) locus has Fast (F) and Slow (S) allozymes distinguishing species A+D from species B+C with a probability of c . 95%.     

印度疟疾媒介库态按蚊卵黄蛋白原基因的克隆与生物信息学分析（英文）

卵黄蛋白原（vitellogenin, Vg)是主要的卵黄蛋白前体, 在雌虫血餐之后在脂肪体内大量合成。卵黄蛋白原的调节元件已经被用于驱动蚊子（与寄生虫发生最大相互作用的场所）中抗寄生基因的组织特异性表达。不过, 迄今为止, 对在印度引起60%~70%疟疾发生的库态按蚊Anopheles culicifacies中的内源启动子尚未进行过分析。本研究通过PCR扩增了包括5′端上游调节区在内的库态按蚊A. culicifacies卵黄蛋白原基因, 并命名为AncuVg (GenBank登录号为JN113091)。它含有一个大约6.2 kb的开放阅读框, 编码2 052个氨基酸, 具有一个16个氨基酸残基的推断的信号肽。也含有一个N_Vitellogenin区和一个VWF型D区, 这两个区在其他昆虫卵黄蛋白原中也保守。估计多肽分子量为238.0 kDa, 含有4个共有的(RXXR/S)切割位点, C端附近有一个GL/ICG基序, 其后是9个半胱氨酸残基和1个位于GL/ICCG基序上游第18个氨基酸残基处的DGXR 基序。在推断的氨基酸序列上发现3个聚丝氨酸区, 其中2个位于氨基端, 1个位于羧基端。根据同义密码子相对使用概率值, 通过有效密码子数, 测定了蚊子卵黄蛋白原基因密码子的偏倚性程度。也预测了库态按蚊A. culicifacies Vg的三维结构。分析了AncuVg基因, 以理解Vg基因的转录调节。对Vg基因5′端上游区进行的系统发育分析表明, 它们聚类于蚊子的3大分枝。也用各种生物信息学工具分析分析了Vg的同源性和特征。     

Anopheles culicifacies sibling species B and E in Sri Lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides

Members of the Anopheles culicifacies Giles complex (Diptera: Culicidae) are well established as the predominant vectors of malaria in Sri Lanka. Until recently, only sibling species B was reported to be present in Sri Lanka, which was surprising as species B is a poor vector of malaria in India. This was clarified by the identification through Y-chromosome morphology that what was reported as B on the island is really a mixture of B and E. The fecundity, longevity and insecticide resistance of B and E are of relevance to malaria transmission and its control and are reported in this study. The mean egg production of these two sibling species did not differ significantly. The mean age of wild mosquitoes was assessed by the Polovodova technique of observing ovarian dilatations. More of species E than B had three or more dilatations, i.e. had reached an age at which sporozoites could have developed to maturity, although the difference between the species was of borderline significance. Following feeding on Plasmodium vivax or Plasmodium falciparum infected blood, some females of species E developed oocysts but none of species B did so. Both sibling species were found fully susceptible in laboratory tests to lambdacyhalothrin and deltamethrin, but resistant to DDT and partially resistant to malathion.     

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.     

Characterization of monoclonal antibodies directed against erythrocytic stage antigens of Plasmodium berghei

Monoclonal antibodies recognizing various facets of the malaria parasite Plasmodium berghei and of the infected erythrocyte were obtained after generation of hybridomas between spleen cells from immunized mice and myeloma cells. The monoclonal antibodies were characterized by enzyme-linked immunosorbent assay, indirect immunofluorescence, immunoprecipitation of [35S]methionine-labeled proteins and immunoblotting. The most readily identified antigen was a parasite surface-associated protein of 230 kDa which is similar to the polymorphic schizont antigen described in a number of malarial species. In addition, three distinct antigens of 13, 31 and 120 kDa, which are external to the parasite, but within the infected erythrocyte were identified.     

Anopheles culicifacies in Baluchistan, Iran

Abstract. Anopheles culicifacies (probably species A) is the main vector of malaria in Baluchistan, southeastern Iran. Adult mosquitoes were collected during 1990-92 by five methods of sampling: knock-down pyrethrum space-spray indoors, human and animal bait (18.00–05.00 hours), pit shelters and CDC light traps, yielding 62%, 3%, 6%, 4% and 25% of specimens, respectively. Whereas spray-catches comprised c. 70% gravid and semi-gravid females, light trap catches were mostly ( c . 60%) unfed females, while females from pit shelters comprised all abdominal stages more equally (13–36%). An. culicifacies populations peaked in April-May and rose again during August-November. Densities of indoor-resting mosquitoes were consistently greater in an unsprayed village than in villages subjected to residual house-spraying with propoxur, malathion or pirimiphos-methyl. Monthly malaria incidence generally followed fluctuations of An. culicifacies density, usually with a peak in May-June.     

Adult anopheline ecology and malaria transmission in irrigated areas of South Punjab, Pakistan

Surface irrigation in the Punjab province of Pakistan has been carried out on a large scale since the development of the Indus Basin Irrigation System in the late 19th century. The objective of our study was to understand how the population dynamics of adult anopheline mosquitoes (Diptera: Culicidae) could be related to malaria transmission in rural areas with intensive irrigation and a history of malaria epidemics. In this paper we present our observations from three villages located along an irrigation canal in South Punjab. The study was carried out from 1 April 1999 to 31 March 2000. Mosquitoes were collected from bedrooms using the pyrethroid spraycatch method and from vegetation and animal sheds using backpack aspirators. Overall, Anopheles subpictus Grassi sensu lato predominated (55.6%), followed by An. stephensi Liston s.l. (41.4%), An. culicifacies Giles s.l. (2.0%), An. pulcherrimus Theobald (1.0%) and An. peditaeniatus Leicester (0.1%). Most mosquitoes (98.8%) were collected from indoor resting-sites whereas collections from potential resting-sites outdoors accounted for only 1.2% of total anopheline densities, confirming the endophilic behaviour of anophelines in Pakistan. Anopheles stephensi, An. culicifacies and An. subpictus populations peaked in August, September and October, respectively. High temperatures and low rainfall negatively affected seasonal abundance in our area. There were interesting differences in anopheline fauna between villages, with An. culicifacies occurring almost exclusively in the village at the head of the irrigation canal, where waterlogged and irrigated fields prevailed. Monthly house-to-house fever surveys showed that malaria transmission remained low with an overall slide positivity rate of 2.4% and all cases were due to infection with Plasmodium vivax. The most plausible explanation for low transmission in our study area seems to be the low density of Pakistan's primary malaria vector, An. culicifacies. The role of other species such as An. stephensi is not clear. Our observations indicate that, in South Punjab, irrigation-related sites support the breeding of anopheline mosquitoes, including the vectors of malaria. As our study was carried out during a year with exceptionally hot and dry climatic conditions, densities and longevity of mosquitoes would probably be higher in other years and could result in more significant malaria transmission than we observed. To assess the overall importance of irrigation-related sites in the epidemiology of malaria in the Punjab, more studies are needed to compare irrigated and non-irrigated areas.     

Differential expression of proteins in the midgut of Anopheles albimanus infected with Plasmodium berghei

The main vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito. The midgut of disease-transmitting mosquitoes carries out a variety of functions that are related to blood feeding. We analyzed the midgut of A. albimanus infected with Plasmodium berghei (resistant mosquito) using a proteomic approach to identify putative short peptides that are enriched in the midgut after blood feeding. Mosquito midguts were analyzed by two-dimensional electrophoresis to determine the changes in protein profiles. We identified 21 spot proteins that are differentially expressed in the blood of mosquitoes during the immune challenge. Molecular weight of the spots varied from 13 to 36 kDa, with a broad isoelectric point range of 3.92–8.90. We identified the differentially expressed proteins using mass spectrometry and constructed a proteomic data base of the A. albimanus midgut with diverse functions, some of them proteins with digestive and immunologic functions. Identification of these proteins may have important implications for understanding the blood meal digestion process, as well as developing novel vector control strategies and understanding parasite vector interactions.     

Distribution of sibling species of Anopheles culicifacies s.l. and Anopheles fluviatilis s.l. and their vectorial capacity in eight different malaria endemic districts of Orissa, India

The study was undertaken in eight endemic districts of Orissa, India, to find the members of the species complexes of Anopheles culicifacies and Anopheles fluviatilis and their distribution patterns. The study area included six forested districts (Keonjhar, Angul, Dhenkanal, Ganjam, Nayagarh and Khurda) and two non-forested coastal districts (Puri and Jagatsingpur) studied over a period of two years (June 2007-May 2009). An. culicifacies A, B, C and D and An. fluviatilis S and T sibling species were reported. The prevalence of An. culicifacies A ranged from 4.2-8.41%, B from 54.96-76.92%, C from 23.08-33.62% and D from 1.85-5.94% (D was reported for the first time in Orissa, except for occurrences in the Khurda and Nayagarh districts). The anthropophilic indices (AI) were 3.2-4.8%, 0.5-1.7%, 0.7-1.37% and 0.91-1.35% for A, B, C and D, respectively, whereas the sporozoite rates (SR) were 0.49-0.54%, 0%, 0.28-0.37% and 0.41-0.46% for A, B, C and D, respectively. An. fluviatilis showed a similarly varied distribution pattern in which S was predominant (84.3% overall); its AI and SR values ranged from 60.7-90.4% and 1.2-2.32%, respectively. The study observed that the co-existence of potential vector sibling species of An. culicifacies (A, C and D) and An. fluviatilis S (> 50%) was responsible for the high endemicity of malaria in forested districts such as Dhenkanal, Keonjhar, Angul, Ganjam, Nayagarh and Khurda (> 5% slide positivity rate). Thus, the epidemiological scenario for malaria is dependent on the distribution of the vector sibling species and their vectorial capacity.     

Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development

The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.     

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.