Malaria: influence of Anopheles mosquito saliva on Plasmodium infection

Malaria is caused by Plasmodium protozoa that are transmitted by anopheline mosquitoes. Plasmodium sporozoites are released with saliva when an infected female mosquito takes a blood meal on a vertebrate host. Sporozoites deposited into the skin must enter a blood vessel to start their journey towards the liver. After migration out of the mosquito, sporozoites are associated with, or in proximity to, many components of vector saliva in the skin. Recent work has elucidated how Anopheles saliva, and components of saliva, can influence host–pathogen interactions during the early stage of Plasmodium infection in the skin. Here, we discuss how components of Anopheles saliva can modulate local host responses and affect Plasmodium infectivity. We hypothesize that therapeutic strategies targeting mosquito salivary proteins can play a role in controlling malaria and other vector-borne diseases.     

Inhibition of malaria parasite development in mosquitoes by anti-mosquito-midgut antibodies.

The mosquito midgut plays a central role in the development and subsequent transmission of malaria parasites. Using a rodent malaria parasite, Plasmodium berghei, and the mosquito vector Anopheles stephensi, we investigated the effect of anti-mosquito-midgut antibodies on the development of malaria parasites in the mosquito. In agreement with previous studies, we found that mosquitoes that ingested antimidgut antibodies along with infectious parasites had significantly fewer oocysts than mosquitoes in the control group. We also found that the antimidgut antibodies inhibit the development and/or translocation of the sporozoites. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.     

Antibody-mediated inhibition of Aedes aegypti midgut trypsins blocks sporogonic development of Plasmodium gallinaceum.

The peritrophic matrix (PM) that forms around a blood meal is a potential barrier of Plasmodium development in mosquitoes. Previously, we have shown that to traverse the PM, Plasmodium ookinetes secrete a prochitinase and that an inhibitor of chitinase blocks further parasite development. Here we report that it is the mosquito trypsin that activates the Plasmodium prochitinase. Trypsin was identified as the chitinase-activating enzyme by two criteria: (i) trypsin activity and activating activity comigrated on one-dimensional gels, and (ii) activating activity and penetration of the PM by Plasmodium parasites were both hindered by trypsin-specific inhibitors. Subsequently, we examined the effect of antitrypsin antibodies on the parasite life cycle. Antibodies prepared against a recombinant blackfly trypsin effectively and specifically inhibited mosquito trypsin activity. Moreover, when incorporated into an infective blood meal, the antitrypsin antibodies blocked infectivity of Aedes aegypti mosquitoes by Plasmodium gallinaceum. This block of infectivity could be reversed by exogenously provided chitinase, strongly suggesting that the antibodies act by inhibiting prochitinase activation and not on the parasite itself. This work led to the identification of a mosquito antigen, i.e., midgut trypsin, as a novel target for blocking malaria transmission.     

Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes

Single-chain immunotoxins are ideal tools to selectively kill infectious agents. In applying this technology to block transmission of malaria parasites in the mosquito vector, we have constructed a single-chain immunotoxin composed of a single-chain antibody fragment (scFv) directed to Pbs2l on the surface of Plasmodium berghei ookinetes linked to a lytic peptide, Shiva-1. The single-chain immunotoxin was expressed in Escherichia coli, and the protein was purified by a Ni-NTA column. The single-chain immunotoxin was initially shown to exhibit greater killing properties for P. berghei ookinetes in vitro compared with the scFv or synthetic Shiva-1 peptide alone. In an attempt to block malaria transmission by genetically engineered bacteria, recombinant E. coli harboring the single-chain immunotoxin gene were introduced into the mosquito midgut by membrane feeding. The number of infected mosquitoes and their oocyst densities were significantly reduced when the mosquitoes were subsequently allowed to feed on P. berghei-infected mice. These results indicate not only that a single-chain immunotoxin with enhanced parasiticidal activity could form a basis for the development of more effective malaria therapeutic agents, but also that introduction of genetically engineered bacteria into anopheline mosquitoes may offer a practical approach to the regulation of malaria transmission.     

Immunization of rabbits with mosquito bites: immunoblot analysis of IgG antimosquito antibodies in rabbit and man

Rabbits immunized with 50-60 Aedes communis mosquito bites developed IgG antibodies recognizing a 21.5-kD mosquito antigen detected by the immunoblot technique. This protein seems to be a major immunogen in the saliva of A. communis mosquitoes. Studies on 60 human sera revealed that IgG antibodies recognizing the 21.5-kD antigen occurred in about 50% of young children and adults but were not found in the sera of 20 unexposed infants. The present immunoblot method allows further characterization of antigens and antibodies specific to mosquito saliva and contributes to understanding their role in mosquito bite reactions.     

Family members stick together: multi-protein complexes of malaria parasites

Malaria parasites express a broad repertoire of proteins whose expression is tightly regulated depending on the life-cycle stage of the parasite and the environment of target organs in the respective host. Transmission of malaria parasites from the human to the anopheline mosquito is mediated by intraerythrocytic sexual stages, termed gametocytes, which circulate in the peripheral blood and are essential for the spread of the tropical disease. In Plasmodium falciparum, gametocytes express numerous extracellular proteins with adhesive motifs, which might mediate important interactions during transmission. Among these is a family of six secreted proteins with adhesive modules, termed PfCCp proteins, which are highly conserved throughout the apicomplexan clade. In P. falciparum, the proteins are expressed in the parasitophorous vacuole of gametocytes and are subsequently exposed on the surface of macrogametes during parasite reproduction in the mosquito midgut. One characteristic of the family is a co-dependent expression, such that loss of all six proteins occurs if expression of one member is disrupted via gene knockout. The six PfCCp proteins interact by adhesion domain-mediated binding and thus form complexes on the sexual stage surface having adhesive properties. To date, the PfCCp proteins represent the only protein family of the malaria parasite sexual stages that assembles to multimeric complexes, and only a small number of such protein complexes have so far been identified in other life-cycle stages of the parasite.     

Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes

Malaria transmission begins with the injection of Plasmodium sporozoites into the skin of a vertebrate host by infected anopheline mosquitoes. Although the size of the sporozoite inoculum likely affects the course of the disease, the number of sporozoites injected by infected mosquitoes has not been determined in vivo. Using a quantitative PCR assay, we determined the number of sporozoites injected into mice by single mosquitoes. Analysis of 59 mosquito feedings showed that a single infected mosquito injected between 0 and 1,297 sporozoites, with a mean of 123 and a median of 18. Twenty-two percent of infected mosquitoes injected no sporozoites. The number of sporozoites injected was only weakly correlated to the salivary gland load. To better understand the large variability in sporozoite injection among mosquitoes, we quantified the sporozoites injected by individual mosquitoes on three different days. Approximately 20% of moderately to heavily infected mosquitoes injected few to no sporozoites on all 3 days, suggesting that some mosquitoes are poor transmitters of sporozoites. Other mosquitoes injected high numbers of sporozoites on at least one of the days observed and minimal numbers on the other day(s), supporting the hypothesis that sporozoite injection is discontinuous, a pattern that may aid in the establishment of malaria infection.     

The role of saliva of Anopheles stephensi in inflammatory response: identification of a high molecular weight neutrophil chemotactic factor

Mosquito bites can elicit dermal hypersensitivity reactions, but little is known about the chemotactic factors for host leukocytes in mosquito saliva. In this study, we determined that saliva from a malarial vector mosquito, Anopheles stephensi, possesses intense neutrophil chemotactic activity. In contrast, the midgut extract had only marginal neutrophil chemotactic activity. Eosinophil chemotactic activity was detected in the midgut but not in the saliva. According to the results of size-exclusion HPLC on a G3000SW column and Western blot analysis, the apparent molecular weight (MW) of the main neutrophil chemotactic factor (NCF) was estimated to be 200 kDa. NCF could bind with IgG from the pooled serum of Solomon islanders, whereas not with that of healthy Japanese. NCF activity was increased upon heating to 56 degrees C for 30 min or protease digestion, whereas it was affected by periodate treatment. Protease-digested NCF and naive NCF bound to lentil lectin-Sepharose, and both were eluted with a competitive sugar, methyl-alpha-D-glucoside. These results indicate that A. stephensi saliva-derived NCF is a high MW glycoprotein, and its protein moiety is important for neutrophil chemotactic activity. This NCF is thought to contribute to the inflammatory reactions through the accumulation of neutrophils at the site of the mosquito bite.     

Carboxypeptidases B of Anopheles gambiae as targets for a Plasmodium falciparum transmission-blocking vaccine

Anopheles gambiae is the major African vector of Plasmodium falciparum, the most deadly species of human malaria parasite and the most prevalent in Africa. Several strategies are being developed to limit the global impact of malaria via reducing transmission rates, among which are transmission-blocking vaccines (TBVs), which induce in the vertebrate host the production of antibodies that inhibit parasite development in the mosquito midgut. So far, the most promising components of a TBV are parasite-derived antigens, although targeting critical mosquito components might also successfully block development of the parasite in its vector. We previously identified A. gambiae genes whose expression was modified in P. falciparum-infected mosquitoes, including one midgut carboxypeptidase gene, cpbAg1. Here we show that P. falciparum up-regulates the expression of cpbAg1 and of a second midgut carboxypeptidase gene, cpbAg2, and that this up-regulation correlates with an increased carboxypeptidase B (CPB) activity at a time when parasites establish infection in the mosquito midgut. The addition of antibodies directed against CPBAg1 to a P. falciparum-containing blood meal inhibited CPB activity and blocked parasite development in the mosquito midgut. Furthermore, the development of the rodent parasite Plasmodium berghei was significantly reduced in mosquitoes fed on infected mice that had been immunized with recombinant CPBAg1. Lastly, mosquitoes fed on anti-CPBAg1 antibodies exhibited reduced reproductive capacity, a secondary effect of a CPB-based TBV that could likely contribute to reducing Plasmodium transmission. These results indicate that A. gambiae CPBs could constitute targets for a TBV that is based upon mosquito molecules.     

Knockout of the rodent malaria parasite chitinase pbCHT1 reduces infectivity to mosquitoes

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite, Plasmodium berghei. We show that the gene, named PbCHT1, is a structural ortholog of PgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity in Anopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds-an artificial situation where midgut invasion occurs before PM formation-suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of Plasmodium-mosquito interactions.     

Neither Mosquito Saliva nor Immunity to Saliva Has a Detectable Effect on the Infectivity of Plasmodium Sporozoites Injected into Mice

Malaria infection is initiated when a female Anopheles mosquito probing for blood injects saliva, together with sporozoites, into the skin of its mammalian host. Prior studies had suggested that saliva may enhance sporozoite infectivity. Using rodent malaria models (Plasmodium berghei and P. yoelii), we were unable to show that saliva had any detectable effect on sporozoite infectivity. This is encouraging for plans to immunize humans with washed, attenuated P. falciparum sporozoites because many individuals develop cutaneous, hypersensitivity reactions to mosquito saliva after repeated exposure. If washed sporozoites have no appreciable loss of infectivity, they likely do not have decreased immunogenicity; thus, vaccinees are unlikely to develop cutaneous reactions against mosquito saliva during attempted immunization with such sporozoites. Earlier studies also suggested that repeated prior exposure to mosquito saliva reduces infectivity of sporozoites injected by mosquitoes into sensitized hosts. However, our own studies show that prior exposure of mice to saliva had no detectable effect on numbers of sporozoites delivered by infected mosquitoes, the rate of disappearance of these sporozoites from the skin or infectivity of the sporozoites. Under natural conditions, sporozoites are delivered both to individuals who may exhibit cutaneous hypersensitivity to mosquito bite and to others who may have not yet developed such reactivity. It was tempting to hypothesize that differences in responsiveness to mosquito bite by different individuals might modulate the infectivity of sporozoites delivered into a milieu of changes induced by cutaneous hypersensitivity. Our results with rodent malaria models, however, were unable to support such a hypothesis.The malaria infection is initiated when a female Anopheles mosquito probing for a blood meal injects saliva, together with sporozoites into the skin of its mammalian host (18, 39). Mosquito saliva is known to enhance the ability of the mosquito to locate a blood source and to inhibit hemostasis by any of several mechanisms. These include injection of an anticoagulant factor (34), inhibition of platelet aggregation by salivary apyrase (29) or a salivary factor that inhibits collagen-induced platelet aggregation (43), inhibition of thrombin activity (14), and vasodilation of host blood vessels (30). Arthropod saliva has been shown to enhance the infectivity of several different pathogens introduced into hosts by arthropods; these include sandfly transmission of Leishmania, tick transmission of viruses and spirochetes, and mosquito transmission of viruses (for a review, see reference 36). Enhancement of Plasmodium sporozoite infectivity by mosquito saliva has also been reported (12, 36) based on a prior study (41), but we felt that this study needed to be reassessed.In addition to these studies on the direct effect of arthropod saliva on infectivity of pathogens injected by arthropods into immunologically naive hosts, studies have also been done on the role of prior exposure of hosts to arthropod saliva in modulating pathogen transmission to immunized hosts. Ever since Trager''s classic study (37) showing that immunity to tick bite can lead to host protection against subsequent feedings by ticks, many workers have studied the role of host immunity to arthropod saliva in interfering with feeding by the arthropod and modulating transmission of pathogens to the host (for reviews, see references 6 and 36). Most of these studies have focused on delayed immune responses that in some cases may enhance and in other cases may control infections with arthropod-transmitted pathogens. This is an appropriate approach in circumstances when a host cellular response may interfere with feeding by the arthropod or may recruit host cells that modulate development of the pathogen at the bite site.Mosquitoes, however, feed relatively rapidly. Thus, only an immediate hypersensitivity response is likely to be able to modulate movement of sporozoites from avascular tissue at the bite site to blood vessels, from which the sporozoite can then reach the liver for further development. Many hosts bitten by mosquitoes over a period of time develop an immediate, cutaneous, hypersensitivity response; it is relevant that this develops at the same site and within the same timeframe during which sporozoites are moving into local blood vessels. We have previously studied the kinetics of P. berghei sporozoite movement out of the skin after deposition by mosquitoes into immunologically naive mice (19). We thus set out to compare this to the kinetics of sporozoites introduced by mosquitoes into mice that we attempted to hyperimmunize against mosquito saliva by repeated mosquito bites. Our results have shown that neither the presence of mosquito saliva nor immediate hypersensitivity to saliva had any detectable effects on deposition of sporozoites by mosquitoes or the movement of these sporozoites from the bite site into the blood to induce infection.     

The effect of Plasmodium yoelii nigeriensis infection on ovarian protein accumulation by Anopheles stephensi

Both anopheline and culicine mosquitoes have been shown to incur a reduction in reproductive fitness when infected with malaria parasites. The agent of rodent malaria, Plasmodium yoelii nigeriensis, was used as a laboratory model to investigate changes in the accumulation of protein in the ovaries of Anopheles stephensi when infected with oocysts or when feeding on mice with heavy asexual parasitaemia but no mature gametocytes. Herein we report that during the early phases of the gonotrophic cycle the ovarian protein content increased normally; however, after 16 h post-blood-feeding there was a significant reduction in the total protein content in ovaries from infected mosquitoes. The development of ovaries from mosquitoes undergoing a second gonotrophic cycle and containing maturing oocysts was similarly affected. Ovarian protein profiles produced by sodi- um dodecyl sulfate-polyacrylamide gel electrophoresis showed a depletion of the yolk protein vitellin. Ovaries from mosquitoes feeding on a mouse with 31 % parasitaemia, no detectable gametocytes and a low haematocrit (29 % packed cell volume) also exhibited a reduction in protein content, although this did not occur until much later in the gonotrophic cycle. The role of blood-meal quality and malaria infection in the reduction in egg production is discussed. Received: 18 October 1996 / Accepted: 15 November 1996     

Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice

The number of malaria sporozoites delivered to a host by mosquitoes is thought to have a significant influence on the subsequent course of the infection in the mammalian host. We did studies with Anopheles stephensi mosquitoes with salivary gland infections of Plasmodium berghei sporozoites expressing a red fluorescent protein. After individual mosquitoes fed on an ear pinna or the ventral abdomen of a mouse, fluorescence microscopy was used to count numbers of sporozoites. Mosquitoes allowed to feed on the ear for periods of 3 versus 15 min deposited means of 281 versus 452 sporozoites, respectively, into the skin; this may have epidemiological implications because mosquitoes can feed for longer periods of time on sleeping hosts. Mosquitoes feeding on the ventral abdomen injected sporozoites not only into the skin but also into the underlying peritoneal musculature. Although mosquitoes injected fewer sporozoites into the abdominal tissues, more of these were reingested into the mosquito midgut, probably a consequence of easier access to blood intake from the abdominal area. The most consistent parameter of sporozoite transmission dynamics under all conditions of mosquito probing and feeding was the relatively slow release rate of sporozoites (approximately 1 to 2.5 per second) from the mosquito proboscis. The numbers of sporozoites introduced into the host by mosquitoes and the transmission efficiencies of sporozoite delivery are multifactorial phenomena that vary with length of probing time, skin site being fed upon, and numbers of sporozoites within the salivary glands.     

Evaluation of candidate traps as tools for conducting surveillance for Anopheles mosquitoes in a malaria-endemic area in western Thailand

The effectiveness of five mosquito traps at sampling anopheline mosquitoes was compared with landing/biting (L/B) collections in western Thailand. Traps evaluated included a CDC style light trap (CDC LT) with dry ice, the American Biophysics Corporation (ABC) standard light trap (ABC LT) with dry ice and octenol, the ABC counterflow geometry (CFG) trap with dry ice and octenol, the ABC mosquito magnet (MM) trap with octenol, and the Nicosia and Reinhardt Company Mosquito Attractor Device (N&R trap). Mosquito numbers captured in landing-biting collections were 5.2, 7.0, 7.3, 31.1, and 168.8 times greater than those collected in the ABC LT, MM, CDC LT, CFG, and N&R traps, respectively, for Anopheles minimus Theobald, the predominant malaria vector in the region. Similar results were obtained for the secondary malaria vectors Anopheles maculatus Theobald and Anopheles sawadwongporni Rattanarithikul & Green. Only Anopheles kochi Doenitz was collected in significantly greater numbers in the CDC LT, ABC LT, and MM traps compared with L/B collections. Although none of the traps were as effective as L/B collections, the ABC LT, MM, and CDC LT were the best alternatives to human bait for the collection of anopheline malaria vectors in Thailand.     

Current status of malaria and potential for control

Malaria remains one of the world's worst health problems with 1.5 to 2.7 million deaths annually; these deaths are primarily among children under 5 years of age and pregnant women in sub-Saharan Africa. Of significance, more people are dying from malaria today than 30 years ago. This review considers the factors which have contributed to this gloomy picture, including those which relate to the vector, the female anopheline mosquito; to human activity such as creating new mosquito breeding sites, the impact of increased numbers of people, and how their migratory behavior can increase the incidence and spread of malaria; and the problems of drug resistance by the parasites to almost all currently available antimalarial drugs. In a selective manner, this review describes what is being done to ameliorate this situation both in terms of applying existing methods in a useful or even crucial role in control and prevention and in terms of new additions to the antimalarial armory that are being developed. Topics covered include biological control of mosquitoes, the use of insecticide-impregnated bed nets, transgenic mosquitoes manipulated for resistance to malaria parasites, old and new antimalarial drugs, drug resistance and how best to maintain the useful life of antimalarials, immunity to malaria and the search for antimalarial vaccines, and the malaria genome project and the potential benefits to accrue from it.     

Relationships between occurrence of Anopheles gambiae s.l. (Diptera: Culicidae) and size and stability of larval habitats

One potentially important target of malaria vector control is the immature stages of anopheline mosquitoes. To design efficient larval control methods, mechanisms regulating mosquito productivity in natural habitats must be understood. We examined the relationships between pupal occurrence of Anopheles gambiae s.l. and size and stability of larval habitats for a period of 1 yr in western Kenya. We also examined relationships between abundance of indoor resting anophelines and habitat availability. Habitat size was measured by the total water volume (cubic meters). Habitat stability was defined as the number of occurrences when water was continuously present in a habitat for 6 d. Pupal occurrence was indicated by the number of days that pupae were observed in a habitat during the study period. We found that habitat stability and pupal occurrence were positively correlated with habitat size. When habitat size fell below approximately 1 m3, habitat stability and pupal occurrence decreased rapidly. Habitat availability was significantly correlated with the density of indoor resting mosquitoes in houses near to larval sites. These results suggest that habitat size is an important determinant of habitat stability, pupal occurrence, and adult mosquito abundance.     

对约氏疟原虫不同易感性的大劣按蚊和斯氏按蚊基因组RAPD序列比较

目的分析对约氏疟原虫不易感的大劣按蚊和易感的斯氏按蚊基因组RAPD谱带并测序，探讨媒介按蚊基因型与疟原虫基因型间的相互关系。方法用已筛选的一条随机引物，随机扩增大劣按蚊和斯氏按蚊成蚊的基因组DNA，扩增产物经琼脂糖凝胶电泳后，对相同迁移率的DNA条带克隆、测序，并采用相关在线程序及软件进行序列比较分析。结果大劣按蚊和斯氏按蚊RAPD谱带具有明显的种间差异，但有4对相同迁移率的条带。序列分析显示4对DNA条带在序列长度和组成上呈现多态性，序列的GC含量和简单重复序列存在差异，序列相似性介于48％～52％之间。结论大劣按蚊和斯氏按蚊之间存在不同的遗传背景，其基因多态性可能与产生对约氏疟原虫的易感性不同有关，为进一步研究按蚊与疟原虫之间的相互作用奠定了基础。     

Monoclonal antibody MG96 completely blocks Plasmodium yoelii development in Anopheles stephensi

In spite of research efforts to develop vaccines against the causative agent of human malaria, Plasmodium falciparum, effective control remains elusive. The predominant vaccine strategy focuses on targeting parasite blood stages in the vertebrate host. An alternative approach has been the development of transmission-blocking vaccines (TBVs). TBVs target antigens on parasite sexual stages that persist within the insect vector, anopheline mosquitoes, or target mosquito midgut proteins that are presumed to mediate parasite development. By blocking parasite development within the insect vector, TBVs effectively disrupt transmission and the resultant cascade of secondary infections. Using a mosquito midgut-specific mouse monoclonal antibody (MG96), we have partially characterized membrane-bound midgut glycoproteins in Anopheles gambiae and Anopheles stephensi. These proteins are present on the microvilli of midgut epithelial cells in both blood-fed and unfed mosquitoes, suggesting that the expression of the protein is not induced as a result of blood feeding. MG96 exhibits a dose-dependent blocking effect against Plasmodium yoelii development in An. stephensi. We achieved 100% blocking of parasite development in the mosquito midgut. Preliminary deglycosylation assays indicate that the epitope recognized by MG96 is a complex oligosaccharide. Future investigation of the carbohydrate epitope as well as gene identification should provide valuable insight into the possible mechanisms of ookinete attachment and invasion of mosquito midgut epithelial cells.     

High levels of human chitotriosidase hinder the formation of peritrophic membrane in anopheline vectors

In the Anopheles midgut, Plasmodium falciparum produces a specific chitinase able to penetrate the blood meal surrounding the chitin-containing peritrophic membrane (PM). High levels of an analogous chitinase, chitotriosidase (CHIT), may be found in human blood, being the markers of macrophage activation. To verify the hypothesis that CHIT present in malaria patient blood could help parasite to overcome PM, we carried out a bioassay by feeding Anopheles stephensi females on an artificial apparatus that contained human blood from four different sources and with different chitinase concentrations: (1) healthy donors, as negative controls; (2) patients with malaria; (3) patients with Gaucher disease; and (4) whole blood enriched with commercial P. falciparum chitinase, as positive controls. After 16, 20 and 24 h of bloodfeeding, mosquitoes were dissected to extract the midgut and assess the effect of the different chitinases on membrane structure. Optical microscopy showed that formation of PM was clearly complete after 16 h in the posterior midgut from Anopheles already fed with healthy donor bloods. By contrast, PM formation was visible after 16 h in the posterior midgut of mosquitoes fed with malaria and Gaucher patient bloods but appeared clearly damaged at 20 and 24 h. At the same time, the PM formation was almost completely inhibited in the midgut of Anopheles fed with P. falciparum chitinase-enriched bloods. These alterations were clearly confirmed by transmission electronic microscopy. In the present paper, we demonstrate that human CHIT from different sources is active on anophelines’ PM.     

Prevalence of avian malaria parasite in mosquitoes collected at a zoological garden in Japan

Several species of captive birds at zoological gardens of Japan were found to be infected with avian Plasmodium. However, incriminated vector mosquito species have not been identified yet. To indicate the competent vectors of avian malaria parasite, we collected mosquitoes at a zoological garden in Japan and examined for the avian malaria parasite DNA. Totally, 1,361 mosquitoes of 11 species were collected in the zoological garden of Kanagawa, the south of Tokyo in Japan in 2005. Captured mosquitoes were pooled by each species, date collected, and location and used for DNA extraction. Eight out of 169 DNA samples were positive for the nested PCR of avian Plasmodium cyt b gene. Estimated minimum infection rates of mosquitoes were 5.9 per 1,000. The PCR positive mosquito species were Culex pipiens group and Lutzia vorax. Some DNA sequences amplified from collected mosquitoes were identical to avian Plasmodium lineages detected from captive birds in the same zoological garden studied. Our results suggest that C. pipiens group and L. vorax could be incriminated vectors of avian malaria parasite transmitting in captive birds kept in the zoological garden in Japan.