The role of the mosquito peritrophic membrane in bloodmeal digestion and infectivity of Plasmodium species.

Secretion and luminal formation of the peritrophic membrane (PM) were induced in female Anopheles stephensi and Aedes aegypti by feeding the mosquitoes on a warmed suspension of latex particles in Ringer's solution. The PM in A. stephensi was produced from apical secretion vesicles stored in the midgut epithelial cells and secreted into the lumen during feeding. In A. aegypti, the PM was formed de novo. When the latex feeding was followed 24 hr later by a meal of lyophilized pig blood, the 2 mosquito species exhibited very different modifications to their PM structure; in A. stephensi no PM was formed around the blood meal, whereas de novo synthesis of the PM in A. aegypti continued during the blood meal, with the resulting PM greatly thickened compared to the normal feeding. This artificial induction of PM formation was used as the basis to study the role of the PM in blood meal digestion and in infectivity of mosquitoes by the appropriate species of Plasmodium. The feeding of a latex suspension alone had no stimulatory effect on the 2 major midgut proteases, trypsin and aminopeptidase, in either species. After a blood meal alone, proteases rose to maximum activity at 30 hr and 24 hr after feeding in A. stephensi and A. aegypti, respectively. After double feeding, protease activities in both species were almost identical to those in blood-fed mosquitoes. Neither the absence of a PM (in A. stephensi) nor the presence of a thickened PM (in A. aegypti), therefore, has any effect on the ability of mosquitoes to digest a blood meal. Malaria infectivity, measured by oocyst counts, also was compared after normal and double feeding using infective blood meals. Infectivity of A. stephensi by Plasmodium berghei was unaffected by the presence or absence of the PM. The thickened PM produced by double feeding in A. aegypti caused a reduction of midgut infectivity by Plasmodium gallinaceum. These results suggest that the PM may act as a partial, but not an absolute, barrier to invasion of the midgut by the ookinete.     

Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells

Background

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.

Methodology/Principal Findings

Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta''), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.

Conclusions

alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.     

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (P < 0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme.     

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.     

Blood‐feeding and immunogenic Aedes aegypti saliva proteins

Mosquito‐transmitted pathogens pass through the insect's midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar‐fed and blood‐fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar‐ or blood‐based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar‐fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up‐regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2‐DE profiles of immunogenic Ae. aegypti SG BF‐related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.     

Effect of cibarial armature of mosquitoes (Diptera,Culicidae) on blood-meal haemolysis

Cibarial armatures of mosquitoes act mechanically on the ingested erythrocytes producing the haemolysis of part of the blood meal. The extent of haemolysis, in blood meals examined immediately after ingestion, is less than 5% in mosquitoes without cibarial armature or with “primitive” types of cibarial armature, while up to 50% haemolysis is observed in mosquitoes with more highly evolved types of cibarial armature. Such high levels of haemolysis are produced by the passage of the blood through the mosquito head but not by the incubation of the blood with salivary gland and midgut homogenates from the same mosquito or by injection of the blood into the intestine via the anus.     

Influence of the endocrine system on tryptic activity in female Aedes aegypti

In MNC-ablated or ovariectomized mosquitoes, blood-fed by enema, the tryptic activity of midgut homogenates was reduced by half. By sealing the anus after the blood meal, we showed that the reduction in enzyme activity was not due to premature or increased excretion of active enzyme in operated females. The enzyme activity was restored to the level of unoperated controls by either reimplantation of an ovary or injection of 20-hydroxy-ecdysone in a large, single dose shortly before the blood meal.The reduction in tryptic activity in MNC-ablated or ovariectomized females did not alter the rate of protein digestion as measured by the decrease of protein in midgut homogenates. We conclude that the neurosecretory system and the ovaries are required for maximal tryptic activity in normal females, and that the enzyme apparently is secreted in excess of that required for complete digestion of the blood meal.     

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.     

Cell death and regeneration in the midgut of the mosquito, Culex quinquefasciatus

Haematophagy, the utilization of blood as food, has evolved independently among insects such as mosquitoes, bedbugs, fleas, and others. Accordingly, several distinct biological adaptations have occurred in order to facilitate the finding, ingestion and digestion of blood from vertebrate sources. Although blood meals are essential for survival and reproduction of these insects, mechanical and chemical stresses are caused by the ingestion of a sizable meal (frequently twice or more times the weight of the insect) containing large amounts of cytotoxic molecules such as haem. Here we present data showing that the stresses caused by a blood meal induce cell death in the midgut epithelium of Culex quinquefasciatus mosquitoes. The process involves apoptosis, ejection of dead cells to the midgut lumen and differentiation of basal regenerative cells to replace the lost digestive cells. The basal cell differentiation in blood-fed mosquito midguts represents an additional mechanism by which insects cope with the stresses caused by blood meals. C. quinquefasciatus adult females are unable to replace lost cells following a third or fourth blood meal, which may have a significant impact on mosquito longevity, reproduction and vectorial capacity.     

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.     

Neuropeptide F and its expression in the yellow fever mosquito,Aedes aegypti

A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal.     

Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement

Gametocytes are the intraerythrocytic stages of malaria parasites that infect mosquitoes. When gametocytes of the chicken malaria parasite Plasmodium gallinaceum are ingested by a mosquito they become extracellular in the mosquito midgut, form gametes, and fertilize within 10 to 15 min after the insect has taken a blood meal. Gametocytes of P. gallinaceum were infectious when fed to Aedes aegypti mosquitoes in blood meals containing native serum from chickens or from the non-host species, man or sheep. Gametocytes stimulated to undergo gametogenesis and to fertilize in vitro were also infectious when fed to mosquitoes in native chicken serum. However, native serum from most non-host species, including sheep and man, suppressed the infectivity of newly fertilized zygotes to mosquitoes and lysed the zygotes in vitro. These effects were shown to be due to the activity of the alternative pathway of complement (APC) in the serum of the non-host species. After mild trypsin treatment, the zygotes of P. gallinaceum no longer infected mosquitoes in the presence of native chicken serum, although in heat-inactivated chicken serum their infectivity was normal. We conclude that trypsin-sensitive components on the zygotes surface protect them from destruction by the APC of their native host. The ability of gametocytes of P. gallinaceum to infect mosquitoes in the presence of native human serum is probably due to proteases that inactivate the APC of human serum before the gametes and zygotes emerge as extracellular parasites in the blood meal.     

Characterization and identification of exflagellation-inducing factor in the salivary gland of Anopheles stephensi (Diptera: Culicidae)

Gamete activation factor (GAF) induces exflagellation of Plasmodium microgametes. We found GAF in the salivary glands of female mosquitoes, Anopheles stephensi. The exflagellation was induced in a concentration-dependent manner in the supernatant of salivary gland's crude homogenate. The exflagellation-inducing activity in the salivary gland was higher than that in the midgut and the head. GAF in the salivary glands was found to be heat stable and low molecular weight (<3000 molecular weight). Analysis of the supernatant by capillary electrophoresis and UV absorbance profile showed that the salivary glands contained xanthurenic acid, which was previously identified as GAF in the head of A. stephensi. The exflagellation-inducing activity in the salivary gland declined immediately after a blood meal, implying that GAF was in the saliva, and was delivered into the midgut together with the blood and induced exflagellation in the midgut.     

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.     

Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium

The involvement of reactive oxygen species (ROS) in mosquito immunity against bacteria and Plasmodium was investigated in the malaria vector Anopheles gambiae. Strains of An. gambiae with higher systemic levels of ROS survive a bacterial challenge better, whereas reduction of ROS by dietary administration of antioxidants significantly decreases survival, indicating that ROS are required to mount effective antibacterial responses. Expression of several ROS detoxification enzymes increases in the midgut and fat body after a blood meal. Furthermore, expression of several of these enzymes increases to even higher levels when mosquitoes are fed a Plasmodium berghei-infected meal, indicating that the oxidative stress after a blood meal is exacerbated by Plasmodium infection. Paradoxically, a complete lack of induction of catalase mRNA and lower catalase activity were observed in P. berghei-infected midguts. This suppression of midgut catalase expression is a specific response to ookinete midgut invasion and is expected to lead to higher local levels of hydrogen peroxide. Further reduction of catalase expression by double-stranded RNA-mediated gene silencing promoted parasite clearance by a lytic mechanism and reduced infection significantly. High mosquito mortality is often observed after P. berghei infection. Death appears to result in part from excess production of ROS, as mortality can be decreased by oral administration of uric acid, a strong antioxidant. We conclude that ROS modulate An. gambiae immunity and that the mosquito response to P. berghei involves a local reduction of detoxification of hydrogen peroxide in the midgut that contributes to limit Plasmodium infection through a lytic mechanism.     

Complement effects of the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. II. Changes in sensitivity to complement-like factors during zygote development

During transformation into ookinetes, the zygotes of Plasmodium gallinaceum are initially resistant to lysis by heat-labile and EDTA-sensitive factors in the serum of their natural host, the chicken. Between 6 and 8 hr postgametogenesis, zygotes cultured in vitro lose their resistance to these factors. Loss of resistance to these factors in vitro is reflected by loss of infectivity of the zygotes to Aedes aegypti mosquitoes in the presence of native chicken serum. These factors are probably components of the alternative pathway of complement (APC) of chicken serum. Gametocytes of P. gallinaceum in chicken blood are able to infect A. aegypti mosquitoes apparently due to inactivation of the APC in a blood meal within 3-4 hr after ingestion, i.e., several hours before the zygotes lose their resistance to chicken APC. In addition to the heat-labile factors (APC) in chicken serum, the zygotes are transiently sensitive to other factor(s) in the mosquito blood meal. These factor(s) are not destroyed by prior heating of the chicken serum given in a blood meal and therefore cannot be complement components. The antiparasitic effects of the factors are neutralized by addition of EDTA to the blood meal and could be due to an EDTA-sensitive metalloprotease present in the mosquito midgut.     

The transmission-blocking effects of antimalarial drugs revisited: fitness costs and sporontocidal effects of artesunate and sulfadoxine-pyrimethamine

Assays used to evaluate the transmission-blocking activity of antimalarial drugs are largely focused on their potential to inhibit or reduce the infectivity of gametocytes, the blood stages of the parasite that are responsible for the onward transmission to the mosquito vector. For this purpose, the drug is administered concomitantly with gametocyte-infected blood, and the results are evaluated as the percentage of reduction in the number of oocysts in the mosquito midgut. We report the results of a series of experiments that explore the transmission-blocking potential of two key antimalarial drugs, artesunate and sulfadoxine-pyrimethamine, when administered to mosquitoes already infected from a previous blood meal. For this purpose, uninfected mosquitoes and mosquitoes carrying a 6 day old Plasmodium relictum infection (early oocyst stages) were allowed to feed either on a drug-treated or an untreated host in a fully factorial experiment. This protocol allowed us to bypass the gametocyte stages and establish whether the drugs have a sporontocidal effect, i.e. whether they are able to arrest the ongoing development of oocysts and sporozoites, as would be the case when a mosquito takes a post-infection treated blood meal. In a separate experiment, we also explored whether a drug-treated blood meal impacted key life history traits of the mosquito relevant for transmission, and if this depended on their infection status. Our results showed that feeding on an artesunate- or sulfadoxine-pyrimethamine-treated hosts has no epidemiologically relevant effects on the fitness of infected or uninfected mosquitoes. In contrast, when infected mosquitoes fed on an sulfadoxine-pyrimethamine-treated host, we observed both a significant increase in the number of oocysts in the midgut, and a drastic decrease in both sporozoite prevalence (?30%) and burden (?80%) compared with the untreated controls. We discuss the potential mechanisms underlying these seemingly contradictory results and contend that, provided the results are translatable to human malaria, the potential epidemiological and evolutionary consequences of the current preventive use of sulfadoxine-pyrimethamine in malaria-endemic countries could be substantial.