Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Invasion of mosquito salivary glands by malaria parasites: Prerequisites and defense strategies

The interplay between vector and pathogen is essential for vector-borne disease transmission. Dissecting the molecular basis of refractoriness of some vectors may pave the way to novel disease control mechanisms. A pathogen often needs to overcome several physical barriers, such as the peritrophic matrix, midgut epithelium and salivary glands. Additionally, the arthropod vector elicites immune responses that can severely limit transmission success. One important step in the transmission of most vector-borne diseases is the entry of the disease agent into the salivary glands of its arthropod vector. The salivary glands of blood-feeding arthropods produce a complex mixture of molecules that facilitate blood feeding by inhibition of the host haemostasis, inflammation and immune reactions. Pathogen entry into salivary glands is a receptor-mediated process, which requires molecules on the surface of the pathogen and salivary gland. In most cases, the nature of these molecules remains unknown. Recent advances in our understanding of malaria parasite entry into mosquito salivary glands strongly suggests that specific carbohydrate molecules on the salivary gland surface function as docking receptors for malaria parasites.     

Salivary gland proteins of the mosquito Culex quinquefasciatus

Several properties of the salivary glands of Culex quinquefasciatus mosquitoes were analysed. The amount of protein in female salivary glands increased from 0.26 microg on day one after emergence to about 1.4 microg on day seven. The major polypeptides found in the female salivary glands had molecular weights of 35.7, 28.3, and 20.5 kDa. Antibodies produced by mice immunized by bites of Culex quinquefasciatus female mosquitoes reacted with the 35.7 and 28.3 kDa polypeptides, showing that these molecules were secreted by mosquitoes during blood feeding. The salivary glands of C. Quinquefasciatus females displayed the same morphological and biochemical organization as that of Aedes aegypti mosquitoes, accumulating apyrase in the distal portions and alpha-glucosidase in the proximal portions of the gland. Arch.     

The effect of Plasmodium yoelii nigeriensis infection on the feeding persistence of Anopheles stephensi Liston throughout the sporogonic cycle.

Vector-borne parasites such as malaria have been shown to modify the feeding behaviour of their invertebrate hosts so as to increase the probability of transmission. However, evolutionary consideration of developmental changes in malaria within Anopheles mosquitoes suggests that the nature of altered feeding by mosquitoes should differ depending on the developmental stage of the parasite. We present laboratory evidence that the feeding persistence of female Anopheles stephensi towards a human host is decreased in the presence of Plasmodium yoelii nigeriensis oocysts (which cannot be transmitted), but increased when the malaria has developed into transmissible sporozoites in the salivary glands. In ten-minute trials, 33% of uninfected mosquitoes gave up their feeding attempt before the test period had ended, 53% of those harbouring oocysts had given up, but only 20% of those infected with sporozoites gave up by this time. We conclude that changes in feeding behaviour of mosquitoes mediated by parasite infection are sensitive to the developmental stage of the parasite and that these changes have important implications for malaria epidemiology.     

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.     

斯氏按蚊生长发育中唾液腺配子体激活因子的变化

为探讨斯氏按蚊生长发育过程中唾液腺抽提物配子体激活因子的消长,应用体外雄配子体出丝观察方法比较羽化后未吸血及吸血组雌性斯氏按蚊唾液腺抽提物中配子体激活因子对柏氏疟原虫雄配子体出丝诱导活性的动态变化。羽化后未吸血组按蚊唾液腺配子体激活因子活性与按蚊生长、发育呈同步变化。羽化后当日至羽化后第6 d,吸血组按蚊唾液腺抽提物的GAF活性变化与未吸血组相似,吸血后该组GAF活性下降,羽化后14 d恢复到吸血前水平。吸血后斯氏按蚊唾液腺配子体激活因子活性的降低与蚊卵发育可能相关。     

Salivary amylase activity of the phlebotomine sand fly, Lutzomyia longipalpis

Both male and female adult stages of the sand fly Lutzomyia longipalpis have detectable amylase activity in their salivary glands, as indicated by formation of p-nitrophenyl-alpha-D-maltoside from p-nitrophenyl-alpha-D-octoside and by hydrolysis of 4-nitrophenyl-alpha-D-maltoheptaoside-4,6,-O-ethylidene. No salivary alpha-glucosidase was detected. Amylase activity was also found in the crop and midgut of female flies, although in a smaller amount. Salivary amylase is significantly reduced from the salivary glands immediately after a blood meal, as is the case with salivary alpha-glucosidases in mosquitoes. Presence of salivary gland amylase in these sand flies, and absence of salivary alpha-glucosidase, indicates that in nature these insects may have a significant intake of carbohydrates in the form of starch, as suggested by their plant-feeding behavior, previously demonstrated by Schlein and Warburg (Schlein, Y., Warburg, A., 1986. Phytophagy and the feeding cycle of Phlebotomus papatasi (Diptera: Psychodidae) under experimental conditions. Journal of Medical Entomology 23, 11-15), and Alexander and Usma (Alexander, B., Usma, M.C., 1994. Potential sources of sugar for the phlebotomine sandfly Lutzomyia youngi (Diptera: Psychodidae) in a Columbia coffee plantation. Ann. Trop. Med. Parasitol. 88, 543-549).     

Aedes aegypti: model for blood finding strategy and prediction of parasite manipulation

Aedes aegypti mosquitoes salivate during intradermal probing of vertebrate prey before ingesting blood (Griffiths and Gordon 1952). Nonsalivating mosquitoes locate blood more slowly; this difference was ascribed to an anti-platelet activity found in the mosquito's saliva (Ribeiro et al. 1984). Mosquitoes infected with Plasmodium gallinaceum suffer pathology that specifically impairs saliva anti-hemostatic activity but without reducing volume of output (Rossignol et al. 1984). The complexity of the feeding apparatus of mosquitoes provides opportunity for a variety of strategies in which pathogens may produce specific lesions that enhance their transmission, but the variables that affect the duration of probing by mosquitoes have not been defined. We sought to resolve this complexity by identifying and quantifying relevant parameters of probing behavior. Mosquitoes thrust their mouthparts repeatedly through their host's skin while searching for blood. Female A. aegypti thrust at 7-sec intervals. If this search results in success, feeding ensues. Alternatively, the mosquito "desists," the mouthparts stylets are withdrawn, and the mosquito attempts to feed at another site. Even after previous desistance, the probability of finding blood remains undiminished. Functions for the probability of feeding success and desistance over time were derived using data from observations on 300 mosquitoes. The probability of feeding success was interpreted as being a function of the density of vessels in the skin, their geometric distribution, and the conditions locally affecting hemostasis. During each probe, the probability of desisting increased linearly with time, and after desisting once, mosquitoes tended to desist more rapidly. A model was developed incorporating Monte Carlo simulation which closely fit observed data. By changing values for the several parameters of the probability functions, we predicted modes in which parasites may manipulate their hosts to enhance transmission, both to and from the vector. In particular, parasite strategies in the vector would include induced salivary pathology; increased duration of probing thrusts; decreased desistance time; and inhibited phagoreception. Predicted parasite strategies in the reservoir host would include increased skin vascular volume and impaired host hemostasis. Our model supports the hypothesis of a mutualistic interaction of malaria and mosquitoes.     

Characterization of the salivary apyrase activity of three rodent flea species

1. Salivary gland lysates of the adult female fleas Oropsylla bacchi, Orchopea howardi and Xenopsylla cheopis hydrolyse ATP and ADP, but not AMP, thus characterizing the existence of a salivary apyrase activity. 2. In all species Mg++ or Ca++ function as activators, and a pH optimum between 7 and 8 is observed. 3. Salivary gland lysates of male fleas contain significantly smaller amounts of the enzyme activity than do those of female fleas. 4. Immediately following a blood meal, apyrase activity and protein content of female X. cheopis salivary glands are 2-3-fold less than that of unfed fleas, indicating that salivary apyrase activity is secreted during feeding. 5. It is suggested that, as in other arthropods, salivary apyrase may facilitate blood location and blood feeding by preventing ADP-induced platelet aggregation at the site of the bite.     

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.     

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.     

Salivary gland apyrase determines probing time in anopheline mosquitoes

Salivary gland homogenates of adult female anopheline mosquitoes, of three different species, hydrolysed ATP and ADP, thereby demonstrating an apyrase activity. Total enzyme activity was greatest in the vector species A. freeborni (20.7 ± 2.4 mU/pair of glands) and least in the autogenous mosquito A. sp. nr. salbaii (3.0 ± 0.4 mU/pair of glands); another vector species, A. stephensi, produced intermediate levels of the enzyme (7.8 ± 0.7 mU/pair of glands). In all cases, the reaction was activated by divalent cations and maximal at pH 9.0 and in the presence of 2-mercaptoethanol. Apyrase activity in each salivary gland correlated with the degree of inhibition of ADP-induced platelet aggregation in vitro. Duration of probing correlated inversely with salivary apyrase content. We conclude that salivary apyrase largely determines a mosquito's ability to locate blood. Differential selective pressures for facility of blood location would have influenced the level of salivary apyrase in these mosquitoes.