斑须按蚊唾液腺中疟原虫配子体激活因子的活性动态

目的　探讨斑须按蚊生长、发育过程唾液腺中疟原虫配子体激活因子的活性动态。方法　应用体外雄配子体出丝分析方法 ,检测雌性斑须按蚊羽化后、吸血及产卵前后唾液腺中疟原虫配子体激活因子对柏氏疟原虫雄配子体出丝诱导活性的变化。结果　羽化后未吸血组斑须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 ;吸血后未产卵组按蚊唾液腺抽提物的配子体激活因子活性在吸血后显著下降 ,吸血后第 8天恢复到吸血前水平 ;而吸血后产卵组按蚊唾液腺抽提物的配子体激活因子活性在吸血后第 4天恢复到吸血前水平。结论　吸血后斑须按蚊唾液腺配子体激活因子的消长与按蚊产卵相关。     

吸血前后斯氏按蚊不同器官配子体激活因子的变化

目的探讨斯氏按蚊头、唾液腺和卵巢中配子体激活因子的活性动态与按蚊吸血的相互关系。方法应用体外雄配子体出丝观察方法检测雌性斯氏按蚊吸血前后唾液腺抽提物、头及卵巢匀浆上清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。结果吸血前后蚊头部匀浆上清的GAF活性为104．6％～122．1％，差异无显著性（P〉0．05）；吸血后1h内唾液腺抽提物和卵巢匀浆上清OAF活性分别降至41．4％和20．4％，吸血后第8d唾液腺GAF活性恢复到吸血前水平；吸血后第2d，卵巢的OAF活性急剧上升至96．0％，并持续到吸血后第8d。结论斯氏按蚊吸血前后唾液腺抽提物和卵巢匀浆上清中的配子体激活因子活性显著不同，其变化与蚊卵发育相关。     

双氢青蒿素阻断食蟹猴疟原虫孢子增殖期发育研究

用双氢青蒿素在食蟹猴疟原虫－大劣按蚊猴疟模型中的试验结果显示：给感染猴一次性口服４０ｍｇ／ｋｇ双氢青蒿素之后，５ｈ内疟原虫总数变化不明显，配子体数量仍继续增加，各期原虫形态观察无改变。但用药后１．５ｈ吸血感染蚊胃内平均卵囊感染数开始明显下降，５ｈ后蚊胃未发现卵囊。结果提示：双氢青蒿素不仅具有速效杀灭疟原虫无性体、复燃率低、剂量少的特点，而且用药后能快速降低食蟹猴疟原虫传疟按蚊蚊胃卵囊感染数，用药５ｈ后可阻断吸血蚊疟原虫孢子增殖期的发育。     

青蒿琥酯对食蟹猴疟原虫孢子增殖期发育影响的研究

血传感染食蟹猴疟原虫的恒河猴,在用青蒿琥酯治疗前和治疗后4h与10h分别以大劣按蚊叮咬吸血.结果治疗前1h按蚊吸血后第13d唾腺子孢子阳性率为2.56％,治疗后4h与10h按蚊吸血后第6～10d蚊胃壁有卵囊形成,但吸血后第11～13d唾腺解剖均未查见子孢子。治疗后受感染猴血内疟原虫无性体比治疗前迅速减少,治疗后10h,未查见裂殖体。结果提示,青蒿琥酯不仅对食蟹猴疟原虫无性体具有明显杀灭作用.而且对配子体蚊体内孢子增殖也具有抑制作用。     

双氢青蒿对约氏疟原虫在蚊体内发育的影响

目的：观察双氢青蒿素（dihydroqinghaosu，DQHS）对约氏疟原虫在斯氏按蚊体内发育的影响。方法：受染疟原虫小鼠一次性经口灌喂不同浓度DQHS药液后，供蚊虫吸血，应用光镜和电镜观察对照组和用药组的疟原虫在蚊体内的发育情况。结果：DQHS对疟原虫配子体有一定的抑制作用，其作用强度与配子体成熟程度和用药剂量不同有关。未成熟配子体对药物较敏感；随着药物剂量的增加，卵囊和子孢子的阳性率和密度随之逐渐下降；但180mg或240mg／kg用药组对子孢子密度的影响的差别无显著性意义。电镜观察60mg／kg作用16h后，用药组的蚊胃上卵囊（12d－13d解剖蚊），出现膜受损，甚至胞质空泡化。120mg／kg药量对蚊体内3日龄卵囊作用16h，卵囊仍继续发育，比较对照组和用药组的卵囊和子孢子密度, 两组间差别无显著意义(P > 0. 05) ; 两组卵囊超微结构形态亦无明显差异。结论: DQHS 影响约氏疟原虫配子体感染性而减少蚊媒传播, 但对蚊体内子孢子增殖期不起直接的抑制作用。     

斯氏按蚊和白纹伊蚊中肠蛋白及蛋白酶分析

取出吸血前后不同时间的雌蚊中肠,分别测定其蛋白含量、组分和蛋白酶活性。所有雌蚊吸血后中肠蛋白含量随时间增加,感染疟原虫的雌蚊中肠蛋白含量均高于同期正常雌蚊。中肠蛋白 SDS—PAGE 表明:斯氏按蚊羽化时和吸血后 d7正常蚊及感染蚊均为15条蛋白区带,吸血后 d10正常蚊为16条区带,感染蚊为14条区带。白纹伊蚊吸血后 d6和 d9正常蚊和感染蚊均为19条蛋白区带。感染雌蚊中肠蛋白酶活性均高于同期正常雌蚊。     

大劣按蚊丝氨酸蛋白酶与疟原虫感染相关性研究

目的探讨大劣按蚊血淋巴中丝氨酸蛋白酶与约氏疟原虫感染的相关性。方法首先利用二维电泳分离感染约氏疟原虫的和吸食正常血的大劣按蚊血淋巴蛋白;然后进行Western blot,用丝氨酸蛋白酶抗体进行识别,并比较感染组与正常对照组间的区别;选择感染后不同时相点对大劣按蚊蚊胃和唾液腺进腺镜检,以观察蚊体内疟原虫情况。结果Western blot显示,正常对照组无阳性蛋白点,感染组有2个阳性蛋白点,分子质量单位分别为44ku和27ku,等电点分别为8.0和5.0;镜检显示,感染7d时可见卵囊颗粒样病变,11d时卵囊发育不同步且黑化明显,之后检出的卵囊数逐渐减少。蚊唾液腺内未见子孢子。结论大劣按蚊血淋巴中的丝氨酸蛋白酶与约氏疟原虫感染相关,可能参与蚊抗疟原虫感染的黑化包被反应。     

双氢青蒿素阻断食蟹猴疟原虫孢子增殖期发育研究

用双氢青蒿素在食猴疟原虫－大劣按蚊猴疟模型中的试验结果提示，给感染猴一次性口服４０ｍｇ／ｋｇ双氢青蒿素之后，５ｈ内疟原虫总数变化不明显，配子体数量仍继续增加，各期原虫形态观察无改变，但用药后１．５ｈ吸血感染蚊胃内平均卵囊感染数开始明显下降，５ｈ蚊胃未发现卵囊，结果提示，双氢青蒿素不仅具有速效杀灭疟原无性体，复燃率低，剂量少的特点，而且用药后能快速降低食蟹猴疟原虫传疟按蚊蚊胃卵囊感染数，用药５ｈ后可     

培养的恶性疟原虫配子体在人血清和按蚊胃中的出丝作用

成熟的雄配子体在按蚊胃中出丝即雄配子形成的过程,除受气体张力、pH及温度等关键因素的影响外,在蚊体内可能还受一些附加因素的作用。例如,当恶性疟原虫配子体培养物中加入不同组无疟史志愿者混合血清时,每个视野中可见到至少为1个多至20个的出丝现象。此外,还发现在6种按蚊体内的动合子感染率和密度均存在差异,可能与这些蚊胃的“出丝因子”不同有关。 每5个视野(400×)有8个或更多出丝体的成熟恶性疟原虫(NF54株)配子体培养物被用来测定人血清和蚊胃匀浆物对出丝的影响。血清来自30名从未接触过疟疾的美国健康成人,30名来自疟疾低度传播区的儿童     

斯氏按蚊血淋巴游离氨基酸和蛋白的研究

用氨基酸自动分析仪测定斯氏按蚊血淋巴游离氨基酸的变化,用紫外吸收差法测定其蛋白浓度。感染约氏疟原虫蚊与正常蚊相比,吸血后第4d,6种氨基酸含量降低,5种氨基酸含量增加;吸血后第7d,4种氨基酸含量降低,7种氨基酸含量增加;吸血后第11d,9种氨基酸含量降低,4种氨基酸含量增加。感染蚊血淋巴蛋白浓度均高于同期正常蚊。     

Interrupting malaria transmission by genetic manipulation of anopheline mosquitoes

Malaria ranks among the deadliest infectious diseases that kills more than one million persons every year. The mosquito is an obligatory vector for malaria transmission. In the mosquito, Plasmodium undergoes a complex series of developmental events that includes transformation into several distinct morphological forms and the crossing of two different epithelia--midgut and salivary gland. Circumstantial evidence suggests that crossing of the epithelia requires specific interactions between Plasmodium and epithelial surface molecules. By use of a phage display library we have identified a small peptide-SM1--that binds to the surfaces of the mosquito midgut and salivary glands. Transgenic Anopheles stephensi mosquitoes expressing a SM1 tetramer from a blood-inducible and gut-specific promoter are substantially impaired in their ability to sustain parasite development and transmission. A second effector gene, phospholipase A2, also impairs parasite transmission in transgenic mosquitoes. These findings have important implications for the development of new strategies for malaria control.     

A putative receptor for dengue virus in mosquito tissues: localization of a 45-kDa glycoprotein

Dengue virus (DENV) infects target cells by attaching to various cell receptors, many of which are still unknown. In C6/36 cells (Aedes albopictus cell line), DENV-4 bound to two glycoproteins of 40 and 45 kDa, located on the cell surface. Preincubation of cells with polyclonal antibody against the 45-kDa protein specifically blocked DENV-4 infection of C6/36 cells. The antibody and purified DENV-4 detected the 45-kDa molecule in total extracts from eggs, larvae, and pupae as well as from the midgut, ovary, and salivary glands from adult-stage Aedes aegypti mosquitoes, whereas in malphigian tubules it was absent. This suggests that the distribution of the 45-kDa protein correlates with tissue tropism of DENV infection in mosquitoes. The 45-kDa molecule was not detected in Anopheles albimanus mosquito. The relevance of our findings is discussed from the pathogenetic and vector competence viewpoints.     

斯氏按蚊血细胞对约氏疟原虫卵囊黑化的影响

目的　探讨斯氏按蚊血细胞及血细胞PPO对疟原虫卵囊黑化的影响。方法　利用电镜观察蚊血细胞在卵囊黑化中的作用、RT -PCR检测蚊血细胞、蚊胃中PPOmRNA的表达、原位杂交检测血细胞中PPOmRNA的表达。结果　通过RT -PCR和原位杂交 ,发现斯氏按蚊PPOmRNA在血细胞中有表达 ,颗粒细胞可能是其主要的表达细胞 ;硝喹可使约氏疟原虫卵囊发生退行性变 ,卵囊黑化比率明显增加。结论　说明血细胞能够合成PPO ,进一步证实了蚊血细胞 ,特别是硝喹对卵囊的发育有抑制和阻断作用时 ,颗粒细胞在疟原虫卵囊黑化中起重要作用。     

Plasmodium infection-induced changes in salivary gland proteins of malaria vector Anopheles stephensi (Diptera:Culicidae)

Parasitism by Plasmodium yoelii yoelii induced 18 polypeptides in the salivary glands of aging malaria vector Anopheles stephensi. A polypeptide of low molecular size (30 kDa) could generally be induced at all infected stages. On day 5 post blood feeding (PBF), no new polypeptide could be found in the salivary glands. Seven polypeptides of low molecular size and 3 of high molecular size could be induced on day 11 PBF, which inducibility coincided with the invasion of the salivary glands by the sporozoites. Quantitatively, soluble proteins decreased in the salivary glands by about one-third in females that had consumed infected or uninfected blood meal on day 9 (oocysts stage) as compared to nonfeeding females. However, on day 15, in the salivary glands invaded by sporozoites, the amount of proteins obtained from infected females was approximately 26% lower than that obtained from uninfected females. A similar reduction was also observed in aged (20 days PBF) salivary glands of infected mosquitoes. These proteins could confer parasite tolerance to the females and enhance parasite transmission potential.     

Natural human humoral response to salivary gland proteins of Anopheles mosquitoes in Thailand

During blood feeding, arthropod vectors inject saliva into vertebrate hosts. The saliva is biochemically complex and pharmacologically active, and may play an important role in pathogen transmission. To examine whether mosquito saliva could elicit humoral immune response in humans under natural conditions, we have collected sera from malaria patients, healthy villagers, and people from a non-malarious region in Thailand. Here we have demonstrated that anti-Anopheles salivary protein antibodies occurred predominantly in patients with acute Plasmodium falciparum or P. vivax malaria, whereas people from a non-malarious area had no such antibodies. Besides, antibody levels against mosquito salivary proteins in malaria patients were highly variable, which may be related to the levels of mosquito exposure. Despite variability, patients' sera with high IgG titers consistently detected several proteins in Anopheles dirus salivary gland protein extracts. Immunohistochemical staining of Anopheles salivary glands with human sera showed that the salivary gland-specific IgGs reacted strongly with the median lobe. Comparison using Anopheles and Aedes salivary proteins suggests that the anti-salivary protein antibodies detected in malaria patients were Anopheles-specific, consistent with the major malaria vector status of An. dirus in this area.     

A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi.

The full development of Plasmodium falciparum in Anopheles stephensi mosquitoes was studied by scanning electron microscopy. Ookinetic development was described from in vitro cultures. Growing oocysts beneath the basal lamina of the midgut wall mechanically stretch this lamina until it is torn and displaced by day 7. In young oocysts the wall appears smooth. In older oocysts wrinkles in the wall are visible after routine fixation. Osmium tetroxide postfixation greatly reduced the occurrence of these wrinkles. Intracapsular development of sporozoites was visualized after mechanical manipulation of the oocysts during sample preparation. In contrast to P. berghei, no ectopic development was seen in P. falciparum in the mosquito midgut. The mechanism of sporozoite escape from the oocyst appears to be similar to that described for rodent malaria. Fracturing of salivary glands provided the first view by scanning electron microscopy of sporozoites located in proximal and distal gland cells and in the draining duct.     

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3--10 days was approximately 1.08 +/- 0.04 microg/female and 0.1 +/- 0.05 microg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.     

硝喹对斯氏按蚊体内不同时期约氏疟原虫发育的影响

目的　观察硝喹对斯氏按蚊体内不同时期约氏疟原虫发育的影响。方法　在感染约氏疟原虫1 d前给予常规蔗糖水或硝喹蔗糖水（含100 μmol/L硝喹）供斯氏按蚊吸食,停糖水24 h后用感染约氏疟原虫的昆明鼠血餐,观察硝喹处理后约氏疟原虫在蚊胃内的卵囊数量变化。感染6、14 d后停常规蔗糖水24 h,随后给予常规蔗糖水或硝喹蔗糖水供斯氏按蚊吸食,观察斯氏按蚊血淋巴及唾液腺中的约氏疟原虫子孢子数量变化。结果　感染前1 d将斯氏按蚊暴露于硝喹蔗糖水后,感染第7天蚊胃中约氏疟原虫卵囊数量[（119.2 ± 16.1）只]较常规蔗糖水组[（207.3 ± 21.8）只]显著降低（t = 3.207,P < 0.05）。感染第6天停常规蔗糖水24 h,将斯氏按蚊暴露于硝喹蔗糖水后,按蚊血淋巴中约氏疟原虫子孢子数量峰值[（952.3 ± 22.7）只]在感染第14天出现,常规蔗糖水组按蚊血淋巴中子孢子数量峰值[（1 287.0 ± 39.0）只]在感染第12天出现;感染第17天,硝喹蔗糖水和常规蔗糖水组按蚊唾液腺中子孢子数量分别为（9 467.0 ± 1 304.0）只和（10 533.0 ± 758.7）只,差异无统计学意义（t = 0.707,P = 0.506）。感染第14天停常规蔗糖水24 h,将斯氏按蚊暴露于硝喹蔗糖水后,按蚊唾液腺中约氏疟原虫子孢子数量[（21 900.0 ± 2 613.0）只]较常规蔗糖水处理组[（10 533.0 ± 732.3）只]显著增加（t = 4.188,P < 0.05）。结论　在斯氏按蚊感染疟原虫不同时期给予硝喹处理对其体内约氏疟原虫发育影响不一,未感染斯氏按蚊经硝喹处理后可减少疟原虫传播。     

云南湄公河流域上游河谷地区疟疾媒介传疟作用研究

目的通过观察蚊媒季节消长、嗜血习性、叮咬行为和疟疾子孢子率以研究出它们在云南湄公河上游河谷地带传疟作用的关系. 方法村内采取诱蚊灯和人工诱捕方法捕蚊;现场采取显微镜解剖蚊虫的方法观察唾腺子孢子,实验室采用ELISA技术检测蚊虫体内环子孢子蛋白(CSP). 结果村内共捕获16种按蚊.现场显微镜解剖7种按蚊,1 010只经产蚊中,发现了7只唾液腺感染子孢子;ELISA方法检测现场捕回的8种按蚊,5 154只各龄期蚊中,发现11只环子孢子蛋白阳性蚊.微小按蚊、中华按蚊、多斑按蚊子孢子阳性率(包括ELISA环子孢子蛋白阳性和镜检唾腺子孢子感染蚊)分别是0.37%、0.22%、0.32%, 昆虫接种率分别估算为9.82、3.97、2.69.这些蚊媒叮咬活动起始于黄昏,并整夜有活动,但微小按蚊夜间活动高峰出现于子夜,中华按蚊和多斑按蚊的高峰则在2100之前.它们的人血指数都在80%以上. 结论以上结果显示微小按蚊、中华按蚊和多斑按蚊是湄公河上游河谷地带的重要传疟媒介.