抗纹媒中肠抗体对冈比亚按蚊生殖率和死亡率影响的研究

目的 探讨冈比亚按蚊中肠抗原抗媒介免疫的作用。进一步研究抗媒介疫苗抗原。方法 解剖室内饲养的冈比亚按蚊雌蚊中肠，立即置于冷的pH7.4PBS溶液中，用SDS-PAGE方法获得分子质量为43ku中肠蛋白抗原，皮下注射免疫大白兔3次，获得的免疫血浆膜饲冈比亚按蚊成蚊，与对照组比较，观察其对冈比亚按蚊死亡率和生殖率的影响。结果 膜饲免疫血浆的冈比亚按蚊的死亡率与对照组相比显著增高，死亡率增加56%，生殖率明显下降，实验组蚊虫平均产卵率为71.4个，对照组为88.1个，下降18.7%。结论 采用有效的方法分离蚊媒中肠的关键性抗原一中肠蛋白抗原可能成为抗蚊媒和其它抗媒介疫苗的靶抗原。     

斯氏按蚊和中华按蚊中肠免疫活性抗原的研究

目的　鉴别斯氏按蚊和中华按蚊中肠具有阻断疟疾传播作用的免疫活性蛋白， 并对斯氏按蚊雌蚊中肠免疫活性蛋白在中肠的分布进行定位。方法　应用酶联免疫电转移印迹技术 （ Ｅ Ｉ Ｔ Ｂ） 分析斯氏按蚊雌、雄蚊和中华按蚊雌蚊中肠的免疫活性抗原； 用间接荧光抗体技术 （ Ｉ Ｆ Ａ） 对斯氏按蚊雌蚊中肠免疫活性抗原进行定位。结果　斯氏按蚊雌、雄蚊蚊中肠抗原及中华按蚊雌蚊中肠抗原与斯氏按蚊雌蚊中肠抗血清作用， 分别显示９ 、９ 和６ 条蛋白带； 斯氏按蚊雌蚊中肠壁外侧出现较强的亮绿色荧光， 肠壁细胞核仅出现样弱的亮绿色荧光。结论　上述三种抗原间既有相同的免疫活性蛋白带， 也有各自特异的蛋白带， ７９ 、７６ 、６０ 、４６ｋ Ｄａ 的蛋白带为斯氏按蚊雌蚊中肠特异性免疫活性蛋白带； 斯氏按蚊雌蚊中肠免疫活性蛋白主要分布在中肠肠壁外侧， 仅有少量分布在肠壁细胞核。     

人环子孢子抗体对感染疟原虫按蚊的影响

蚊媒吸血时获得的人体环子孢子(CS)抗体能否影响子孢子的感染性目前尚有争议。作者对西肯尼亚恶性疟高发地区的媒介——冈比亚按蚊(An.gambiae)和催命按蚊(An.funestus)作了调查,包括野栖按蚊     

试纸条法与PCR法比较：赤道几内亚一个沿海村庄全黑按蚊自然感染的检测[英]／Moreno M，Cano J，Nzambo S，et al//Malaria J

疟疾是赤道几内亚高发病率和死亡率的主要因素之一。以往的调查表明有冈比亚按蚊复合体的5种按蚊分布，冈比亚按蚊复合体和催命按蚊是非洲热带地区主要的传疟媒介。传统方法通过解剖单个按蚊的唾液腺获得按蚊感染疟原虫子孢子率，但这种方法费力费时。以后发展了血清学检测方法和分子检测方法。试纸条法是将针对疟原虫环子孢子蛋白的单抗固定到试纸条，快速简便，按蚊不需要特殊保存条件。     

试纸条法与PCR法比较:赤道几内亚一个沿海村庄全黑按蚊自然感染的检测

疟疾是赤道几内亚高发病率和死亡率的主要因素之一。以往的调查表明有冈比亚按蚊复合体的5种按蚊分布,冈比亚按蚊复合体和催命按蚊是非洲热带地区主要的传疟媒介。传统方法通过解剖单个按蚊的唾液腺获得按蚊感染疟原虫子孢子率,但这种方法费力费时。以后发展了血清学检测方法和分子检测方法。试纸条法是将针对疟原虫环子孢子蛋白的单抗固定到试纸条,快速简便,按蚊不需要特殊保存条件。近年发展起来的聚合酶链反应(PCR)被用于扩增感染按蚊中恶性疟原虫特异DNA序列,PCR法有极高的敏感性,蚊唾液腺中子孢子少至10个时能被检测到,而试纸条法…     

抗斯氏按蚊中肠蛋白组分的抗体对约氏疟原虫卵囊的作用

目的 观察抗斯氏按蚊中肠蛋白组分的抗体对约氏疟原虫卵囊的抑制作用。 方法 解剖实验室饲养斯氏按蚊雌蚊，取中肠（胃）制备中肠蛋白抗原并免疫BALB/c小鼠（8只， 100 μg/只)， 共免疫4次， 每次间隔7～10 d，末次免疫后10 d，腋窝动脉取血，分离血清。用蛋白质印迹（Western blotting）分析中肠蛋白的免疫活性抗原。用葡聚糖凝胶过滤法获得相对分子质量（Mr）为38 000~50 000的蛋白。用该中肠蛋白免疫小鼠（12只， 100 μg/只)， 共免疫4次，每次间隔7~10 d。同时设PBS对照组。末次免疫后7 d，ELISA检测小鼠血清中的抗体，抗体效价≥1 : 2 560时，该免疫组小鼠和对照组小鼠经腹腔接种感染约氏疟原虫（约含2×10⁷个感染疟原虫的红细胞），感染后3 d取小鼠尾血镜检，雌配子体数＞2/10个视野的小鼠作为供血鼠，斯氏按蚊成蚊吸血后9 d解剖，计数中肠的卵囊数量。 结果 Western blotting显示斯氏按蚊中肠蛋白抗原的显色区带有8条，其中Mr 38 000~50 000的区带显色较清晰；实验组和对照组中肠卵囊感染率分别为28.70%（62/216）和51.09%（47/92）（P＜0.05），中肠卵囊指数分别为14.14（1 541/109）和26.02（1 223/47），两者差异有统计学意义（P＜0.01）。 结论 斯氏按蚊Mr 38 000~50 000中肠蛋白有免疫活性；针对该中肠蛋白的抗体对约氏疟原虫卵囊的发育有明显的抑制作用。     

蚊与疟原虫相互关系Ⅱ:支持疟原虫在按蚊体内发育的因子

疟疾是一种严重危害人类健康和阻碍流行区国家经济、社会发展的寄生虫病,随着多抗药虫株的出现和媒介按蚊对杀虫剂产生抗性,疟疾的防治面临新的挑战。对按蚊与疟原虫相互关系的研究,特别是寻找按蚊支持疟原虫发育因子,弄清疟原虫在蚊体内的配子生殖和孢子生殖机制,可为疟疾防治研究提供有益线索。近年来,这方面的研究已经取得了一些进展,这些进展不但促进了人们对疟原虫和按蚊相互关系的理解,丰富了疟原虫生物学知识,更重要的是可为寻找新型杀虫剂靶点、发现新的抗疟疫苗的合适候选抗原、完善疟原虫的体外培养技术、实现遗传改造蚊媒的防疟…     

冈比亚按蚊和阿拉伯按蚊在塞内加尔具有相似的吸血嗜性

评价一种媒介传疟能力即媒介能量的主要指标是人蚊接触强度,特别是叮人率和人血指数。以往大多数的研究认为,同样作为非 洲的主要传疟媒介,冈比亚按蚊比阿拉伯按蚊更嗜人血。作者曾在塞内加尔对室内栖息的按蚊进行调查,发现冈比亚按蚊和阿拉伯按蚊具有相似的人血指数,为了证实这一点,作者又在室外在相同条件下对这两种按蚊的吸血嗜性进行了观察。     

阿拉伯按蚊遗传的性分离和不育性杂种的产生

冈比亚按蚊复合体是非洲传播疟疾和丝虫病的主要媒介。阿拉伯按蚊(A.arabiensis)即冈比亚按蚊 B,因其野栖性,室内滞留喷洒杀虫剂无效。因而对于这种按蚊特别值得考虑遗传学的防制方法。一种叫做 R70的遗传性系统,是以常染色体上的抗狄氏剂半显     

肯尼亚西部按蚊幼虫的空间分布和孳生地特点

在肯尼亚当地恶性疟在人群中的流行率大于50％。重要的传播媒介有冈比亚按蚊种团和催命按蚊,其中在肯尼亚同一地区出现了3种冈比亚种团的按蚊,包括冈比亚按蚊,阿拉伯按蚊和纯净按蚊。许多研究表明,在肯尼亚西部和沿海地带媒介密度有明显差异。     

Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion

Malaria transmission entails development of the Plasmodium parasite in its insect vector, the Anopheles mosquito. Parasite invasion of the mosquito midgut is the critical first step and involves adhesion to host epithelial cell ligands. Partial evidence suggests that midgut oligosaccharides are important ligands for parasite adhesion; however, the identity of these glycans remains unknown. We have identified a population of chondroitin glycosaminoglycans along the apical midgut microvilli of Anopheles gambiae and further demonstrated ookinete recognition of these glycans in vitro. By repressing the expression of the peptide-O-xylosyltransferase homolog of An. gambiae by means of RNA interference, we blocked glycosaminoglycan chain biosynthesis, diminished chondroitin sulfate levels in the adult midgut, and substantially inhibited parasite development. We provide evidence for the in vivo role of chondroitin sulfate proteoglycans in Plasmodium falciparum invasion of the midgut and insight into the molecular mechanisms mediating parasite-mosquito interactions.     

Increased melanizing activity in Anopheles gambiae does not affect development of Plasmodium falciparum

Serpins are central to the modulation of various innate immune responses in insects and are suspected to influence the outcome of malaria parasite infection in mosquito vectors. Three Anopheles gambiae serpins (SRPN1, -2, and -3) were tested for their ability to inhibit the prophenoloxidase cascade, a key regulatory process in the melanization response. Recombinant SRPN1 and -2 can bind and inhibit a heterologous phenoloxidase-activating protease and inhibit phenoloxidase activation in vitro. Using a reverse genetics approach, we studied the effect of SRPN2 on melanization in An. gambiae adult females in vivo. Depletion of SRPN2 from the mosquito hemolymph increases melanin deposition on foreign surfaces such as negatively charged Sephadex beads. As reported, the knockdown of SRPN2 adversely affects the ability of the rodent malaria parasite Plasmodium berghei to invade the midgut epithelium and develop into oocysts. Importantly, we tested whether the absence of SRPN2 from the hemolymph influences Plasmodium falciparum development. RNAi silencing of SRPN2 in an An. gambiae strain originally established from local populations in Yaoundé, Cameroon, did not influence the development of autochthonous field isolates of P. falciparum. This study suggests immune evasion strategies of the human malaria parasite and emphasizes the need to study mosquito innate immune responses toward the pathogens they transmit in natural vector-parasite combinations.     

蚊媒感染疟原虫后应答性表达增高基因的富集和筛选

目的　分离和识别蚊媒疟原虫感染相关基因并探讨其机制。方法　以斯氏按蚊 约氏疟原虫为实验模型 ,分别将刺叮约氏疟原虫感染鼠血和正常鼠血后 2 4h的饱血斯氏按蚊作为T组和D组 ,经抑制性差减杂交和选择性PCR扩增 ,建立一个T组表达特异增高基因的差减cDNA库。将此差减库分别与T组和D组的cDNAs混合探针作斑点杂交 ,筛选出差异表达基因 ,测定其序列并做基因库BLAST搜索。结果　T组相对于D组的差异表达基因得到有效富集 ,5 8个重组克隆中有 2 4个表达增高 ,阳性率为 4 1.4 %。所代表的 2 3个基因搜索结果显示 ,12个与功能已知的基因同源 ,4个与功能未知的基因同源 ,7个为新发现的基因 ;其中 9个与LPS诱导的冈比亚按蚊免疫反应性细胞系EST同源 ,占 39.1% (9/ 2 3)。结论　建立了蚊媒感染疟原虫早期 (2 4h)直接导致的全基因组应答性表达增高cDNA库 ,筛选结果显示蚊虫对疟原虫感染的反应涉及多种生化途径及机制 ,尤其与天然免疫系统和能量代谢关系密切     

抗白纹伊蚊组织抗体在蚊体内的动态分布

目的　研究抗白纹伊蚊组织抗体在蚊体内的动态分布情况。方法　采用直接法免疫酶组织化学染色法对吸食免疫小鼠血后不同时间的白纹伊蚊组织切片染色，在镜下观察并摄片。结果　白纹伊蚊吸食免疫小鼠血后，0 h肠腔内呈阳性染色，肠壁及其它组织无着色反应；12 h肠壁细胞、马氏管和卵巢均出现阳性反应，蚊围食膜开始出现，但不完整；24 h围食膜发达，膜壁很厚。卵巢反应较强，马氏管和肠壁细胞反应较弱；36 h围食膜消失，血液完全被消化。卵巢呈阳性染色，肠壁及马氏管无明显反应；48 h除卵巢小管壁呈阳性反应外，仅少量卵巢管内部出现着色反应。马氏管和肠壁无反应。结论　（1）白纹伊蚊吸血后12 h围食膜开始出现，到24 h时最发达，36 h后消失；（2）抗白纹伊蚊小鼠抗体可穿过蚊肠壁进入到蚊体腔，并与相应的靶组织结合；（3）抗蚊抗体在蚊吸血后12 h进入蚊体腔的量最多，抗体与蚊卵巢组织的作用时间最长。     

A cell surface mucin specifically expressed in the midgut of the malaria mosquito Anopheles gambiae

An invertebrate intestinal mucin gene, AgMuc1, was isolated from the malaria vector mosquito Anopheles gambiae. The predicted 122-residue protein consists of a central core of seven repeating TTTTVAP motifs flanked by hydrophobic N- and C-terminal domains. This structure is similar to that of mucins that coat the protozoan parasite Trypanosoma cruzi. Northern blot analysis indicated that the gene is expressed exclusively in the midgut of adult mosquitoes. A length polymorphism and in situ hybridization were used to genetically and cytogenetically map AgMuc1 to division 7A of the right arm of the second chromosome. The subcellular localization of the encoded protein in tissue culture cells was examined by using a baculovirus vector to express AgMuc1 protein tagged with the green fluorescent protein (GFP). The results indicated that this protein is found at the cell surface and that both hydrophobic domains are required for cell surface targeting. We propose that AgMuc1 is an abundant mucin-like protein that lines the surface of the midgut microvilli, potentially protecting the intestinal epithelium from the proteinase-rich environment of the gut lumen. An intriguing possibility is that, as an abundant surface protein, AgMuc1 may also interact with the malaria parasite during its invasion of the mosquito midgut.     

The presence of Plasmodium falciparum gametocytes in human blood increases the gravidity of Anopheles gambiae mosquitoes

We conducted a field study in an area of endemic malaria transmission in western Kenya to determine whether mosquitoes that feed on gametocyte-infected blood but do not become infected have reduced or enhanced fecundity in comparison to mosquitoes fed on uninfected blood. Fifteen paired membrane-feeding experiments were conducted in which two strains of Anopheles gambiae mosquitoes were simultaneously fed on either Plasmodium falciparum-infected blood from children or uninfected control blood from adults. The presence of noninfecting gametocytes in blood increased the probability that An. gambiae would produce eggs after one blood meal by sixfold (odds ratio for control relative to infected blood group 0.16; 95% CI 0.10-0.23). This result could not be explained by variation in blood meal size or hemoglobin content between hosts. When children cleared their infections, the difference in gravidity between mosquitoes fed on their blood and uninfected adults disappeared, suggesting this phenomenon is due to the presence of Plasmodium gametocytes in blood and not to host-specific factors such as age. This result was observed in two mosquito strains that differ in their innate fecundity, suggesting it may apply generally. To our knowledge, this is the first time that Plasmodium has been implicated as enhancing vector gravidity.     

Contribution of Anopheles funestus, An. gambiae and An. nili (Diptera: Culicidae) to the perennial malaria transmission in the southern and western forest areas of Côte d'Ivoire

The involvement of members of the Anopheles gambiae complex Giles and An. funestus Giles and An. nili Theobald groups in the transmission of Plasmodium falciparum was recently investigated in the villages of Gbatta and Kpéhiri, which lie, respectively, in forest areas in the west and south of C?te d'Ivoire. Adult female mosquitoes were collected, using human landing catches, inside and outside dwellings. After identification and dissection, the heads and thoraces of all the anopheline mosquitoes were tested, in an ELISA, for circumsporozoite protein (CSP). All the female anopheline mosquitoes collected and identified to species using PCR were found to be An. gambiae s.s., An. nili s.s. or An. funestus s.s., with An. gambiae s.s. and An. funestus s.s. predominant in Gbatta but An. nili s.s. the most common species in Kpéhiri. In Gbatta, 3·1% of the female An. gambiae collected, 5·0% of the female An. funestus and 1·8% of the female An. nili were found CSP-positive. The corresponding values in Kpéhiri were even higher, at 5·9%, 6·2% and 2·4%, respectively. The estimated entomological inoculation rates (EIR) were very high: 302 infected bites (139 from An. gambiae, 146 from An. funestus and 17 from An. nili)/person-year in Gbatta and 484 infected bites (204 from An. gambiae, 70 from An. funestus and 210 from An. nili)/person-year in Kpéhiri. In Gbatta, An. gambiae s.s. was responsible for most of the rainy-season transmission while An. funestus became the main malaria vector in the dry seasons. In Kpéhiri, however, An. nili appeared to be the main vector throughout the year, with An. gambiae of secondary importance and An. funestus only becoming a significant vector during the rainy season. Although, in both study sites, intense transmission was therefore occurring and the same three species of anopheline mosquito were present, the relative importance of each mosquito species in the epidemiology of the human malaria at each site differed markedly.     

中华按蚊抗菌肽defensinA编码基因cDNA克隆与序列分析

目的克隆中华按蚊defensin编码基因,并对其序列进行分析。方法提取中华按蚊总RNA。据文献报道合成1对引物def1、def2,RT-PCR扩增中华按蚊defensin编码基因,克隆于T-载体,序列测定与分析。结果RT-PCR扩增中华按蚊cDNA获大约308 bp片段,测定目的片段序列,结果显示,克隆基因cDNA序列长度为308 bp,推导编码64个氨基酸,序列分析显示中华按蚊defensin编码基因与冈比亚按蚊defensin编码基因有高度同源性(95%)。分析结果显示,中华按蚊defensin编码基因为新基因,定名为中华按蚊defensinA。结论克隆出中华按蚊defensinA编码基因,为进一步研究该基因打下了基础。     

Immune interactions between mosquitoes and their hosts

The intimate contact between mosquitoes and the immune system of their hosts is generally not considered important because of the transient nature of mosquito feeding. However, when hosts are exposed to many feeding mosquitoes, they develop immune responses against a range of salivary antigens. Understanding the importance of these responses will provide new tools for monitoring vector populations and identifying individuals at risk of mosquito-borne diseases, and allow the development of novel methods for monitoring control and mosquito-release programmes. Antibodies targeting the mosquito midgut are also important in the development of mosquito vaccines. The feasibility of this approach has been demonstrated and future research opportunities are considered in this review. The potential impact of mosquito vaccines is also discussed. Our understanding of the interplay between mosquitoes and the immune system of their hosts is still in its infancy, but it is clear that there is great potential for exploiting this interplay in the control of mosquito-borne diseases.     

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.